WO2013173337A2 - Self-stabilizing linker conjugates - Google Patents

Self-stabilizing linker conjugates Download PDF

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Publication number
WO2013173337A2
WO2013173337A2 PCT/US2013/040951 US2013040951W WO2013173337A2 WO 2013173337 A2 WO2013173337 A2 WO 2013173337A2 US 2013040951 W US2013040951 W US 2013040951W WO 2013173337 A2 WO2013173337 A2 WO 2013173337A2
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WO
WIPO (PCT)
Prior art keywords
unit
ligand
drug
linker
conjugate
Prior art date
Application number
PCT/US2013/040951
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French (fr)
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WO2013173337A3 (en
Inventor
Robert Lyon
Svetlana Doronina
Timothy Bovee
Original Assignee
Seattle Genetics, Inc.
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Publication date
Priority claimed from US13/799,244 external-priority patent/US9504756B2/en
Priority to JP2015512756A priority Critical patent/JP6423340B2/en
Priority to KR1020227042635A priority patent/KR102557309B1/en
Priority to EA201401254A priority patent/EA037203B1/en
Priority to KR1020237024059A priority patent/KR20230113821A/en
Priority to CN201810349815.9A priority patent/CN108465112B/en
Priority to CN201380025771.XA priority patent/CN104822656B/en
Priority to EP13791383.6A priority patent/EP2850094B1/en
Priority to AU2013263002A priority patent/AU2013263002C1/en
Priority to KR1020217023061A priority patent/KR102475777B1/en
Priority to KR1020207022850A priority patent/KR102433686B1/en
Priority to KR1020147034652A priority patent/KR102144069B1/en
Application filed by Seattle Genetics, Inc. filed Critical Seattle Genetics, Inc.
Priority to NZ630870A priority patent/NZ630870A/en
Priority to SG11201406252WA priority patent/SG11201406252WA/en
Priority to BR112014028222A priority patent/BR112014028222A2/en
Priority to CA2869777A priority patent/CA2869777C/en
Priority to MX2014013807A priority patent/MX368678B/en
Publication of WO2013173337A2 publication Critical patent/WO2013173337A2/en
Priority to ZA2014/07369A priority patent/ZA201407369B/en
Publication of WO2013173337A3 publication Critical patent/WO2013173337A3/en
Priority to HK15108030.8A priority patent/HK1207388A1/en
Priority to AU2018201190A priority patent/AU2018201190B2/en
Priority to AU2020202805A priority patent/AU2020202805B2/en
Priority to AU2021201636A priority patent/AU2021201636B2/en
Priority to AU2023203005A priority patent/AU2023203005A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • A61K47/6801Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • A61K47/6801Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • A61K47/6801Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • A61K47/6835Medicinal 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
    • A61K47/6851Medicinal 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/68Medicinal 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
    • A61K47/6889Conjugates 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
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00Drugs for immunological or allergic disorders

Definitions

  • ADC antibody-drug conjugate
  • the invention provides inter alia, Linkers, Drug-Linkers, Ligand-Drug Conjugates, Ligand-Linker Conjugates, Ligand-Functional Agent Conjugates, and Functional Agent-Linkers, and methods of preparing and using them.
  • the Ligand-Drug Conjugates are stable in circulation, yet capable of inflicting cell death once released in the vicinity or within tumor cells.
  • Figure 1 provides a reaction scheme illustrating the reduction of the interchain disulfides of a human IgGl , followed by conjugation of the resulting thiols with a self-stabilizing linker and subsequent hydrolysis of the succinimide ring (Top); and the use of mass spectrometry to monitor the change in the molecular weight of the antibody conjugates due to hydrolysis (Bottom).
  • FIG. 2 illustrates the timecourse of succinimide ring hydrolysis of a self-stabilizing antibody conjugate by electrospray mass spectrometry.
  • Conjugation of fully reduced cACI O with maieimido-DPR-val-cit-PAB-MMAE was performed at pH 7.2 and 22°C, then samples were subjected to analysis by LC-MS at the indicated times (Top).
  • the resulting data of % hydrolysis was plotted versus time and fit to an exponential equation to determine kinetic parameters (Bottom).
  • Figure 3 provides the hydrolysis kinetic profiles for bioconjugates prepared with an IgGl antibody and self-stabilizing linkers with varying spacing between the maleimide and the basic group (a primary amine). Conjugation was performed at pH 8 and 37°C, then hydrolysis of the IgGl light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation.
  • Figure 4 provides kinetic profiles of the hydrolysis of bioconjugates prepared with an IgG l antibody and self-stabilizing maleimide linkers with varying spacing between the maleimide and the basic group (a primary amine). Conjugation was performed at pH 8 and 37°C, then hydrolysis of the IgG l light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation.
  • Figure 5 provides hydrolysis kinetic profiles for bioconjugates prepared with an IgGl antibody and various N-substituted maieirnides. Conjugation was performed at pH 7.4 and 22°C, then hydrolysis of the IgG l light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation. Hydrolysis of the maleimido-caproyl conjugate (bottom structure) is too slow to produce any detectable hydrolysis in 24 hours under these conditions.
  • Figure 6 provides hydrolysis kinetic profiles for self-stabilizing maleimido drug- linkers prepared with et-diaminopropionic acid (a-DPR, open circles) and with ⁇ - diaminopropionic acid ( ⁇ -DPR, filled circles). Although isomers of each other, the positioning of the basic amino group and the electron withdrawing carboxamide relative to the succinimide results in a 17-fold difference in the rate of succinimide hydrolysis.
  • Figure 7 illustrates the change in drug loading over time for an ADC prepared with a self-stabilizing maleimido-DPR drug-linker versus one prepared with a maieimido-caproyl drug linker when incubated in a buffer containing excess thiol.
  • the reversed -phase chromatograms of the two ADCs at lime zero and time 14 days after incubation is shown in the top panel.
  • Chromatographic peak assignments L0, LI , HO, HI, H2, and H3 correspond to unconjugated light chain, light chain with one drug, unconjugated heavy chain, and heavy chain with 1 , 2, or 3 drugs, respectively.
  • the self-stabilizing maleimido-DPR drug-linker is represented with open circles versus one prepared with a maieimido-caproyl drug linker (open squares). Drug loading remains constant at 8 per antibody for the self-stabilizing drug-linker (open circles), but falls to 4 drugs per antibody over 14 days for the maieimido-caproyl drug linker (open squares), reflecting loss of drug by maleimide elimination.
  • ADC samples at each timepoint were purified by Ig Select affinity resin and their drug loading evaluated by reversed-phase HPLC analysis of the ADCs.
  • ADCs were captured on Protein A affinity resin at each timepoint and the drug released enzymatically via its protease-cleavable linker. The released drug was then quantified by LC- MS/MS and normalized to the initial value. Each timepoint reflects the percent of the conj ugated drug that was observed at tO.
  • ADCs were dosed i.v. and plasma samples from each timepoint were purified by Ig Select affinity resin and their drug loading evaluated by reversed-phase HPLC analysis of the ADCs.
  • FIG 11 illustrates the antitumor activity of ADCs in a murine xenograft model of ALCL (Karpas-299 cell line).
  • ADCs were prepared with the anti-CD3G antibody cACl O and drug linkers containing the val-cit-PAB-MMAE cytotoxic payload linked to the antibody via either a maleimido-caproyl group (closed circles) or a self-stabilizing maleimido-DPR group (open circles).
  • Tumors were allowed to reach a volume of approximately 250 mm J before dosing at 1 mg/kg weekly for three doses (six mice per dose group).
  • the self-stabilizing ADC dose group experienced complete responses (no detectable tumor) in all six animals, with five animals experiencing durable regressions, while the maleimido-caproyl ADC experienced no complete responses.
  • trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.
  • electron-withdrawing group refers to a functional group that draws electrons away from a reaction center.
  • electron withdrawing groups can also include aryl groups (e.g., phenyl) and certain heteroaryj groups (e.g., pyridine).
  • electron withdrawing groups includes aryls or heteroaryls further substitued with electron withdrawing groups.
  • base refers to a functional group that deprotonates water to produce a hydroxide ion.
  • exemplary bases are amines and nitrogen containing heterocycles.
  • Representative bases include -N(R 3 )(R 4 ) wherein R 3 and R 4 are independently selected from H or Ci-6 alkyl, preferably H or methyl,
  • R 5 , R & , R 7 and R 8 are, at each occurrence, independently selected from hydrogen or Ci-g alky], preferably H or methyl, and e is 0-4.
  • the base is a nitrogenous base.
  • antibody herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, muitispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity.
  • An intact antibody has primarily two regions: a variable region and a constant region.
  • the variable region binds to and interacts with a target antigen.
  • the variable region includes a complementary determining region (CDR.) that recognizes and binds to a specific binding site on a particular antigen.
  • CDR. complementary determining region
  • the constant region may be recognized by and interact with the immune system (see, e.g., Janeway et al, 2001, Immuno, Biology, 5th Ed., Garland Publishing, New York).
  • An antibody can be of any type (e.g. , IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, lgG3, IgG4, IgAl and IgA2) or subclass.
  • the antibody can be derived from any suitable species.
  • the antibody is of human or murine origin,
  • An antibody can be, for example, human, humanized or chimeric,
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • an "intact antibody” is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, C H 1 , C H 2, Q arid C H , as appropriate for the antibody class.
  • the constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
  • an "antibody fragment” comprises a portion of an intact antibody, comprising the antigen-binding or variable region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragments) produced by a Fab expression library, or an epitope-binding fragments of any of the above which immunospecificaily bind to a target antigen (e.g., a cancer ceil antigen, a viral antigen or a microbial antigen).
  • a target antigen e.g., a cancer ceil antigen, a viral antigen or a microbial antigen.
  • An "antigen” is an entity to which an antibody specifically binds.
  • the terms “specific binding” and “specifically binds” mean that the antibody or antibody derivative will bind, in a highly selective manner, with its corresponding target antigen and not with the multitude of other antigens.
  • the antibody or antibody derivative binds with an affinity of at least about IxlO "7 M, and preferably 10 "8 M to iO "9 M, 10 "10 M, 10 ⁇ n M, or 10 "!2 M and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen.
  • the term “inhibit” or “inhibition of means to a reduce by a measurable amount, or to prevent entirely.
  • the term "therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a mammal.
  • the therapeutically effective amount of the drug may reduce the number of cancer ceils; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer.
  • the drug may inhibit growth and/or kill existing cancer ceils, it may be cytostatic and/or cytotoxic.
  • efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
  • substantially refers to a majority, / ' , e. >50% of a population, of a mixture or a sample, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% of a population.
  • intracellulariy cleaved and “intracellular cleavage” refer to a metabolic process or reaction inside a ceil on a Ligand Drug conjugate (e.g., an Antibody Drug Conjugate (ADC) or the like), whereby the covalent attachment, e.g., the linker, between the Drug moiety (D) and the Ligand unit (e.g., an antibody (Ab)) is broken, resulting in the free Drug, or other metabolite of the conjugate dissociated from the antibody inside the cell.
  • ADC Antibody Drug Conjugate
  • the cleaved moieties of the Drug-Linker-Ligand conjugate are thus intracellular metabolites.
  • cytotoxic activity refers to a cell-killing, a cytostatic or an anti-proliferative effect of a Drug-Linker-Ligand con jugate compound or an intracellular metabolite of a Drug- Linker-Ligand conjugate. Cytotoxic activity may be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half the cells survive.
  • cytotoxic agent refers to a substance that inhibits or inhibits the function of cells and/or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., 211 At, 13i I, 125 1, 90 Y, f 86 Re, 188 Re, 1S3 Sm, 2i2 Bi, 32 P, 60 C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enz raatically active toxins of bacterial fungal, plant or animal origin, including synthetic analogs and derivatives thereof.
  • radioactive isotopes e.g., 211 At, 13i I, 125 1, 90 Y, f 86 Re, 188 Re, 1S3 Sm, 2i2 Bi, 32 P, 60 C, and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., chemotherapeutic agents, and toxins such as small molecule toxins or enz
  • cancer and “cancerous” refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth.
  • a “tumor” comprises one or more cancerous cells.
  • autoimmune disease herein is a disease or disorder arising from and directed against an individual's own tissues or proteins.
  • Examples of a "patient” include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl.
  • the patient is a human.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • treating includes any or all of: inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.
  • the term “treating” includes any or all of: inhibiting replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease.
  • the term "Detection unit” refers to refers to any molecule which produces, or can be induced to produce, a detectable signal. Detection units having reporter molecules that can be detected by imaging equipment include, but are not limited to, radioactive, paramagnetic, fluorescent or radtoopaque chemical entities. In some embodiments, the
  • Detection unit will be a radioactive compound, a chemilummescent agent, a fluorescent agent, or a chromogen. In some embodiments, the Detection unit will be a fluorescent molecule such as a fluorophore.
  • Stability unit refers to a compound that promotes the stability of the conjugate, e.g., by increasing systemic retention of the Ligand when administered to a patient.
  • a Stability unit can also increase the water solubility of the conjugate.
  • An exemplary Stability unit is polyethylene glycol
  • Th compound can contain at least one amino group, and accordingly acid addition salts can be formed with the amino group.
  • Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tarmate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1 ,1 '-methyl ene-bis - (2-hydroxy-3- naphthoate)) salts.
  • pamoate i.e., 1 ,
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterfoil may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. " Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
  • alkyl by itself or as part of another term refers to a substituted or unsubstituted straight chain or branched, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "-CfCg alkyl” or “-Cf-Cio alkyl refer to an aikyl group having from I to 8 or 1 to 10 carbon atoms, respectively). When the number of carbon atoms is not indicated, the aikyl group has from 1 to 8 carbon atoms.
  • Representative straight chain "-Cj -Cg aikyl” groups include, but are not limited to, -methyl, -ethyl, -n-propyl, - n-butyl, -n-pentyl, -n-hexyl, -n-heptyi and -n-octyl; while branched -Ci-Cg alkyis include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -fert-butyl, -isopent l, and -2-methylbutyl;
  • unsaturated -C 2 -C 8 alkyis include, but are not limited to, -vinyl, -allyl, -1 -butenyl, -2-butenyl, -isobutyienyl, -1-pentenyl, -2-pentenyl, -3-methyI- 1 -butenyl, -2-methyl-2 ⁇ butenyl,
  • an aikyl group is unsubstituted.
  • An aikyl group can be substituted with one or more groups.
  • an aikyl group will be saturated,
  • alkylene by itself of as part of another term, refers to a substituted or unsubstituted saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-10 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent, alkane.
  • Typical alkylene radicals include, but are not limited to:
  • an alkylene is a branched or straight chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).
  • aryl by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
  • Some aryl groups are represented in the exemplary structures as "Ar".
  • Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like.
  • An exemplary aryl group is a phenyl group as follows:
  • an "arylene,” by itself or as part of another term, is an aryl group as defined above which has two covalent bonds (i.e., it is divalent) and can be in the ortho, meta, or para configurations as shown in the following structures, with phenyl as the exemplary group:
  • a C 3 -C S heterocycle refers to a monovalent substituted or unsubstiiuted aromatic or non-aromatic monocyclic or bicyclic ring system having from 3 to 8 carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P or S, and derived by removal of one hydrogen atom from a ring atom of a parent ring system.
  • One or more N, C or S atoms in the heterocycle can be oxidized.
  • the ring that includes the heteroatom can be aromatic or nonaromatic.
  • the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
  • Representative examples of a C Cg heterocycle include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpbolinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, inclolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazoiyl, imidazolyl, pyrazoiyl, pyrimidinyl, pyridinyl, pvrazinyl, pyridazinyl. isothiazolyi, and isoxazolyl.
  • A"C 3 -Cs Iieteroaryi is an aromatic C 3 -C 8 heterocycle.
  • C 3 -Q heterocyclo refers to a C 3 -Cg heterocycle group defined above wherein one of the heterocycle group's hydrogen atoms is replaced with a bond (i.e., it is divalent).
  • C 3 -C 3 carbocycle by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7- or 8-menibered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system.
  • 32 include, but are not limited to, cyclopropyi, cydobutyl, cyelopentyl, cydopentadienyl, cyelohexyl, cyelohexenyi, 1 ,3-cyclohexadienyl, 1,4-cyclohexadienyl, eyeloheptyl, 1,3- cycioheptadienyl, 1 ,3,5-cydoheptatrieny I, cyciooctyl, and cyclooctadienyl.
  • C 3 -Cg carbocyclo refers to a Q Cs carbocycle group defined above wherein another of the carbocycle groups' hydrogen atoms is replaced with a bond (i.e., it is divalent),
  • heteroalkyi by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 3 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to ten, preferably one to three, heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized.
  • the heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyi group or at the position at which the alky! group is attached to the remainder of the molecule.
  • a Cj to C 4 heteroalkyi or heteroaikylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms and a Q to C 3 heteroalkyi or heteroaikylene has 1 to 3 carbon atoms and 2 or 2 heteroatoms.
  • a heteroalkyi or heteroaikylene is saturated.
  • heteroaikylene by itself or as part of another subsiituent means a divalent group derived from heteroalkyi (as discussed above), as exemplified by -CH 2 -CH 2 -S-CH 2 -CH 2 - and - H2-S-CH2-CH2-NH-CH2-.
  • heteroaikylene groups heteroatoms can also occupy either or both of the chain termini.
  • alkylene and heteroaikylene linking groups no orientation of the linking group is implied,
  • Substituted alkyl and “substituted aryl” mean alkyl and aryl, respectively, in which one or more hydrogen atoms are each independently replaced with a subsiituent.
  • Typical substiiuerrts include, but are not limited to, -X, -R, -O " , -OR, -SR, -S " , -NR 2 , -NR 3 ,
  • each X is independently a halogen: -F, -CI, -Br, or -I; and each R. is independently -H, -C; ⁇ C 2 o alkyl, -
  • R.G is a reactive group that contains a reactive site (RS) that is capable of forming a bond with either the components of the Linker unit (i.e., A, W, Y) or the Drug unit D.
  • RS is the reactive site within a Reactive Group (RG).
  • Reactive groups include sulfhydryj groups to form disulfide bonds or thioether bonds, aldehyde, ketone, or hydrazine groups to form hydrazone bonds, carboxylic or amino groups to form peptide bonds, carboxylic or hydroxy groups to form ester bonds, sulfonic acids to form sulfonamide bonds, alcohols to form carbamate bonds, and amines to form sulfonamide bonds or carbamate bonds.
  • the following table is illustrative of Reactive Groups, Reactive Sites, and exemplary functional groups that can form after reaction of the reactive site. The table is not limiting.
  • R' may represent one or more components of the self-stabilizing linker or optional secondary linker, as the case may be, and R" may represent one or more components of the optional secondary linker, Drug unit, Stability unit, or Detection unit, as the ease may be.
  • the reactive site RS can form a new bond with components of the Linker unit or the Drug unit, as the case may be.
  • the reactive site, RS, once linked to the remainder of the Linker unit has typically lost its reactivity,
  • dibenzyl sulfonylurea refers to a cyclic amide that forms from a macro- cyclicization reaction with a thio-substituted succinimide and base present on the self-stabilizing linker assembly.
  • Hydrolysis of a maieimide represents a nucieophi!ic addition reaction in which water, acting as the nucleophile, attacks one of the electrophi!ic carbonyl carbon atoms of the maieimide ring (or succinimide ring).
  • the rate of this reaction is influenced by eleciroph iicity of the carbonyls, which can vary with the substitution of electron- donating or electron- withdrawing groups present on the nitrogen of the imide group.
  • the rate of the hydrolysis reaction is also influenced by the pH of an aqueous solvent, which effectively increases the nucleopliilicity of water with increasing pH.
  • the present invention provides, inter alia, N-substituted maleimides with hydrolysis rates that fall within a useful range wherein their reaction with thiols occurs mors quickly than their hydrolysis to the maieie acid derivative, but which yield thio-substituted succinimides with hydrolysis rates that are sufficiently rapid to achieve complete hydrolysis under gentle conditions that are very suitable for the manufacture of protein-based bioconjugates.
  • the present invention is based, in part, on the discovery that a basic functional group proximal to a maleimide will catalyze the hydrolysis of a thio-substituted succinimide which is formed upon conjugation of the maleimide and a protein thiol leading to a stable bioeonjugate.
  • a proximal basic group with an electron withdrawing group
  • the rate of thio-substituted succinimide ring hydrolysis can be tuned to a desirable level.
  • Design parameters that affect the rate of hydrolysis include the p a of the basic group, the strength of the electron withdrawing group when present, and the proximity of both groups to the maleimide carbonyl carbons.
  • Design parameters that affect the percentage hydrolysis include the nature and proximity of the base to the maleimide carbonyl carbons.
  • a Linker unit comprising a self-stabilizing linker assembly is referred to herein as a Self-Stabilizing Linker or Self-Stabilizing Linker unit.
  • the Self-Stabilizing Linker prior to conjugation with the Ligand unit comprises a maleimide group.
  • the Self-Stabilizing Linker is self-stabilizing by virtue of the proximity of the maleimide group to a base within the linker unit which catalyzes the hydrolysis of its own thio-substituted succinimide after conjugation to the Ligand unit. This is represented schematically below:
  • Self-Stabilizing Linker refers to the Linker unit both prior to and post stabilization
  • a Ligand-Functional Agent Conjugate comprising a Ligand unit and at least one Functional Agent selected from a. Drug unit, a Detection Unit, or a Stabilizing Unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide ring directly conjugated to the Ligand unit via a thioether linkage; and a base and an electron withdrawing group (conjugated to the Ligand unit via the succinimide) operably linked to stabilize the conjugate in plasma relative to a ligand drug conjugate lacking the self-stabilizing linker assembly (i.e.
  • the electron withdrawing group is positioned to increase the electrophilicity of the succinimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide ring (e.g., by an intramolecular base cataly sis mechanism).
  • the succinimide ring in place of the succinimide ring is a dilactam formed when the base reacts with the succinimide ring.
  • Functional Agent- Linker units are provided wherein the Linker portion comprises a self-stabilizing linker assembly
  • Ligand-Linker conjugates are provided, wherein the Linker portion comprises a self-stabilizing linker assembly.
  • the Linker portion further comprises an optional secondary linker assembly (L°).
  • the Ligand-Functional Agent Conjugate is a Ligand-Drug Conjugate.
  • the present invention provides in one group of embodiments, a Ligand-Drug Conjugate comprising a Ligand unit and at least one Drug unit, wherein the Ligand unit and each of the Drug unit(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide ring directly conjugated to the Ligand unit via a thioether linkage; and a base and an electron withdrawing group (conjugated to the Ligand unit via the succinimide ring) operably linked to stabilize the conjugate in plasma relative to a ligand drug conjugate lacking the self-stabilizing linker assembly (i.e.
  • the electron withdrawing group is positioned to increase the electrophilicity of the succinimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide ring (e.g., by an intramolecular base catalysis mechanism), in some aspects, in place of the succinimide ring is a dilactam formed when the base reacts with the succinimide ring.
  • Drug-Linker units are provided wherein the Linker portion comprises a self-stabilizing linker assembly.
  • Ligand-Linker conjugates are provided, wherein the Linker portion comprises a self-stabilizing linker assembly, In some embodiments, the Linker portion further comprises an optional secondary linker assem bly (L°). In some embodiments, the secondary linker assembly is a releasabie linker assembly (L * ) which comprises a Cleavable unit and optionally one or more of a Stretcher and a Spacer unit. In some other embodiments, the secondary linker assembly is a non-releasable linker assembly (L N ) which comprises one or more of a Stretcher unit and a Spacer unit. In still other embodiments, the invention provides methods of treating cancer, immune disease, infectious diseases and other diseases and disorders using a Ligand-Drug Conjugate comprising a self-stabilizing linker assembly,
  • the Linker unit of the Ligand-Functional Agent Conjugate can further comprise, in addition to a self-stabilizing linker assembly, an optional secondary linker assembly (L°) which joins each Functional Agent (or Drug unit) to the self-stabilizing linker assembly.
  • the secondary linker assembly can be a releasabie linker assembly or a non- releasable linker assembly.
  • Linker unit can be used herein to refer to the linker portion of the Ligand- Functional Agent Conjugate (or Ligand-Drug Conjugate) comprising the self-stabilizing linker assembly and optional secondary linker assembly.
  • the Self-Stabilizing Linkers are designed such that the rate of the post-conjugation hydrolysis of the succinimide ring will be controllable and fall within a desired range.
  • the limits of this range are typically dictated by issues which arise in the manufacture of ligand-drug conjugates.
  • hydrolysis which is too slow would require unacceptable delays in the manufacturing process or aggressive conditions of pH and temperature which may induce damage to the protein backbone,
  • a maleimide which is too reactive with water may be hydrolyzed to the corresponding maleic acid derivative before it can react with available protein thiols (see undesired pathway):
  • maleic acid derivative (2 possible positional isomers)
  • Such maleic acid derivatives are not reactive with thiols, and thus this reaction pathway does not result in a bioconjugate. Therefore, maleimides which undergo hydrolysis faster than thiol addition under applicable conditions are not useful reagents, in general, structural features which increase the hydrolysis rate of a thio-substituted succinimide will also increase the hydrolysis rate of the parent maleimide.
  • the p a of the basic group, the strength of the electron withdrawing group(s), and the proximity of both groups to the maleimide are inter-related variables and will affect the hydrolysis rate of both the maleimide and corresponding thio-substituted succinimide product. Accordingly, positioning of the electron withdrawing group and base will be dependent upon the pKa of the base and the strength of the electron withdrawing group(s).
  • the skilled artisan will understand that for particularly strong electron withdrawing groups such as fluoro,
  • the group can be further from the maleimide.
  • the hydrolysis reaction may compete with a macro-cyclization reaction such that the resultant conjugates comprise a heterogenous mixture of hydrolyzed thio-substituted succinimide conjugates and cyclized thio-substituted dilactam conjugates.
  • a dilactam will not be formed.
  • the Ligand-Functionai Agent Conjugate is represented by Formula 1:
  • L is a Ligand unit
  • D' is a Drug unit, a Detection unit, or a Stability unit:
  • is the optional secondary linker assembly
  • L ss is the self-stabilizing linker assembly, wherein
  • M ! is a succinimide ring or a hydroiyzed succiniraide or together with BIJ forms a dilactam
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ci -8 alkylene, C 1-8 heteroalkylene, Ce-io arylene, or Quo heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
  • the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C 6- io arylene or C 4 _; 0 heteroarylene;
  • r is zero, in some aspects, a is 1 and b is zero. In other aspects, a is zero and b is 1.
  • the scaffold is a C 6 -io arylene or C4.10 heteroarylene and acts as the electron withdrawing group.
  • exemplary aryls and heteroaryls include phenyl and pyridinyl,
  • m + q + r is 1 or 2.
  • the Conjugate is represented by Formula I or a sal t thereof wherein a is 1 and r is zero.
  • the Conjugate is represented by Formula I or a salt thereof wherein L,° is present and is a releasable linker assembly
  • the circle represents a scaffold that is Cj.g alkylene or Cj.g heteroalkylene (preferably C 1-4 alkylene or C1.4 heteroalkylene), a is 1, r is zero, and the sum of m+q is 1.
  • the scaffold is Ci -3 alkylene or d-3 heteroalkylene.
  • the alkylene is straight chain or branched.
  • the Conjugate is represented by Formula I or a salt thereof wherein V " is present and is a releasable linker assembly, the circle represents a scaffold that is C 1-8 alkylene or Ci-8 heteroalkylene (preferably C].4 alkylene or C heteroalkylene), a is 1 , and m and r are zero.
  • the scaffold is C 1 -3 alkylene or C1.3 heteroalkylene.
  • the alkylene is straight chain or branched.
  • the Conjugate is represented by Formula I or a salt thereof wherein L° is present and is a releasable linker assembly, the circle represents a scaffold that is Ci , C 2 , C3 or C4 straight or branched chain alkylene, a is i, r is zero, and the sum of m+q is 1.
  • the Conjugate is represented by Formula 1 or a salt thereof wherein L° is present and is a releasable linker assembly, the circle represents a scaffold that is Q, C 2 , C 3 or Q straight or branched chain alkylene, a is 1 , and m and r are zero.
  • the alkylene or heteroalkylene chain can be straight or branched, n some aspects, the alkylene or heteroalkylene chain will be a straight chain. In other aspects, it will be branched.
  • p can range from 1 to 20, preferably 1 to 12, even more preferably i to 10, or 1 to 8.
  • M 1 is preferably a succinimide ring (i.e., non- hydrolyzed) or a hydroiyzed succinimide ring (also referred to herein as hydroiyzed
  • D' can be a Drug unit
  • D and the Ligand-Functional Agent Conjugate can be a Ligand-Drug conjugate.
  • th scaffold will comprise a reactive site suitable for attachment to the optional secondary linker assembly or D'.
  • the self-stabilizing linker assembly (L 3 ⁇ 4 ) is represented by Formula ⁇ :
  • M 1 represents a succinimide ring or a hydroiyzed succiniraide ring or a diiactam formed when the base reacts with the succinimide ring
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ci.g alkylene, Cj.s heteroalkyiene, C& io arylene, or C4-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly, D !
  • the subscripts m, q and r are each 0 or J , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C 6- ;o arylene or C4.10 heteroarylene.
  • the self-stabilizing linker assembly is represented by Formula 11 wherein r is zero.
  • m + q + r is 0.
  • the Cg-jo arylene or C 4 .]o heteroarylene act as the electron withdrawing group.
  • Exemplary aryls and heteroaryis include phenyl and pyridinyl.
  • m + q + r is 1 or 2.
  • the self-stabilizing linker assembly is represented by Formula 11 or a salt thereof wherein the circle represents a scaffold that is C f .g aikylene or Cj.g heteroalkylene (preferably C aikylene or heteroalkylene), r is zero, and the sum of m+q is i .
  • the scaffold is C1.3 aikylene or Cj. 3 heteroalkylene.
  • the aikylene is a straight chain or branched aikylene.
  • the self-stabilizing linker assembly is represented by Formula II or a sait thereof wherein, the circle represents a scaffold that is C 1-8 aikylene or C]- heteroalkylene (preferably C aikylene or heteroalkylene) and m and r are zero.
  • the scaffold is C1.3 aikylene or Cj.3 heteroalkylene.
  • the aikylene is a straight chain or branched aikylene.
  • the self-stabilizing linker assembly is represented by Formula II or a salt thereof wherein the circle represents a scaffold that is Ci , C 2 , C 3 . or C4 straight or branched chain aikylene, r is zero, and the sum of m+q is I .
  • the self-stabilizing linker assembly is represented by Formula II or a salt thereof wherein the circle represents a scaffold that is C C 2 , C 3 , or C4 straight or branched chain aikylene, and m and r are zero.
  • the aikylene or heteroalkylene chain will preferably be a straight or branched chain. In some aspects, the aikylene or heteroalkylene chain will be a straight chain. In other aspects, it will be a branched chain.
  • M 1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
  • D' is preferably D, a Drug unit.
  • the Ligand-Fursctional Agent Conjugate has the Formula (I);
  • each of the scaffold, L, M ! , HE, BU, L°, D', and the subscripts p, m, q and r have the meanings provided above, selected embodiments include those wherein
  • r is 1 , and m and q are 0;
  • r is 1, m and q are 0, and a is 1 ;
  • r is 1, m and q are 0, and D' is a i ug unit, D;
  • the Basic unit comprises a primary, a secondary amine, or a tertiary amine.
  • the Basic unit is selected from the group consisting of-(C(R )( R !
  • the Basic unit is selected from the group consisting of -(CH 2 ) X NH 2 , -(CH 2 ) x NHR a , and -(CH 2 ) x NR a 2 , wherein x is an integer of from 0 to 6 (preferably 0 to 4, or 1 to 4) provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each R a is independently selected from the group consisting of C 1-6 alkyl and C 1-6 haloaikyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or pipe
  • x is an integer of from 1 to 4.
  • the Basic unit is Ni k -CH 2 NH 2 , -CH 2 CH 2 NH 2 , - CH2CH2CH2 NH2, or -- 3 ⁇ 4CH 2 CH 2 CH 2 NH 2 provided that if the Basic unit is -NH 2 , there are no less than 2 intervening atoms between the base and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam.
  • HE preferably comprises a carbonyl, sulfonyl or phosphoryl moiety.
  • 1 is a succinimide ring or hydrolyzed succinimide.
  • the circle represents a scaffold that is Cj -8 alkylene or Ci..g heteroalkylene (preferably Cj. 4 alkylene or C heteroalkylene).
  • the alkylene is a straight or branched chain alkylene
  • the Ligand-Functional Agent Conjugates have the formula
  • each of the scaffold, L, M , HE, BIJ, L , D', and the subscript p has the meaning provided above, selected embodiments include those wherein: 1) the Basic unit (BU) comprises a primary, a secondary amine, or a tertiary amine, and D' is preferably a Drug unit D.
  • BU Basic unit
  • the Basic unit is selected from the group consisting of -(C(R 9 )( R. 1 )) X NH 2) -(C(R 9 )( R 10 )) x NHR a , and -(C(R 9 )( R 10 )) x NR a 2 , wherein x is an integer of from 0-4 (or 1-4) and each R a is independently selected from the group consisting of C 1-6 alk l and Q.6 haloalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that if x is zero, there are no less than 2 intervening- atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and R 9 and R lU are independently selected from H or C 1 .3 alky
  • the Basic unit is selected from the group consisting of-(CH 2 ) X NH2, ⁇ (CH 2 ) X NHR ⁇ and - ⁇ (CH 2 ) x NR a 2 , wherein x is an integer of from 0 to 6 (preferably 0 to 4 or 1 to 4) provided that if x is zero, there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each R a is independently selected from the group consisting of C e alkyl and .6 haloalkyl.
  • R 8 groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, and D' is preferably a Drug unit D.
  • X is an integer of from 1 to 4.
  • the Basic unit is -N3 ⁇ 4, -CH 2 N3 ⁇ 4 ⁇ CH 2 CH 2 NH 25 -C3 ⁇ 4CH 2 CH 2 NH 2 , or
  • HE comprises a carbonyl, sulfonyl or phosphoryl moiety
  • D' is preferably a Drug unit D.
  • M 1 is a succinimide ring or hydrolyzed succinimide
  • D' is preferably a Drug unit (D).
  • the circle represents a scaffold that is Q-g alkylene or C-.-g heteroalkylene (preferably CM alkylene or C heteroalkylene), and D' is preferably a Drug unit (D).
  • the alkylene is a straight chain or branched chain alkylene.
  • HE is a carbonyl
  • D' is preferably a Drug unit (D).
  • H E is a carbonyl and the circle represents a scaffold that is a straight chain Ci-8 alkylene or C heteroalkylene (preferably CM alkylene or C heteroalkylene), and D' is preferably a Drug unit (D).
  • H E is a carbonyl and the circle represents a scaffold that is a branched chain C i.g alkylene or Ci-8 heteroalkylene (preferably CM alkylene or CM heteroalkylene), and D' is preferably a Drug unit (D).
  • Ligand-Drug Conj ugates have the formula:
  • is a releasable linker assembly
  • BU is (CI ! ; ⁇ ) v Nf -(CH 2 ) x NHR a , and ⁇ " ! h ), ⁇ ' R : ' 2 , wherein x is an integer of from 1-4 and each R a is independently selected from the group consisting of Q.
  • is a releasable linker assembly
  • BU is C! !-N! k -CH 2 CH 2 NH 2 , -CH 2 CH 2 CH 2 H 2 , or - CH2CH2CH 2 CH2 NH2.
  • the Ab can be replaced by a non-antibody protein.
  • M 1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
  • the Ligand-Drug Conjugates have the formula:
  • is a releasable linker assembly
  • BU is -(OL )xNH 2 , -(CH 2 ) NHR a , and -(Cl3 ⁇ 4 ) x NR a 2 , wherein x is an integer of from 1-4 and each R a is independently selected from the group consisting of Cj. 6 alkyl and C;.
  • is a releasable linker assembly
  • BU is -CH 2 NH 2 , -CH2CH 2 NH2, --CH 2 CH 2 CH 2 NH 2 , or -CH2CH2CH 2 CH2 H 2
  • the Ab can be replaced by a non-antibody protein.
  • M 1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
  • the Ligand-Drug Conjugates have the formula:
  • is a releasable linker assembly
  • BU is -(CH 2 ) X NH 2 , -(CH 2 ) x NHR a , and ⁇ (CH 2 ) x NR a 2 , wherein x is an integer of from 0-4 and each R a is independently selected from the group consisting of Cj.
  • is a releasable linker assembly
  • BU is -NH 2 , -CH 2 NH 2 , -CH 2 CH 2 NH 2 , ( ⁇ LCM K i h L. or -CH2CH 2 CH2CH 2 NH 2 .
  • the Ab can be replaced by a non-antibody protein.
  • M 1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
  • the present invention provides Fuctional Agent-Linker Conjugates (e.g., Drug-Linker Conjugates), Linkers, and Ligand-Linker assemblies.
  • Fuctional Agent-Linker Conjugates e.g., Drug-Linker Conjugates
  • Linkers e.g., Linkers, and Ligand-Linker assemblies.
  • the present invention provides Functional Agent-Linker Conjugates (e.g., Drug-Linker Conjugates) having the formula:
  • D' is a Drug unit, a Detection unit, or a Stability unit
  • is an optional secondary linker assembly
  • L ss is the self-stabilizing linker assembly, wherein BU is a Basic unit;
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ci_g alkylene, Cj.g heteroalkylene, C 6 -io arylene, or C,i.. ]0 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
  • the subscripts rn, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C 6 -io arylene or C4.10 heteroarylene, and the subscript a and b are each 0 or 1 , and the sum of a+b is 1.
  • the Functional Agent-Linker Conjugate is represented by the formula:
  • the Drug-Linker Conjugate is represented by the formulae:
  • RG is a reactive group (comprising a reactive site) at the terminus of L°, suitable for attaching a Drug unit;
  • is an optional secondary linker assembly that is present:
  • L ss is the self-stabilizing linker assembly, wherein
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ci-s alkyiene, C ⁇ .% heteroalkyiene, C 6- i 0 arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit:
  • the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C&io arylene or Gno heteroarylene: and the subscript a and b are each 0 or 1, and the sum of a+b is 1 .
  • RG is a reactive group that contains a reactive site that is capable of forming a bond with a Detection unit or a Stability unit instead of a Drug unit.
  • the Linker is represented by the formula:
  • Linker is represented by the formulae:
  • Ligand-Linker Conjugates having the formula:
  • L is a Ligand unit
  • RG is a reactive group (comprising a reactive site) at the terminus of L° which is suitable for attaching a Drug unit;
  • L u is an optional secondary linker assembly that is present
  • L ss is a self-stabilizing linker assembly, wherein
  • M is a succinimide ring or hydrolyzed succinimkle
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be C 1-8 a!kylene, C].g heteroalkyiene, C 6 -io arylene, or C4.10 heteroaryiene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit;
  • the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C 6 .io arylene or C -io heteroaryiene;
  • RG is a reactive group that contains a reactive site that is capable of forming a bond with a Detection unit or a Stability unit instead of a Drug unit.
  • the Ligand-Linker Conjugate is represented fay the formula:
  • Ligand-Linker Conjugate is represented by the formula:
  • L is an antibody (Ab).
  • the circle, HE, M ⁇ BU, L° and RG have the meanings provided above. Additionally, each of the specifically recited selected embodiments for Ab, M ', BU, L° and RG (for any of the conjugates provided herein) are equally applicable to these Ligand-Linker conjugates.
  • the self-stabilizing linker assembly instead of being represented by the structure for L ss is represented by L' 1 and has Formula (III):
  • M 1 is a non-hydro lyzed or hydrolyzed succinimide or M 1 forms a dilactam with B (e.g., a dilactam is formed when B reacts with the succinimide ring), wherein the succinimide or dilactam is conjugated to the Ligand unit via a thioether linkage;
  • V, Q, T, and G are independently selected from -(C(R 9 )( R 10 ))-;
  • R 1 is H or C
  • R 9 and R'°are in each occurrence, independently selected from H or C1.3 alkyl
  • RS is a reactive site for conjugation to a component of the optional secondary linker assembly or Drug unit;
  • g is 0 to 5;
  • n 0 to 5;
  • n 0 to 5;
  • d is 0 or 1 ;
  • x is 0 to 4, provided that when m is 0, x is 1 to 4;
  • Ligand-Drug Conjugates have the formula:
  • Drug-linkers have the formula:
  • Ligand -Linker Conjugates have the formula:
  • L if present, is a Ligand unit
  • is an optional secondary linker assembly
  • RG if present, is a reactive group (comprising a reactive site) at the terminus of L° which is suitable for attaching a Drug unit;
  • M 1 if present, is a non-hydrolyzed or hydrolyzed succinimide or 1 forms a diiactam with B (e.g., a diiactam is formed when the base reacts with the succinimide ring), wherein the succinimide or diiactam is conjugated to the Ligand unit via a thioether linkage;
  • V, Q, T, and G are independently selected from - ⁇ C(R 9 )( R 10 ))-;
  • R' is H or Ci. 3 alkyl
  • R 9 and R ! ° are, in each occurrence, independently selected from H or Cj. 3 aikyl;
  • RS is a reactive site for conjugation to a component of the optional secondary linker assembly or Drug unit;
  • g O to 5;
  • n 0 to 5;
  • x is 0 to 4, provided that when m is 0, x is 1 to 4;
  • p if present, ranges from 1 to 20, preferably 1 to 12;
  • the electron withdrawing group will either be represented by F (e.g., E 1 , E ⁇ or E : and E z" ) or by the reactive site RS.
  • F e.g., E 1 , E ⁇ or E : and E z"
  • the reactive site will act as an electron withdrawing group.
  • n, d, and g are zero or m
  • Exemplary embodiments wherein the Ligand-Drug Conjugates, Drug-Linkers, Linkers, or Ligand-Linker conjugates are represented by Formula III (or formulae I l ia, illb, Hie, or IIId, as the case may be) or pharmaceutically acceptable salts thereof, include those wherein m is zero; m is zero and n is zero, one, two, or three; x is I ; x is zero and n is zero, one, two, or three; and m is zero, n is zero, and x is 1.
  • Exemplary embodiments include those described herein wherein wherein R and R 1 are hydrogen.
  • E' and E" are independently selected from hydrogen, -CN, -N0 2 , -CX , and -X wherein is halogen or E ! and E: 2 together are (-O);
  • R 3 , R 4 , R 5 , R 6 . R 7 and R 8 are endently selected from H or Cj-6 alkyl and e is 0 to 4;
  • (xiv) B is ⁇ N(R 3 )(R 4 ), wherein R 3 and R 4 are independently selected from H or Ci-g aikyl;
  • (xv) B is as in (xiii) or (xiv) and R 3 , R 4 , R R b , R 7 and R 8 are independently selected from H or Ci -3 alkyl;
  • (xvi) B is as in (xiii) or (xiv) and R 3 , R 4 , R 5 , R B , R and R 8 are independently selected from 1 1 or methyl;
  • (xvii) B is as in (xiii) or (xiv) or (xvi) and R 3 and R 4 are hydrogen;
  • (xviii) B is as in (xiii) or (xiv) or (xvi) and at least one of R J and R 4 are hydrogen;
  • (xix) B is as in (xiii) or (xiv) or (xvi) and at least one of R 3 and R 4 is not hydrogen;
  • R ! , RQ, and R '° are independently selected from H or methyl;
  • R ⁇ R Y , and R ! ° are independently selected from H or methyl;
  • the cleavable unit is present and conjugated directly to the Drug unit via a cleavable peptide, disulfide, or hydrazone bond;
  • the Drug is an auristatin
  • M 1 is a hydrolyzed or non-hydrolyzed succinimide
  • the Ligand unit is an antibody
  • the Ligand unit is an antibody and is attached to the Linker unit though a cysteine residue of an interchain disulfide
  • the Ligand unit is a monoclonal antibody; and any combinations or subcombinations of (i) througli (xxxvii) provided that the combinations or subcombinations do not conflict with each other (e.g., xxx and xxxi conflict because p cannot be both about 4 and about 8).
  • m is zero, and n is zero, one, two, or three.
  • m is zero, n is zero, and x is one.
  • d can be one and g can be from 1 to 5 or d can be one and g can be from 2 to 5.
  • one or more of (i), (iii) or (xi)-(xxxvi) can apply.
  • the optional secondary linker assembly can be represented by the following formula: wherein -A- is an optional Stretcher unit, the subscript a' is 0 or I ;
  • -W- is an optional Cleavable unit, the subscript v' is 0 or 1 ; and - ⁇ - is an optional Spacer unit, and the subscript y' is 0 or 1.
  • V, T, B, I , Q, F, G, m, x, n, d, and g are as defined for formula III and RG is a reactive group comprising a reactive site, RS, for conjugation to the Drug unit D when the secondary linker assembly is absent or to a component of the secondary linker assembly wherein the secondary linker assembly secondary linker has the following formula: wherein -A- is an optional Stretcher unit, the subscript a' is 0 or 1 :
  • -W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or I .
  • Exemplary self-stabilizing linker assemblies prior to conjugation with a Ligand and following conjugation and hydrolysis of the thio-substituted sueccinimide which is formed upon conjugation are as follows:
  • V, Q, m. n, x, and B, are as defined above for Formula III or any other selected embodiment, c is from 1 to 4, and R' ' and R » 1 ! 2 are, at each occurrence, independently selected from H or Ci-C 6 alkyl, in an exemplary embodiment c is 1 or 4,
  • the "S" of the hydrolyzed thio-succinimide represents a sulfur atom of the Ligand (e.g., antibody).
  • the wavy line indicates linkage to the secondary linker assembly or Drug unit. In an exemplary embodiment, the wavy line indicates linkage to the following secondary linker assembly
  • -A- is an optional Stretcher unit, the subscript a' is 0 or 1 ; -W- is an optional Cleavable unit, the subscript w ! is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or 1 . It will be understood that more than one (e.g., 1 to 20) drug-linkers can be attached to each Ligand.
  • a self-stabilizing linker assembly may undergo macro-cyclization to form a dilactam as follows wherein R represents the remainder of the conjugate:
  • the optional secondary linker can comprise a variety of linking groups.
  • can be present and have the formula: wherein
  • -A- is an optional Stretcher unit, the subscript a' is 0 or 1 ;
  • -W- is an optional Cleavable unit, the subscript v' is 0 or 1 ;
  • -Y- is an optional Spacer unit, and the subscript y' is 0 or 1 ;
  • the optional secondary linker assembly can be a releaseabie linker assembly, L R .
  • w is I .
  • the optional secondary linker assembly is a non-reieasable linker assembly.
  • w is 0 and release of drug is via a total protein degradation pathway (i.e., non-cleavabie pathway).
  • a Ligand Unit is present.
  • the Ligand unit (L-) is a targeting agent that specifically binds to a target moiety.
  • the Ligand can specifically bind to a cell component (a Cel l Binding Agent) or to other target molecules of interest.
  • the Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit interacts.
  • Ligands include, but are not limited to, proteins, polypeptides and peptides.
  • Suitable Ligand units include, for example, antibodies, e.g., full-length antibodies and antigen binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substance.
  • the ligand is a non- antibody protein targeting agent.
  • a Ltgand-Functional Agent is provided wherein D* is a Detection Unit or Stability unit and the Ligand unit is a protein (e.g., a non- antibody protein).
  • a Ligand unit forms a bond with the maleirnide of the Self-Stabilizing Basic unit via a sulfhydryl group of the Ligand to form a thio-substituied succinimide.
  • the su!fhydryi group can be present on the Ligand in the Ligand's natural state, for example a naturally-occurring residue, or can be introduced into the Ligand via chemical modification.
  • the site of drug conj ugation can affect a number of parameters including ease of conjugation, drug-linker stability, effects on biophysical properties of the resulting bioconjugates, and in-vitro cytotoxicity.
  • the site of conj ugation of a drug-linker to a ligand can affect the ability of the conjugated drug-linker to undergo an elimination reaction and for the drug linker to be transferred from the ligand of a bioconjugate to an aiternative reactive thiol present in the milieu of the bioconjugate, such as, for example, a reactive thiol in albumin, free cysteine, or glutathione when in plasma.
  • Self-Stabilizing Linkers of the present invention is particularly beneficial when conjugated to thiol residues at sites that are susceptible to the elimination reaction and subsequent transfer of drug-linker if non-self-stabilizing alkyl maleimides are used (e.g., maleimido-eaproyi drug linker).
  • sites include, for example, the interchain disulfides as well as select cysteine engineered sites,
  • Use of the Self-Stabilizing Linkers of the present invention provides a stable linkage and ability to attach multiple drugs to each Ligand unit.
  • the Ligand unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryi groups.
  • the reagents that can be used to modify lysines include, but ar not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2- Iminothiolane hydrochloride (Traut's Reagent).
  • the Ligand unit can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryi groups.
  • the Ligand is an antibody and the sulfhydryi group is generated by reduction of an interchain disulfide. Accordingly, in some embodiments, the Linker unit is conjugated to a cysteine residue of the reduced interchain disulfides.
  • the sulfhydryi group is chemically introduced into tire antibody, for example by introduction of a cysteine residue.
  • the Linker unit is conjugated to an introduced cysteine residue.
  • Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to. transferrin, epidermal growth factors ("EGF”), bombesin, gastrin, gastrin- releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF”), such as TGF-a and TGF- ⁇ , vaccinia growth factor (“VGF”), insulin and insuiin-like growth factors I and II, somatostatin, lectins and apoprotein from low density lipoprotein,
  • EGF epidermal growth factors
  • TGF transforming growth factors
  • VGF vaccinia growth factor
  • insulin and insuiin-like growth factors I and II somatostatin, lectins and apoprotein from low density lipoprotein
  • Particularly preferred ligands are antibodies.
  • Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals.
  • Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g. , a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof).
  • a monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.
  • Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies.
  • the antibodies include full-length antibodies and antigen binding fragments thereof.
  • Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312: Kozbor et al, 1983, Immunology Today 4:72-79; and Olsson et al, 1982, Meth, Enzymol 92:3-16).
  • the antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to target ceils (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies bound to tumor cells or matrix.
  • target ceils e.g., cancer cell antigens, viral antigens, or microbial antigens
  • “functionally active” means that the fragment, derivative or analog is able to elicit anti-anii- idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived.
  • the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence thai specifically recognizes the antigen.
  • synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, e.g., Kabat et al., 1991, Sequences of Proteins of
  • antibodies include fragments of antibodies such as, but not limited to, F(ab') 2 fragments, Fab fragments, Fvs, single chain antibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as the antibody.
  • recombinant antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., U.S. Patent No, 4,816,567; and U.S. Patent No.
  • Humanized antibodies are antibody molecules from non- human species having one or more complementarity determining regions (CDRs) from the non- human species and a framework region from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671 ; European Patent Publication No. 0 184 187; European Patent Publication No.
  • Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
  • Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity.
  • derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc.
  • any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, forrnylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.
  • Antibodies can have modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors.
  • antibodies can have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g.. International Publication No. WO 97/34631, which Is incorporated herein by reference In its entirety ).
  • Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques.
  • immunospecific for a cancer cell antigen can be obtained, e.g., from the GenEiank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • antibodies for the treatment of cancer can be used.
  • Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques.
  • the nucleotide sequence encoding antibodies immunospecific for a cancer ceil antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
  • antibodies for the treatment of an autoimmune disease are used in accordance with the compositions and methods of the invention.
  • Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization ⁇ e.g..
  • useful antibodies are immunospecific for the treatment of autoimmune diseases incl ude, but are not limited to, anti-nuclear antibody; anti-ds DNA; Anti-ss DNA, anti-cardiolipin antibody igM, IgG; anti-phospholipid antibody IgM, IgG; anti-SM antibody; anti-mitochondrial antibody; thyroid antibody; microsomal antibody;
  • thyroglobulin antibody anti-SCL-70 antibody; anti-Jo antibody; anti-U;RNP antibody;
  • anti-La/SSB antibody anti-SSA
  • anti-SSB antibody anti-perital ceils antibody
  • anti-histones antibody anti-RNP antibody
  • C-ANCA antibody C-ANCA antibody
  • P-ANCA antibody anti-centromere antibody
  • Anti-Fibriilarin antibody and anti-GBM antibody anti-La/SSB antibody
  • anti-SSA anti-SSA
  • anti-SSB antibody anti-perital ceils antibody
  • anti-histones antibody anti-RNP antibody
  • C-ANCA antibody C-ANCA antibody
  • P-ANCA antibody anti-centromere antibody
  • Anti-Fibriilarin antibody and anti-GBM antibody anti-GBM antibody
  • useful antibodies can bind to a receptor or a receptor complex expressed on an activated lymphocyte.
  • the receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein.
  • suitable immunoglobulin superfamily members are CD 2, CDS, CD4, CDS, CD 19, CD20, CD22, CD28, CD30, CD70, CD79, CD90, CD 152/CTLA-4, PD-1 , and 1COS.
  • Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD 137/4-1 BB, T F-R 1 , T F -2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3.
  • Non-limiting examples of suitable integrins are CD1 l a, CD i lb, CD i lc, CD 18, CD29, CD41 , CD49a, CD49b, CD49c, CD49d, CD49e, C! ) 49f. ( 1 ) 1 03, and CD 104.
  • Non- limiting examples of suitable lectins are C-type, S-type, and I-type lectin.
  • the drug unit (D) can be any cytotoxic, cytostatic or immunosuppressive drug also referred to herein as a cytotoxic, cytostatic or immunosuppressive agent.
  • the Drug unit has an atom that can form a bond with the Linker Unit, in some embodiments, the Drug unit D has a nitrogen atom that can form a bond with the Linker unit. In other embodiments, the Drug unit D has a carboxylic acid that can form a bond with the Linker unit. In other embodiments, the Drug unit D has a su!fhydryl group that can form a bond with the Linker unit . In other embodiments, the Drug unit D has a hydroxy! group or ketone that can form a bond with the Linker unit.
  • cytotoxic or immunosuppressive agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g. , platinum complexes such as cis-platin, mono(p!atinum), bis(platinum) and tri-nuelear platinum complexes and carbopiatin), anthracyclin.es, antibiotics, antifolates-, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinois, pre-formirsg compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisornerase inhibitors, vinca alkaloids, or the like.
  • alkylating agents e.g. , platinum complexes such as cis-platin, mono
  • cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors.
  • cytotoxic agents include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM! and DM4), taxanes, benzodiazepines (e.g.,
  • PBDs pyrrolo [1 ,4] benzodiazepines
  • oxazolidinobenzodiazepines oxazolidinobenzodiazepines
  • vinca alkaloids oxazolidinobenzodiazepines
  • Select benzodiazepine containing drugs are described in WO 2010/091 150, WO 2012/1 12708, WO 2007/085930, and WO 201 1/023883.
  • Individual cytotoxic or immunosuppressive agents include, for example, an androgen, antbramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busuJfan, buthionine sulfoximine, calicheamicin, camptothecin, carbopiatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin).
  • AMC antbramycin
  • asparaginase 5-azacytidine
  • azathioprine 5-azacytidine
  • azathioprine 5-azacytidine
  • azathioprine 5-azacytidine
  • azathioprine 5-azacytidine
  • azathioprine
  • suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and ⁇ exitropsins, a CBI compound; see also U.S. Patent No. 6,130,237), duocarmycras (see U.S. Publication No.
  • taxanes e.g., paclitaxei and docetaxel
  • puromycins e.g., vinca alkaloids, CC-1065, SN-38, topotecan, morpholino- doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomvcin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermoiide, eleutherobin, and mitoxantrone.
  • taxanes e.g., paclitaxei and docetaxel
  • puromycins e.g., vinca alkaloids, CC-1065, SN-38, topotecan, morpholino- doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomvcin, comb
  • the Drug unit is an anti-tubulin agent.
  • anti-tubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxei), Taxotere ⁇ (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesme, and vinorelbine).
  • antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermoiide, and eleutherobin.
  • the cytotoxic agent is maytansine or a maytansinoid, another group of anti-tubulin agents.
  • ImmunoGen, Inc. see also Chari et ah, 1992, Cancer Res. 52:127- 131 and U.S. Patent No. 8,163,888).
  • the Drug unit is an auristatin.
  • Auristatins include, but are not limited to, AE, AFP, AEB, AEVB, MMAF, and MMAE.
  • the synthesis and structure of auristatins are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005- 0238649 2005-0009751 , 2009-01 1 1756, and 201 1 -0020343: International Patent Publication No. WO 04/010957, international Patent Publication No. WO 02/088172, and U.S. Patent Nos. 7,659,241 and 8,343,928; each of which is incorporated by reference in its entirety and for all purposes.
  • Exemplary auristatins of the present invention bind tubulin and exert a cytotoxic or cytostatic effect on the desired cell line.
  • Exemplary auristatin Drug units have the following formula or a pharmaceutically acceptable sal t thereof wherein the wavy line indicates site of attachment to the linker unit:
  • the Drug is a benzodiazepine (including benzodiazepine containing drugs e.g., pyrrolo[l ,4]benzodiazepiries (PBDs), indolinobenzodiazepines, and oxazo!idino benzodiazepines) .
  • benzodiazepine including drugs e.g., pyrrolo[l ,4]benzodiazepiries (PBDs), indolinobenzodiazepines, and oxazo!idino benzodiazepines.
  • PBDs are of the general structure:
  • PBDs to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents.
  • the biological activity of these molecules can be potentiated by, for example, joining two PBD units together through their C8/C -hydroxy! functionalities via a flexible alkylene linker.
  • the PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link which is thought to be mainly responsible for their biological activity.
  • [0J 4] There are a number of different assays that can be used for determining whether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effect on a cell line. In one example for determining whether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effect on a ceil line, a thymidine incorporation assay is used.
  • Ligand- Drug Conj ugate has a cytostatic or cytotoxic effect on the cell line if the cells of the culture have reduced ⁇ -thymidine incorporation compared to cells of the same cell hne cultured under the same conditions but not contacted with the Ligand-Drug Conjugate.
  • cell viability is measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM blue ⁇ see, e.g.. Page et l. t 1993, Intl. J. of Oncology 3:473-476).
  • a dye such as neutral red, trypan blue, or ALAMAR.TM blue
  • the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically.
  • Preferred Ligand-Drug Conjugates include those with an IC50 value (defined as the mAB concentration that gives 50% cell kill) of less than 1000 ng/ml, preferably less than 500 ng/ml, more preferably less than 100 ng ml, even most preferably less than 50 or even less than 10 ng/rnl on the cell line,
  • a non-hydrolyzed succinimide (also referred to herein as a succinimide ring) conjugated to the Ligand unit via a thioether linkage can be represented as follows wherein R represents the remainder of the Linker unit optionally conjugated to a Drug unit, Detection unit or Stability unit:
  • a hydrolyzed succinimide (also referred to herein as a hydrolyzed succinimide ring) conjugated to the Ligand unit via a thioether linkage can be represented as one of its two positional isomers as follows wherein R represents the remainder of the Linker unit optionally conjugated to a Drug unit, Detection unit or Stability unit:
  • the non-hydrolyzed succinimides and hydrolyzed succinimide representations there can be from 1 to 20, preferably 1 to 12, 1 to 10 or 1 to 8 self-stabilizing linkers conjugated to each Ligand. In some aspects, there are from I to 20, preferably 1 to 12, 1 to 10 or 1 to 8 drug-linkers conjugated to each Ligand. Additionally, for the conjugates described herein where a Ligand is not attached, the succinimide is in an unsaturated form as a maieimide (capable of reactive with a thiol or the Ligand).
  • the Basic unit can be essentially any base capable of facilitating a hydroxide ion (or vvaier) attack to hydrolyze a nearby succinimide group.
  • BU represents any "base” but is typically a group comprising a tethered amine or nitrogen containing heterocycle; the amine or nitrogen containing heterocycle acting as the base of the Basic unit.
  • R epresentative amines include ⁇ N(R 3 )(R 4 ) wherein R 3 and R 4 are independently selected from H or Ci ⁇ alkyl, preferably H or methyl.
  • R 5 , R 6 , R 7 and R 8 are, at each occurrence, independently selected from hydrogen or Ci ⁇ alkyl, preferably H or methyl, and e is 0-4.
  • the wavy line indicates the point of attachment to a tethering group (typically an aikylene linker - ⁇ (C(R )( R 1,J )) ⁇ wherein the subscript x is an integer of from 0 to 6 (or 1 to 6 ) provided that if x is 0 there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydroiyzed) or diiactam and R.
  • y and R'° are independently selected from H or C: .3 a!kyi.
  • the aikylene linker is -(CH 2 ) x - wherein the subscript x is an integer of from 0 to 6 (or 1 to 6) provided that if x is 0 there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non- hydroiyzed) or diiactam.
  • the subscript x is preferably 0 to 4, 1 to 4, or from 1 to 3, or from 2 to
  • the Basic unit will in some embodiments, be selected from the group consisting of -( €3 ⁇ 4 ) X NH 2 , -( 3 ⁇ 4 ) x NHR a , and --( €3 ⁇ 4 ) X NR " 2, wherein x i s an integer of from 0 to 4, 1 to 4 , or from 1 to 3, or from 2 to 3 , or from 2 to
  • each R a is independently seiected from the group consisting of Cue alkyl and Q.6 haloalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group.
  • the base of the Basic unit will be a nitrogenous base.
  • the hydrolysis enhancers (HE) of Formula I, as well as the other formulae comprising a self-stabilizing linker (L Ss> ), can be essentially any electron-withdrawing group capable of facilitating the hydrolysis of a nearby succinimide group,
  • the hydrolysis is further facilitated by the Basic unit (BU) assisting a hydroxide ion (or water) attack to hydrolyze a nearby succinimide group; or to render the nearby succinimide group more susceptible to hydrolysis.
  • HE can include a functional group that draws electrons away from a reaction center.
  • Exemplary electron withdrawing groups include, but are not limited to, -C( :::: 0), ( () ⁇ .
  • -C -NO?, -CX 3 , - X, -COOR, -CONR2, -COR, -COX, -SO2R, -SO2OR, -SO2NHR, -SO2NR2, -PO3R2, - P(0)(CH 3 )NHR, NO, - R f .
  • -CR CR 2 , and -C ⁇ CR wherein X is F, Br, CI or ⁇ , and R is, at each occurrence, independently selected from the group consisting of hydrogen and C h alky!.
  • Exemplary electron withdrawing groups can also include aryl groups (e.g., phenyl) and certain heteroaryl groups (e.g., pyridine).
  • electron withdrawing groups includes aryls or heteroaryls further substitued with electron withdrawing groups.
  • HE comprises a carbony], sulfonyl or phosphoryl moiety.
  • HE will comprise a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit.
  • the optional secondary linker assembly can be represented by the formula: wherein - A- is an optional Stretcher unit, the subscrip a' is 0 or 1 ; -W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; and ⁇ Y- Is an optional Spacer unit, and the subscript y' is 0 or 1.
  • Hie wavy line adjacent to the optional Stretcher unit indicates the site of attachment to the self- stabilizing linker assembly and the wavy line adjacent, to the optional Spacer unit indicates the site of attachment to the Drue unit.
  • Monomethyi auristatin E is conjugated tbrough a protease cleavable peptide linker to an antibody
  • monomethyi auristatin F is conjugated directly to an antibody through
  • DM1 is conjugated through a disulfide or directly through the heterobifunctional SMCC linker
  • DM4 is conjugated through a disulfide linker.
  • linker systems can be adapted for use with a self-stabilizing linker assembly or modified to include a basic component and/or electron withdrawing group using the teachings described herein and provide release of drug by a cleavable or non-cleavable system depending on the linker system used.
  • Disulfide, thioether, peptide, hydrazine, ester, or carbamate bonds are all examples of bonds that can be used to connect a Drug Unit to a Linker Unit. Stretcher units, Cleavable units, and Spacer units are described in more detail below.
  • the Stretcher unit is designed in such a way to allo branching within the Linker unit, e.g., the attachment of more than one Drug unit or Detection unit or Stability unit to each self-stabilizing linker assembly, as represented by the following formula:
  • -W- is an optional Cleavable unit, the subscript vv' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , u is from 2 to 20 (preferably from 2 to 10);
  • A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; and a' is 0 or 1 .
  • Each A', W, Y, and D' can be the same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit.
  • D' is a Drug unit D.
  • Ligand-Functional Agent Conjugates or Ligand-Drug Conjugates having either branched or non-branched linkers have the following formulae:
  • D is a Drug unit
  • the circle represents a scaffold that can be C !-8 alkylene, Ci.g heteroalkylene, C 6 -io arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D' (or D as the case may be);
  • -W- is an optional Cleavable unit, the subscript W is 0 or 1 ;
  • -Y- is an optional Spacer unit, the subscript y' is 0 or 1, A is a.
  • A' is an optional Stretcher unit component at the terminus of A: a' is 0 or I ; and u is from i to 20 (preferably from I to 10) wherein when u is from 2 to 20 A is present and when u is 1, A can be present or absent.
  • Each A', W, Y, and D' (or D as the case may be) can be the same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit.
  • w' is 1.
  • w' is 1 and a' is 0.
  • the circle can represent a scaffold that is Ci.g alkyiene or Q-g heteroalkyiene (preferably C alkyiene or C heteroalkyiene) or C-..3 alkyiene or C1.3 heteroalkyiene.
  • the alkyiene is straight chain or branched
  • Ligand-Functional Agent Conjugates having either branched or non-branched linkers can be represented by the following formulas:
  • the circle represents a scaffold that can be C;.g alkyiene, C 1-8 heteroalkylene, C 6-1 o aryiene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D', -W- is an optional Cieavable unit, the subscript w' is 0 or 1; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and 11 is from 1 to 20 (preferably from 1 to 10), wherein when u is from 2 to 20 A is present and when u is 1 , A can be present or absent.
  • a scaffold that can be C;.g alkyiene, C 1-8 heteroalkylene, C 6-1 o aryiene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D',
  • Each A', W, Y, and D' can be the same or different.
  • Each Cieavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, ' is 1.
  • w' is I and a' is 0.
  • the linker isn't branched u is 1
  • a' is 0, and A can be present or absent.
  • the linker is branched u is from 2 to 20 (preferably from 2 to 10).
  • BU is selected from the group consisting of -(CH 2 ) X N!3 ⁇ 4, -iC3 ⁇ 4 ) x NHR a , and
  • x is an integer of from 0-4 and each R a is independently selected from the group consisting of Q -6 alkyl and C 1-6 haloalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidiny) group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrol zed).
  • X is 0-4 and each R a is Ci. 6 alkyl.
  • Ligand-Drug Conjugates having either branched or non-branched linkers can be represented by the following formulas:
  • D is a Drug unit
  • the circle represents a scaffold that can be Ct-g alkylene, C 1-8 heteroalkylene, C io arylene, or C4.10 heteroaryiene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D
  • -W- is an optional Cleavable unit
  • the subscript W is 0 or 1
  • -Y- is an optional Spacer unit
  • the subscript y' is 0 or 1
  • A is a Stretcher unit
  • A' is an optional Stretcher unit component at the terminus of A
  • a' is 0 or 3
  • u is from 3 to 20 (preferably from 1 to 10), wherein when u is from 2 to 20 A is present and when u is 1 , A can be present or absent.
  • Each A', W. Y, and D can be th same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit.
  • w' is 1.
  • w ! is 1 and a' is 0.
  • the linker isn't branched u is 1
  • a' is 0, and A can be present or absent.
  • the linker is branched u is from 2 to 20 (preferably from 2 to 10).
  • BU is selected from the group consisting of --(CHb ) ⁇ ⁇ 2 .
  • x is an integer of from 0-4 and each R a is independently selected from the group consisting of Cj.. 6 alky! and haloalkyl, or two R a groups are combined with the nitrogen to which they are aitached to form an azetidinyi, pyrrol idinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydro !yzed or non-hydrolyzed).
  • X is 0-4 and each R a is C 1-6 alkyl.
  • the circle represents a scaffold that can be Ci-g alkyiene, C 1-8 heteroalkylene, C 6- J O arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D';
  • W- is an optional Cleavable unit, the subscript w* is 0 or 1 ;
  • -Y- is an optional Spacer unit, the subscript y !
  • A is 0 or 1
  • A is a Stretcher unit
  • A' is an optional Stretcher unit component at the terminus of A
  • a' is 0 or 1
  • u is from 1 to 20 (preferably from 1 to 10, wherein when u is from 2 to 20, A is present and when u is 1, A can he present or absent.
  • Each A', W, Y, and D ! can be the same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 1 . in some aspects, w' is i and a' is 0.
  • the linker isn't branched u is 1, a' is 0, and A can be present or absent.
  • the linker is branched u is from 2 to 20 (preferably from 2 to 10)
  • the circle can represent a scaffold that is Q.g alkylene or Q.g heteroalkylene (preferably CM alkylene or C heteroalkylene) or Q.3 alkylene or C1..3 heteroalkylene.
  • the alkylene is straight chain or branched.
  • D' can he D.
  • D' is D.
  • BU is selected from the group consisting of -(CH 2 ) X NH 2 , ---(CH 2 ) x NHR , and -(CH 2 ) x NR a 2, wherein x is an integer of from 0- 4 and each R a is independently selected from the group consisting of Cj-e alkyl and Ci-g haloa!kyl, or two R 3 groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed).
  • X is 0-4 and each R a is C 1-6 alkyl.
  • Ligand-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
  • the circle represents a scaffold that can be C 1-8 alkylene.
  • RG is a reactive group (comprising a reactive site) at
  • W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y* is 0 or 1 ,
  • A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2 to 20, A is present and when u is 1, A can be present or absent.
  • Each A', W, Y, and D can be the same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit.
  • W is 1 .
  • w' is 1 and a' is 0.
  • the linker isn't branched u is I , a! is 0, and A can be present or absent.
  • the linker is branched u is from 2 to 20 (preferably from 2 to 10).
  • the circl can represnet a scaffold that is C .$ alkylene or Ci dem heteroalkylene (preferably C
  • BU is selected from the group consisting of - ⁇ €3 ⁇ 4 ) X NH 2 , - (CH 2 ) X NHR ⁇ and -(CH 2 ) x NR a 2 , wherein x is an integer of from 0-4 and each R a is
  • Ci -6 alkyl and C 1-6 haloalkyl independently selected from the group consisting of Ci -6 alkyl and C 1-6 haloalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyi group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non- bydrolyzed).
  • X is 0-4 and each R R is Ci -6 alkyl.
  • Branched or non-branched Linkers can be represented by the following formulas:
  • each of the scaffold, HE, BU, and the subscripts a, b, m, q, and r have the meanings provided for Formula ⁇ and any of the selected embodiments for Formula I, the circle represents a scaffold that can be d. « alkylene, Ci-8 heteroalkylene, C 6- i 0 arylene, or C4.10 heteroarylene, and optionally comprises a reactive site- suitable for attachment to A, W, Y or D; RG is a reactive group (comprising a reactive site) at
  • W- is an optional Cleavable unit
  • the subscript w' is 0 or 1 :
  • - Y- is an optional Spacer unit, the subscript y' is 0 or 1
  • A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and
  • u is from 3 to 20
  • each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 3. In some aspects, w' is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1 , a' is 0, and A can be present or absent, h other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10).
  • the circle can represent a scaffold that is Cj.8 alkylene or Ci.g
  • heteroalkylene preferably C alkylene or Q.4 heteroalkylene
  • heteroalkylene In some such aspects, the alkylene is straight chain or branched. 1.85] In some aspects j s represented
  • BU is selected from the group consisting of --(C3 ⁇ 4 ) X NH 2 , - (CH 2 ) x NHR a , and (CI 1 2 ) x ' NR a 2 , wherein x is an integer of from 0-4 and each R a is independently selected from the group consisting of C f .g alkyl and Q-e haioalkyl, or two R a groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrroiidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the suceinimide (hydrolyzed or non- hydrolyzed),
  • X is 0-4 and each R a is Ci-6 alkyl.
  • exemplary Ligand-Drug Conjugates having either branched or non-branched linkers have the following formulae:
  • L is a Ligand unit
  • W- is an optional Cleavable unit
  • the subscript w' is 0 or 1
  • -Y- is an optional Spacer unit
  • the subscript y' is 0 or 1
  • A is a Stretcher unit.
  • A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1; and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2 to 20, A is present and when u is 1 , A can be present or absent.
  • Each A', W, Y. and D can be the same or different.
  • Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 1. in some aspects, w' is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1, a' is 0, and A can be present or absent, in other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10).
  • Stretcher units Cleavable units, and Spacer units are described in more detail below.
  • a Stretcher unit is capable of linking the self-stabilizing linker assembly to the Cleavable unit when the Cleavable unit is present, the self-stabilizing linker assembly to the Spacer unit when the Cleavable unit is absent but the Spacer unit is present and the self-stabilizing linker assembly to the Drug unit when both the Cleavable unit and the Spacer unit are absent.
  • a Stretcher unit is capable of attaching to more than one Cleavable unit, Spacer unit, and/or Drug unit.
  • the Stretcher unit can also act to alter the physiochemical properties of the Drug- Linker depending on components of the Stretcher unit, in some aspects, the Stretcher unit will be added in order to increase the solubility of the Drug-Linker and will comprise one or multiple solubility-enhancing groups such as ionic groups or water-soluble polymers.
  • Water-soluble typically includes any segment or polymer that is soluble in water at room temperature and includes poly(ethylene)g!yeol groups as well as other polymers such as polyethyleneimines.
  • a Stretcher unit can comprise one or multiple stretcher groups.
  • Exemplary stretcher groups include, for example, -NH-C
  • a representative stretcher group having a carbonyl group for linkage to the remainder of the Linker unit or the Drug unit is as follow: wherein R !
  • j is -Ci-Cio alkylene-, -C 3 -Cgcarbocyclo-, -aryiene-, -Ci-Cjoheteroalkylene-, -C 3 - Cgheterocyclo-, -Cj -Chalky lene-arylene-.
  • R 13 is -Ci-Cio alkylene- or -C j -C 3 oheteroalkylene-.
  • R 13 is -Ci ⁇ Ci 0 alkylene-, -(CH 2 CH 2 O) 1-10 (-CH 2 )i -3 -, or
  • R is -Ci-Cio alkylene- polyethyleneglycol, or polyethyjeneimine.
  • Non-cleavable drug release systems are known in the art and can be adapted for use with the self-stabilizing linker assemblies of the present invention as Stretcher units and/or Spacer units.
  • a non-cleavab!e linker in capable of Unking a Drug unit to a Ligand in a generally- stable and covalent manner and is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase- or esterase-induced cleavage, and disulfide bond cleavage.
  • Drug is released from Ligand Drug Conjugates containing non-cieavable linkers via alternative mechanisms, such as proteolytic ligand degradation.
  • Cross-linking reagents that form non-cleavable linkers between maytansinoid drugs and ligands are well known in the art and can adapted for use herein.
  • Exemplary cross-linking reagants that form non-cleavable linkers between the maytansinoid drugs and ligands comprise a maleimido or haloacetyl-based moiety.
  • the Cleavable unit (- W-), when present, is capable of linking the self-stabilizing linker assembly to the Spacer unit when the Spacer unit is present or the self-stabilizing linker assembly to the Drug unit when the Spacer unit is absent.
  • the linkage from the self-stabilizing linker assembly to the Spacer unit or to the Drug unit can be directly from the self-stabilizing linker assembly when the Stretcher unit is absent or via the Stretcher unit if the Stretcher unit is present.
  • the Cleavable unit will be directly conjugated to the self- stabilizing linker assembly on one end and to the Drug unit on the other end. in other embodiments, the Cleavable unit will be directly conjugated to the Stretcher unit on one end and to the Drug unit on the other end. In yet other embodiments, the Cleavable unit will be directl conjugated to the Stretcher unit on one end and to the Spacer unit on the other end. in even yet other embodiments, the Cleavable unit will be directly conjugated to the self-stabilizing linker assembly on one end and to the Spacer unit on the other end. Any of specifically described self- stabilizing linker assemblies described herein can be used in these embodiments.
  • the Cleavable unit is capable of forming a cleavable bond with a Drug unit or a Spacer unit.
  • Reactive groups for forming cleavable bonds can include, for example, sulfhydryi groups to form disulfide bonds, aldehyde, ketone, or hydrazine groups to form hydrazone bonds, carboxylic or amino groups to form peptide bonds, and carboxylic or hydroxy groups to form ester bonds.
  • cleavable linkers include disulfide containing linkers that are cleavable through disulfide exchange, acid-labile linkers that are cleavable at acidic pH, and linkers that are cleavable by hydrolases, peptidases, esterases, and glucuronidases.
  • the structure and sequence of the Cleavable unit is such that the unit is cleaved by the action of enzymes present at the target site.
  • the Cleavable unit can be cleavable by other mechanisms.
  • the Cleavable unit can comprise one or multiple cleavage sites.
  • the Cleavable unit will comprise one amino acid or one or more sequences of amino acids.
  • the Cleavable unit can comprise, for example, a monopeptide, a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
  • each amino acid can be natural or unnatural and/or a D- or L-isomer pro vided of course that there is a cleavable bond, in some embodiments, the Cleavable unit will comprise only natural amino acids. In some aspects, the Cleavable unit will comprise 1 to 12 amino acids in contiguous sequence.
  • each amino acid is Independently selected from the group consisting of alanine, arginine, asparlic acid, asparagine, histidme, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, ⁇ -alanine, aminoalkanoic acid, aminoalkynoic acid, aminoalkanedioic acid, arainobenzoic acid, amino- heterocyclo-alkanoic acid, heterocycio-carboxyiic acid, citrulline, statine, diaminoalkanoic acid, and derivatives thereof.
  • each amino acid is independently selected from the group consisting of alanine, arginine. asparlic acid, asparagine, hisiidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, and selenocysteine.
  • each amino acid is independently selected from the group consisting of alanine, arginine, aspartic acid, asparagine, histidme, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, and valine, in some embodiments, each amino acid is selected from the proteinogenic or the non-proteinogenic amino acids.
  • each amino acid is independently selected from the group consisting of the following L-(natural) amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan and valine.
  • each amino acid is independently selected from the group consisting of the following D-isomers of these natural amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan and valine.
  • the bond between the Cleavable unit and the Drug unit can be enzymaticaliy cleaved b one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
  • Useful Cleavable units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease.
  • a linkage (or bond) between the Cleavable unit and the Drug unit or Spacer unit is that which cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.
  • the Cleavable unit can comprise only natural amino acids. In other embodiments, the Cleavable unit can comprise only non-natural amino acids. In some embodiments, the Cleavable unit can comprise a natural amino acid linked to a non-natural amino acid. In some embodiments, the Cleavable unit can comprise a natural amino acid linked to a D ⁇ isomer of a natural amino acid.
  • An exemplary Cleavable unit is the dipeptide -Val-Cit-, -Phe-Lys- or -Val-Ala.
  • the Cleavable unit will comprises a peptide and will comprise from 1 to 22 amino acids.
  • the peptide will be conjugated directly to the Drug unit and the Spacer unit will be absent.
  • the Stretcher unit and Spacer unit will be absent.
  • the peptide will be a dipeptide.
  • the Cleavable unit -TMW W - will be represented by -(- ⁇ ) ⁇ .. ⁇ 2-, or (- ⁇ ) ⁇ .. ⁇ wherein AA is at each occurrence independently selected from natural or non- natural amino acids, in one aspect, AA is at each occurrence independently selected from natural amino acids.
  • amino acids are typically linked to the Drug unit or Spacer unit through functional units present in the amino acid, e.g., its earboxylie acid or amino termini.
  • the Ligand Drug Conjugates and Drug-Linkers are represented by the following formulae or salts thereof wherein L, L bS , L 1 r A, a', AA, Y, y', D, and p are as defined in any of the embodiments described herein and f is an integer from 1 to 12:
  • the Cieavable unit will comprise a glucoronide unit, preferably 1 or 2 glucoronide units.
  • the Glucuronide unit comprises a sugar moiety (Su) linked via a glycoside bond (- ( )'-) to a self-immolative Spacer:
  • glycosidic bond ( ⁇ 0' ⁇ ) is typically a ⁇ -glucuronidase-cleavage site, such as a bond cieavable by human, lysosomal ⁇ -glucuronidase,
  • Su is a Sugar moiety,_-0'- represents a glycosidic bond; each R is independently hydrogen, a halogen, -CN, or -N0 2 ; wherein the wavy bond adjacent to the nitrogen atom indicates covalent attachment to the Stretcher unit or to the Ligand and the wavy bond adjacent to the oxygen indicates covalent attachment to the Spacer unit or to the Drug unit.
  • An exemplary Linker unit comprising a glucoronide prior to conjugation to an antibody and post conjugation is as follows wherein the wavy line indicates attachment to a Drug unit or Spacer unit and Ab represents an antibody and S is a sulfur atom of the antibody, It will be understood that more than one self-stabilizing assembly can be attached to each antibody:
  • the Cleavable unit itself will comprise a sulfur atom that is capable of forming a bond with a sulfur atom of a Spacer unit or Drug unit to form a disulfide or hindered disulfide. Cleavage occurs between the two sulfur atoms of the disulfide.
  • one of the sulfur atoms is cleaved from the Drug unit and, provided there is no further release mechanism, the other sulfur atom remains attached to the Drug unit.
  • a Linker unit comprising a Cleavable unit having a sulfur atom is capable of forming a bond with a sulfur atom of a Spacer unit or Drug unit to form a disulfide or hindered disulfide
  • Exemplary linkers include, for example, the following Drug-Linker wherein the wavy line indicates the site of attachment to the remainder of the Linker unit, D is a mavtansinoid drug, and R a and 3 ⁇ 4 are independently selected from H or meth l.
  • a variety of disulfide linkers are known in the art and can adapted for use in the present invention, including, for example, those that can be formed using SATA (N-succirdraidyl-S- aeetyithioacetaie), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) > SPDB (N- succinimidyl-3-(2 ⁇ pyridyldithio)butyrate), SMPT (N-succiniraidyl-oxycarbonyl-alpha-methyl- alpha-(2-pyridyl-dithio)toluene), and SPP (N-succinimidyl 4-(2-pyridyIdithio)pentanoate).
  • SATA N-succirdraidyl-S- aeetyithioacetaie
  • SPDP N-succinimidyl-3-
  • the cleavable linker is pH-sensitive and will comprise, for example, an acid-labile linker that is hydrolyzable in the iysosome (e.g., a hydrazone, seraicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetai, or ketal group) can be used.
  • an acid-labile linker that is hydrolyzable in the iysosome
  • Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the Iysosome.
  • the Cleavable unit will be conjugated directly to the Drug unit and the Spacer unit will be absent and the Cleavable unit will be linked to the Drug unit via a cleavable peptide, disulfide, or hydrazone bond.
  • the Spacer unit when present, links a Cleavable unit to the Drug unit or a Stretcher unit to the Drug unit or a self-stabilizing linker assembly to a Drug unit.
  • the Spacer unit when present can act to extend the framework of the Linker unit.
  • the Spacer unit can comprise multiple self-immolative or non-self immolative groups.
  • the Spacer unit comprises one or more self-immolative groups, in this context, the term "self-immolative group” refers to a Afunctional chemical moiety that is capable of covalentiy linking together two spaced chemical moieties into a normally stable tripartite molecule.
  • the Spacer unit is not self-imraolative. In these embodiments, part, or all of the Spacer unit remains attached to the Drug unit.
  • -Y - is a self-immolative group and is linked to a Cleavable unit via the methylene carbon atom of the self-immolative group, and linked connected directly to the Drug unit via a carbonate, carbamate or eiher group.
  • -Yy- is a p-aminobenzyl alcohol (PAB) unit whose phenylene portion is optionally substituted with -C]-Cg alkyl, -0-(C t ⁇ Cg alkyl), -halogen,- nitro or -cyano.
  • PAB p-aminobenzyl alcohol
  • -Yy- can be a carbonate group.
  • An unsubstituted PAB unit is as follows:
  • self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-arainoitnidazol-5-methanol derivatives (see, e.g., Hay et ah, 1999, Bioorg. Med. Chem, Lett. 9:2237) and ortho or para- arninobenzyiaeetals.
  • Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (see, e.g., Rodrigues et ah, 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (s e, e.g.. Storm et ah, 1972, J. Amer. Chem. Soc. 94:5815) and 2-arninophenylpropionic acid amides (see, e.g., Amsberry et ah, 1990, J. Org. Chem. 55:5867).
  • Elimination of amine- containing drugs that are substituted at the a-position of glycine are also examples of self-immolative groups.
  • the optional Secondary Linker Assembly will typically be absent.
  • the Stretcher unit will generally be present but the Cleavable unit and the Spacer unit will be absent.
  • the Stretcher unit will extend the framework of the Linker unit to provide more distance between the self- stabilizing assembly and the Detection unit or Stability unit, in such aspects, the Stretcher unit is capable of linking the self-stabilizing linker assembly to the Detection unit or the Stability unit.
  • the number of self-stabilizing linkers per Ligand is represented by p.
  • p represents the number of drug-linker molecules (or detection-linker or stability-linker molecules) per Ligand molecule (e.g., antibody).
  • p can represent the average number of self-stabilizing linkers per Ligand (or in embodiments where the linkers are not branched, the average number of drug-linker molecules (or detection-linker or stability-linker molecules) per Ligand (e.g., antibody)).
  • the variable p ranges from 1 to 20, typically 1 to 12, 1 to 10 and is preferably from 1 to 8.
  • p when p represents the average, number of self-stabilizing linkers per antibody, p ranges from about 2 to about 5. in some embodiments, p is about 2, about 4, or about 8. in some preferred embodiments, when p represents the average number of drug-linker molecules per antibody, p ranges from about 2 to about 5. In some embodiments, p is about 2, about 4, or about 8.
  • the number of D' per self-stabilizing linkers is represented by u. u ranges from 1 to 10.
  • the average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, HiC and HPLC.
  • the quantitative distribution of Drug-Linker-Ligand conjugates in terms of p may also be determined.
  • separation, purification, and characterization of homogeneous Ligand-Drug Conjugates, where p is a certain value from Ligand -Drug Conj gate with other drug loadings may be achieved by means such as reverse phase HPLC or
  • the Self-Stabilizing linker assembly links the Ligand unit to a Stretcher unit if the Stretcher unit is present, links the Ligand unit to a Cleavabie unit if the Stretcher unit is absent and a Cleavabie unit is present, links the Ligand unit to a Spacer unit if the Stretcher unit and the Cleavabie unit are absent and the Spacer unit is present, or links the Ligand Unit to D' (e.g., a Drug Unit) if the Stretcher unit. Cleavabie unit and Spacer unit are absent.
  • D' e.g., a Drug Unit
  • the Stretcher unit, Cleavabie unit, and Spacer unit will be absent and the self- stabilizing linker assembly will be conjugated directly to D' (e.g., a Drug Unit). In other embodiments, one or more of the Stretcher unit, Cleavabie unit, and Spacer unit will be present.
  • tl/2 of hydrolysis refers to the time taken for half of the compound of interest to hydrolyze, i.e., undergo a ring opening, under stated conditions (e.g., pH 7.4 and 22°C).
  • the tl/2 of hydrolysis of the thio-substituted succinimide of the Self-Stabilizing Linker unit is less than 4 hours, preferably less than 3 hours, even more preferably, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes using the following assay and sta ed conditions.
  • the hydrolysis reaction rates of maleimido drug linkers following conjugation to antibody cysteines can be determined by mass spectrometry, as the hydrolyzed product has a molecular weight 18 daltons greater than the unhydrolyzed conj ugate. Reduction of the interchain disulfides of a human IgGl creates a single reduced cysteine on the light chain and three reduced cysteines on the heavy chain.
  • the self-stabilizing raaleimide drug-linker can then be conjugated to the reduced antibody at pH 7,4 and 22°C and introduced to a high-resolution electrospray mass spectrometer via a reversed-phase HPLC column which separates the conjugated light and heavy chains.
  • the masses of the conjugated light and heavy chains can thus be measured, and the peak intensities determined by standard mass spectrometry data processing software (e.g., MassLynx).
  • standard mass spectrometry data processing software e.g., MassLynx
  • the disappearance of the peak corresponding to the mass of the original, unhydrolyzed conjugate and the appearance of the peak corresponding to the mass of the hydrolyzed conjugate can be monitored, the intensities of the peaks determined, and the percentage of hydrolyzed conjugate calculated at each timepoint.
  • a curve is generated (e.g., using PRISM) which can be fit to a standard equation for exponential phenomena which includes a parameter for tl/2.
  • the Self-Stabilizing Linker will be designed such that the maleimide component of the Self-Stabilizing Linker does not substantially undergo hydrolysis prior to conjugation to the Ligand unit,
  • the tl 12 of hydrolysis of the thio- substituted succinimide of the Self-Stabilizing Linker is from about 5 or about 10 minutes to about 24 hours, preferably from about 5 or about 10 minutes to about 12 hours, more preferably from about 5 or about 10 minutes to about 5 hours, more preferably from about 5 or about 10 minutes to about 2.5 hours, even more preferably from about 5 or about 10 minutes to about 1 hour, even more preferably from about 5 or about 10 minutes to about 30 minutes, even more preferably from about 5 or about 10 minutes to about 20 minutes, and even more preferably from about 10 minutes to about 15 minutes at a ⁇ of about 7 to about 7.5 (e.g., 7.4) and a temperature of about 22°C,
  • the hydrolysis goes to completion. Complete hydrolysis is considered to be achieved if 90% of the thio-substiiuted succinimide hydroiyzes. Preferably, 95% or greater, 96%. 97%, 98%, 99% or 100% hydrolysis will be achieved.
  • the hydrolysis reaction will compete with a dilactam formation and will not achieve completion.
  • at least 90% of the reaction product will be a combination of either a hydrolyzed thio-substituted succinimide Ligand-Drug Conjugate or a thio-substituted dilactam Ligand-Drug Conjugate.
  • the reaction product will be a combination of either a hydrolyzed thio-substituted Ligand-Drug Conjugate or a thio- substituted dilactam Ligand-Drug Conjugate.
  • the percentage of hydrolysis can be calculated from the mass spectrometrie data of the conjugate at the final timepoint by determining the intensity of the peak corresponding to the mass of the original, unhydrolyzed conjugate and the intensity of the peak corresponding to the mass of the hydrolyzed conjugate, and using the sum of the peak intensities to determine the percentage hydrolyzed and percentage unhydrolyzed,
  • the stability of the Ligand-Drug Conjugate can he characterized by the ability of the Ligand-Drug Conjugate to undergo an elimination reaction and for the Drug-Linker to be transferred from the Ligand unit to a alternative reactive thiol present in the milieu of the Ligand-Drug Conjugate, in some embodiments, the Drug-Linker will exhibit no or substantially no disassociation from the Ligand under the following assay and stated conditions.
  • substantially no disassociation from the Ligand is considered to be achieved if less than 40%, preferably less than 20%, even more preferably less than 10%, or even more preferably less than 5% or less than 2% of the Drug-Linker in a sample disassociates from the L gand.
  • the elimination of a drug-linker containing an enzyme-cleavable linker from an antibody can be measured in ex vivo plasma by the following method.
  • the conjugate is placed in sterile plasma and incubated at 37°C.
  • an aliquot is removed at fro/en at -80°C.
  • the samples are passed over a protein A affinity resin to capture the antibody, the resin is washed with buffer, and then drug is released from the captured antibody by treatment with an appropriate enzyme (e.g. papain or proteinase K for peptide-based cleavable linkers).
  • an appropriate enzyme e.g. papain or proteinase K for peptide-based cleavable linkers.
  • the released drug can then be quantified by standard LC-MS methodology, and the quantity of drug measured at each timepoint divided by the q uantity of drug measured for the pre-incubation aliquot to determine the percentage of drug remaining conjugated to the antibody at each timepoint.
  • the precision of this assay can be improved by including an internal standard antibody-drug conjugate which is prepared using an isotopically labeled version of the same drug-linker, such that the drug which is released from it can be detected independently in the LC-MS assay from the drug released from the test drug-linker by virtue of its mass difference.
  • This isotopically labeled internal standard antibody-drug conjugate is added to each sample in equal amounts immediately prior to the protein A capture step.
  • An alternative method for evaluating the elimination of a maieimide drug-linker from an antibody (or other ligand) is to incubate the conjugate in buffer (e.g., phosphate-buffered saline) at slightly elevated pH (e.g., pH 8.0) in the presence of a large excess of a small-molecule thiol (e.g., N-acetyl cysteine, NAC) which will react with any maleimide that eliminates from the parent conjugate.
  • buffer e.g., phosphate-buffered saline
  • slightly elevated pH e.g., pH 8.0
  • a small-molecule thiol e.g., N-acetyl cysteine, NAC
  • LC-MS assays can be performed to detect and quantify the drag-linker conjugated to NAC, or the parent !igand-eonjugate. In the latter case, the ratio of the ligatid- conjugate to unconjugated ligand can be measured and will remain constant over time if the ligand-conjugate is stable. Additional methods are provided in the examples section.
  • the Ligand-Drug Conjugates are useful for inhibiting the multiplication of a tumor ceil or cancer cell, causing apoptosis in a tumor or cancer ceil, or for treating cancer in a patient.
  • the Ligand-Drug Conjugates can be used accordingly in a variety of settings for the treatment of cancers.
  • the Ligand-Drug Conjugates can be used to deliver a drug to a tumor cell or cancer cell.
  • the Ligand unit of a Ligand-Drug Conjugate binds to or associates with a cancer-ceil or a tumor-cell-associated antigen, and the Ligand-Drug Conjugate can be taken up (internalized) inside a tumor cell or cancer cell through receptor-mediated endocytosis or other internalization mechanism.
  • the antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell.
  • the drug is released within the cell, in an alternative embodiment, the Drug or Drug unit is cleaved from the Ligand-Drug Conjugate outside the tumor ceil or cancer cell, and the Drug or Drug unit subsequently penetrates the cell,
  • the Ligand-Drug Conjugates can provide conjugation-specific tumor or cancer drug targeting, thus reducing genera! toxicity of the drug.
  • the Linker units stabilize the Ligand-Drug Conjugates in blood, yet are capable of liberating drug once inside the cell.
  • the Ligand unit binds to the tumor cell or cancer cell.
  • the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor eel! or cancer cell.
  • the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
  • the specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated.
  • a ligand drug conjugate haying a BR96 Ligand unit can be useful for treating antigen positive carcinomas including those of the lung, breast colon, ovaries, and pancreas.
  • Ligand- Drug Conjugates having an anti-CD30 or an anti-CD70 binding Ligand unit can be useful for treating hematologic malignancies,
  • Solid tumors including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangtosarcoma, lymphangioendotheliosareoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papil lary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
  • ALL acute lymphoblastic leukemia
  • B-eell leukemia acute lymphoblastic B-eell leukemia
  • T-ceil leukemia acute myeloblasts leukemia
  • AML acute myeloblasts leukemia
  • APL ac promyelocyte leukemia
  • APL acute monoblastic leukemia
  • acute erythroleukeraic leukemia acute megakaryoblastic leukemia
  • acute myeiomonocytic leukemia acute nonlymphocyctic leukemia
  • acute undifferentiated leukemia chronic myelocytic leukemia "CML”
  • chronic lymphocytic leukemia chronic lymphocytic leukemia
  • hairy ceil leukemia multiple myeloma acute and chronic leukemias:
  • lymphoblastic, myelogenous, lyraphocytic, myelocytic leukemias Lymphomas
  • Cancers including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or inhibited by administration of a Ligand-Drug Conjugate.
  • methods for treating cancer including
  • a Ligand-Drug Conjugate administering to a patient in need thereof an effective amount of a Ligand-Drug Conjugate and a. chemotherapeutic agent.
  • the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory, In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory.
  • the Ligand-Drug Conjugates can be administered to a patient that has also undergone surgery as treatment for the cancer. [0247] in some embodiments, the patient also receives an additional treatment, such as radiation therapy.
  • the Ligand-Drug Conjugate is administered concurrently with the chemotherapeutic agent or with radiation therapy, In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a ligand drug conjugate,
  • a chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents, such a standard of care chemotherapeutic agent(s), can be administered,
  • methods of treatment of cancer with a Ligand-Drug Conj ugate are provided as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated.
  • the patient being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
  • the Ligand-Drug Conjugates are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease.
  • the Ligand-Drug Conjugates can be used accordingly in a variety of settings for the tTeatment of an autoimmune disease in a patient.
  • the Ligand-Drug Conjugates can be used to deliver a drug to a target ceil.
  • the Ligand-Drug Conjugate associates with an antigen on the surface of a target cell, and the ligand drug conjugate is then taken up inside a target-cell through receptor-mediated endocytosis.
  • the Linker unit binds to an autoimmune antigen.
  • the antigen is on the surface of a ceSi involved in an autoimmune condition.
  • the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.
  • the Ligand unit binds to activated lymphocytes that are associated with the autoimmune disease state.
  • the Ligand-Drug Conjugate kills or inhibit the multiplication of cells that produce an autoimmune antibody associated with a particular autoimmune disease.
  • Th2 lymphocyte related disorders e.g., atopic dermatitis, atopic asthma, rhinocorijunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease
  • Thl lymphocyte-related disorders e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave ' s disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis
  • activated B lymphocyte-related disorders e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type 1 diabetes
  • Polyglandular Syndromes Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome, Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome, Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosus, Takayasu's Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis, Type B insulin Resistance, Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia, Wegener's
  • Methods for treating an autoimmune disease including administering to a patient in need thereof an effective amount of a Ligand-Drug Conjugate and another therapeutic agent known for the treatment of an autoimmune disease.
  • the Ligand-Drug Conjugates are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease.
  • the Ligand-Drug Conjugates can be used accordingly in a variety of settings for the treatment of an infectious disease in a patient.
  • the Ligand-Drug Conjugates can be used to deliver a drug to a target cell.
  • the Ligand unit binds to the infectious disease cell.
  • the conjugates kill or inhibit the multiplication of cells that produce a particular infectious disease.
  • Particular types of infectious diseases that can be treated with the Ligand-Drug Conjugates include, but are not limited to, those disclosed in Table 3.
  • Measles, Subacute sclerosing panencephalitis Common Cold, Mumps, Rubella, Roseola, Fifth Disease, Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis, infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot- and-Mouth Disease, Bornholm Disease, Genital Herpes, Genital Warts, Aseptic Meningitis, Myocarditis, Pericarditis. Gastroenteritis, Acquired
  • AIDS immunodeficiency Syndrome
  • HSV Human Immunodeficiency Virus
  • Reye's Syndrome Kawasaki Syndrome
  • influenza Bronchitis
  • Viral "Walking" Pneumonia Acute Febrile Respiratory Disease
  • Acute pharyngoconj uncti val fever Epidemic keratoconjunctivitis
  • Herpes Simplex Virus i HSV-1
  • Herpes Simplex Virus 2 HSV-2
  • Shingles Cytomegalic Inclusion Disease
  • Rabies Progressive Multifocal Leukoencephalopathy
  • Kuru Fatal Familial Insomnia
  • Creutzfeldt- Jakob Disease Gerstraann-Straussler-Scheinker Disease
  • Tropical Spastic Paraparesis Western Equine Encephalitis, California Encephalitis, St.
  • Methods for treating an Infectious disease including administering to a patient in need thereof a Ligand-Drug Conjugate and another therapeutic agent that is an anti- infectious disease agent.
  • a Ligand-Drug Conjugate and another therapeutic agent that is an anti- infectious disease agent.
  • the present invention provides pharmaceutical compositions comprising the Ligand- Drug Conjugates described herein and a pharmaceutically acceptable carrier.
  • the Ligand-Drug Conjugates can be in any form that allows for the compound to be administered to a patient for treatment of a disorder associated with expression of the antigen to which the Ligand unit binds.
  • the conjugates can be in the form of a liquid or solid.
  • the preferred route of administration is parenteral. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques.
  • the compositions are administered parenterally.
  • the compounds are administered intravenously.
  • the present invention also provides pharmaceutical compositions comprising the Ligand-Functional Agent Conjugates described herein and a pharmaceutically acceptable carrier.
  • the Ligand-Drug Conjugates can be in any form that allows for the compound to be
  • compositions can be formulated so as to allow a compound to be bioavailable upon administration of the compositi on to a patient.
  • Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit.
  • compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the compound, the manner of administration, and the composition employed.
  • the composition can be, for example, in the form of a liquid.
  • the liquid can be useful for delivery by injection, in a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.
  • the liquid compositions can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digyleerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution
  • a parenteral composition can be enclosed in ampoule, a disposable syringe or a mul tiple-dose vial made of glass, plastic or other material.
  • Physiological saline is an exemplary adjuvant.
  • An injectable composition is preferably sterile.
  • the amount of the conjugate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques, in addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges,
  • the precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • compositions comprise an effective amount of a compound such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a compound by weight of the composition.
  • the composition can comprise from about 0.01 to about 100 mg of a Ligand-Drug Conjugate per kg of the animal's body weight, in one aspect, the composition can include from about I to about 100 mg of a Ligand-Drug Conjugate per kg of the animal's body weight, in another aspect, the amount administered will be in the range from about 0.1 to about 25 mg/kg of body weight of a compound.
  • the dosage of a compound administered to a patient is typically about 0.01 mg/kg to about 100 mg/kg of the subject's body weight. In some embodiments, the dosage administered to a patient is between about 0.01 mg/kg to about 35 mg/kg of the subject's body- weight. In some embodiments, the dosage administered to a patient is between about 0.1 mg/kg and about 15 mg/kg of the subject's body weight. In some embodiments, the dosage
  • the administered to a patient is between about 0.1 mg/kg and about 20 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 0.1 mg/kg io about 5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's body weight. In some
  • the dosage administered is between about 1 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg/kg to about 10 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 0.1 to 4 mg/kg, even more preferably 0.1 to 3.2 mg/kg, or even more preferably 0.1 to 2.7 mg/kg of the subject's body weight over a treatment cycle.
  • Ligand-Functional Agent Conjugates e.g., Ligand-Drug Conjugates
  • the Ligand-Functional Agent Conjugates can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa). Administration can be systemic or local.
  • Various delivery systems are known, e.g.,
  • encapsulation in liposomes, microparticles, microcapsules, capsules can be used to administer a compound.
  • more than one compounds or composition is administered to a patient,
  • carrier refers to a diluent, adjuvant or excipient, with which a compound is administered.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil.
  • the carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,.
  • auxiliary, stabilizing, thickening, lubricating and coloring agents can be used.
  • the compound or compositions and pharmaceutically acceptable carriers when administered to a patient, are sterile. Water is an exemplary carrier when the compounds are administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanoi.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the conjugates are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings.
  • the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions.
  • the compositions can also include a solubilizing agent.
  • Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • a conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceuticaf grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • compositions of the present invention comprise the Ligand Drug Conjugates of the present invention and a pharmaceutically acceptable carrier.
  • all, or substantially ail. or more than 50% of the Ligand Drug Conjugates present in the pharmaceutical composition comprises a hydrolyzed thio-substituted succinimide.
  • more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% of the Ligand Drug Conjugates present in the pharmaceutical composition comprises a hydrolyzed thio-substituted succinimide.
  • the present invention provides methods of preparing Ligand- Drug Conjugates or Ligand-Functional Agent Conjugates comprising a Self-Stabilizing Linker.
  • methods of the present invention comprise the steps of providing a Drug-Linker or Linker unit as described herein, conj gating said Drug-Linker or Linker unit to a sulfhydryl group of a Ligand unit to form a conjugate, allowing the resultant conjugate to undergo a hydrolysis reaction to form a Ligand-Drug conjugate comprising a thio- substituted hydrolyzed succinirnide.
  • the rate of the ihio-substitued succinirnide hydrolysis can be manipulated by adjusting the reaction conditions following conjugation of the Drug-Linker to the Ligand, e.g., by adjusting the pH or temperature.
  • all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95% of the thio- substituted succinirnide is hydrolyzed without manipulation of the reaction conditions, i.e., the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction.
  • the conjugation conditions are pH of about 7.4 and a temperature of about 22° C,
  • methods for preparing a Ligand-Drug Conjugate comprises the steps of providing a Drug-Linker or Linker unit comprising a Self-Stabilizing Linker;
  • a Ligand- Drug Conjugate conjugate comprising a non-hydrolyzed thio-substituted succinirnide; al lowing the non-hydrolyzed thio-substituted succinirnide to undergo a hydrolysis reaction, wherein all, substantially all, or at least 50%, 60%, 70%, 80% or even 85% of the succinirnide is hydrolyzed from 10 minutes to 4 hour's following conjugation.
  • all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95 % of the succinirnide is hydrolyzed by 10 minutes, by 20 minutes, 40 minutes 60 minutes, 90 minutes or 120 minutes following conjugation.
  • the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction.
  • the conjugation conditions are pH of about 7.4 and a temperature of about 22° C.
  • the present invention provides, inter alia, Self-Stabilizing Linkers. Methods of preparing Self-Stabilizing Linker units are encompassed within the scope of the present invention.
  • Maleimide compounds are typically prepared from corresponding amines by reaction of the primary amine with maleic anhydride followed by cyclodehydration of the maleamic acid. The overall scheme for the preparation of maleimde compounds is shown in the scheme below.
  • the first step of maleimide preparation the formation of the maleamic acid is very facile and can be usually accomplished in good yield by slow addition of the amine to a suspension containing a stoichiometric excess of the maleic anhydride.
  • the second step, cyclodehydration of the maleamic acid can be accomplished in a number of ways known to skilled in the art.
  • the use of chemical dehydrating agents has been a well established method for accomplishing this step.
  • Carbodiimides in combination with isomerizing alcohols, for example: DCC/HOBt have been used to effect, cyclodehydration of amic acids to maleimides.
  • Thermal cyclodehydration with use of azeotropic distillation in the presence of acid catalyst is another well known method to generate maleimides.
  • the use of an azeotropic solvent permits the efficient removal of the water co-product as it forms, thereby driving reaction to maleimide.
  • Suitable azeotropic solvents include cyclohexane, benzene, toluene, ethylbensene, mesitylene, and the like. Toluene is considered to be the most desirable since it boils at 1 10 °C at atmospheric pressure. Boiling temperatures below 200 °C are preferable to minimize possible thermal isomerization of maleamic acid to the more thermodynamically stable trans (fumaramic acid) structure.
  • the use of polar aprotic co-solvents can be beneficial for overall yield improvement as well as reducing time of cyciodehydration.
  • Several polar aprotic solvents including
  • dimethy!formamide dimethylacetamide, acetonitrile, N-methylpyrrolidone, dimethylsulfoxide, arid sulfonate have been claimed to be useful.
  • the most useful polar aprotic solvent is dimethylformamide.
  • maleimide compounds can be generated from maleimide and appropriate alcohol using, for example, Mitsunobu reaction conditions as shown in the scheme below (M.A. Walker, Tetrahedron Letters, 1994, v. 35, n 5, pp. 665-668),
  • the self-stabilizing linker assembly of the present invention are linked to the Stretcher unit, Cleavable unit, Spacer unit, or Drug unit using the teachings described herein in
  • Linkers and Drug-Linkers are conj gated to Ligand units using teachings described herein in combination with methods known in the art.
  • an antibody ca be treated with a reducing agent, such as dithiothreitol (DTT) to reduce some or all of the interchain disulfide cysteine residues to form highly nucleophilic cysteine thiol groups.
  • DTT dithiothreitol
  • the full reduced antibody or partially reduced antibody can be subsequently conjugated to the maleimide of the Linker Unit.
  • conjugation conditions are gentle ones, pH of about 7 and a temperature of about 22 °C.
  • the present invention provides intermediates for use in making Self-Stabilizirig Linkers. Intermediates include the following wherein T, c, R 31 and R 12 are as previously described.
  • self-stabilizing linkers for increasing the hydrolysis rates of mono thio-substituted succinimides, self-stabilizing linkers can also be used to increase the hydrolysis rate of mono-thio-substituted maleimides, di-thio-substituted maleimides, or di-thio- substituted succinimides.
  • a Ligand-Functionai Agent Conjugate comprising a Ligand unit and at least one Functional Agent selected from a Drug unit, a Detection unit, or a Stability unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring wherein the succinimide ring, maleimide ring, hydrolyzed succinimide ring or hydrolyzed maleimide ring is directly conjugated to the Ligand unit via one or two thioether linkages; and a base and an electron withdrawing group operabiy linked to stabilize the conjugate in plasma relative to a Ligand-Functionai Agent Conjugate lacking the self-stabilizing Sinker assembly (i.e.
  • the electron withdrawing group is positioned to increase the electrophilicity of the succinimide or maleimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide or maleimide ring (e.g., by an intramolecular base catalysis mechanism),
  • the Ligand-Functionai Agent Conjugates are represented by- Formula IV or IVa:
  • L is a Ligand unit
  • LL is a Ligand unit that can be present or absent, wherein L and LL can be the same or different Ligand units;
  • D ! is a Drug unit, a Detection unit, or a Stability unit
  • is an optional secondary linker assembly
  • * is a maleimide ring, a hydrolyzed maieimide, a succinimide ring, or a hydrolyzed succinimide conjugated to at least one of L or L ⁇ via a thioether linkage;
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be C; -8 alkylene, Cj. 3 heteroalkylene, aryiene, or C 4-1 o heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D';
  • the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0.
  • the scaffold is a Ce- ⁇ aryiene or C4.10 heteroarylene;
  • the subscript w' is 0 or 1 ;
  • -A- is an optional Stretcher unit
  • -A'- is an optional Stretcher unit component at the terminus of A
  • a' is 0 or i ;
  • u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present, and when u is L A can be present or absent.
  • L, HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, L, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates.
  • L and L L can be different Ligand units or the same Ligand unit, In embodiments, wherein L and L t , are the same Ligand unit, the succinimide or maleimide can be conjugated to the Ligand unit on the same or different polypeptide chains of the Ligand unit.
  • m + q + r is 0, 1 or 2
  • r is zero.
  • the Ligand-Funciional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein r and m are zero and q is one.
  • the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is C 1-g alkylene or Cj-g heteroalkylene (preferably C 1 .4 alkylene or C heteroalkylene), r is zero, and the sum ofm+q is 1.
  • the scaffold is Q. 3 alkylene or Q.3 heteroalkylene. in some such aspects, the alkylene is straight chain or branched.
  • the Ligand-Functional Agent Conjugate is represented by Formula IV or I Va or a salt thereof wherein the circle represents a scaffold that is C 1-8 alkylene or Ci-s heteroalkylene (preferably C alkylene or C].. 4 heteroalkylene), and m and r are zero.
  • the scaffold is C 1 .3 alkylene or C1.3 heteroalkylene.
  • the alkylene is straight chain or branched.
  • the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is Q, C 2 , C3 or C4 straight or branched chain alkylene, r is zero, and the sum of m+q is 1.
  • the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is Ci , C 2s C 3 or straight or branched chain alkyiene, and m and r are zero.
  • n and r are zero and is represented by
  • M" is a succinimide ring or hydrolyzed succinimide and Lj_, is present.
  • M 2 is a succinimide ring or hydrolyzed succinimide and LL is absent.
  • M is a maleimide ring or hydrolyzed maleimide and L L is present.
  • M is a maleimide ring or hydrolyzed maleimide and LL is absent.
  • when M 2 is a maleimide ring or hydrolyzed maleimide LL is present or absent and when M 2 is a succinimide ring or hydrolyzed succinimide LL is present.
  • L and L L when L and L L are present, L and L L can be the same Ligand unit or different Ligand units. In some aspects wherein L and LL are present and are the same Ligand unit, the maleimide or succinimide can be conjugated to the ligand unit on the same or different polypeptide chains of the Ligand unit.
  • the alkyiene or heteroaikylene chain can be straight or branched, in some aspects, the alkyiene or heteroaikylene chain will be a straight chain. In other aspects, it will be branched.
  • p can range from 1 to 20, preferably 1 to 12, even more 1 to 10 or 1 to 8.
  • the scaffold in some aspects wherein the scaffold itself is directly linked to the optional secondary linker assembly or D', (for example, in select embodiments when q is zero or when q is zero and r is zero), the scaffold will comprise a reactive site suitable for attachment to A or D'.
  • the maleimide ring can be conjugated to the Ligand unit via one or two thioether linkages as illustrated below both in non-hydrolyzed and hydrolyzed form and the succinimi.
  • de ring can be conjugated to the Ligand unit via two thioether linkages as illustrated below in both non-hydrolyzed and hydrolyzed form wherein the wavy line indicates the point of attachment to the remainder of the linker conj ugate or linker-functional agent, conjugate:
  • p typically ranges from 3 to 30, or 1 to 8, or 1 to 4 and the maleimide or succinimide can be conjugated to the same or different polypeptide chains of the Ligand.
  • the Ligand is an antibody. In other aspects, the Ligand is a non-antibody protein.
  • D' is a Drug unit, a Detection unit, or a Stability unit
  • is an optional secondary linker assembly
  • Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen:
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ci.g alkylene, Ci.g heteroalkylene, C$.10 arylene, or C4-S 0 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D';
  • the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C-6-10 arylene or C4.10 heteroarylene;
  • W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or i. ,
  • A is an optional Stretcher unit
  • A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1 , A can be present or absent
  • the halogen is bromine
  • HE and BU, L°, A, W, and Y have the meanings provided for the Drag-Linker Conjugates. Additionally each of the specifically recited selected embodiments for the circle, HE, BU, L°. A, W, and Y are equally applicable to these Conjugates.
  • n and r are zero and is represented by
  • Ligand-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
  • L is a Ligand unit
  • LL is a Ligand unit that can be present or absent, wherein L and L
  • RG is a reactive group (comprising a reactive site) at the terminus of L° or is suitable for attaching a Drug unit, Detection unit or Stability unit;
  • is an optional secondary linker assembly that is present
  • M 1 is a maleimide ring, a hydrolyzed ma!eirmde, a succinimide ring, or a hydrolyzed succinimide conjugated to at least one of L or LL via a thioether linkage;
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Cj -8 alkylene, Ci. heteroalkylene, C 6- jo aryiene, or C4.10 heteroarvlene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or FA;
  • the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a Q.
  • I G aryiene or C4. 10 heteroarylene;
  • the subscript p ranges from 1 to 20.
  • W- is an optional Cleavable unit, the subscript w ! is 0 or 1 ;
  • -Y- is an optional Spacer unit, the subscript y' is 0 or 1 ,
  • A is an optional Stretcher unit
  • A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 3 , A can be present or absent.
  • L, HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, L, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates.
  • r and m are zero and is represented by:
  • Branched or non-branched Linkers can be represented by the following formulas:
  • RG is a reactive group (comprising a reactive site) at the terminus of L
  • L is an optional secondary linker assembly that is present:
  • Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen;
  • BU is a Basic unit
  • HE is a hydrolysis enhancer comprising an electron withdrawing group
  • the circle represents a scaffold that can be Ciippog alkyfene, C;_g heteroalkyiene, C 6- io arylene, or C4- 1 0 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or RG;
  • the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a -io arylene or C 4 . 1 0 heteroarylene;
  • W ⁇ is an optional Cleavable unit, the subscript w' is 0 or i ;
  • -Y- is an optional Spacer unit.
  • the subscript y' is 0 or 1 ,
  • A is an optional Stretcher unit
  • A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1 , A can be present or absent.
  • HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates.
  • r and m are zero and is represented by:
  • [0331 j mDPR-Val-Cit-PAB- MAE was prepared by coupling Boc-protected mDPR to Val- Cit-PAB-MMAE using standard methods for peptide coupling. The Boe group was removed in the final step.
  • MMAE (5.34g, 6,94 mmol), Fmoc-Val-Cit-PAB-OCO-pNP (5.0g, 6.94 mmol) and HOBi (1.4 mmol) were charged to a 250 mi round bottom flask purged with N2 and dissolved in 15 ml of DMA. DIPEA (2.44 ml, 14 mmol) was then added, and the solution was stirred overnight at room temperature under inert atmosphere. The product was isolated by preparative HPLC, using a linear gradient from 30% MeCN (0.05%TFA) to 100% MeCN (0.05% TFA) over 40 min. Fractions containing product were concentrated on the rotovap to a white powder, affording 3.2 g (34%)
  • MMAE 100 mg, 0.089 mmol
  • HATU 41 mg, 0.107 mmol
  • DIPEA 34 uL
  • the reaction mixture was diluted with 1 ml DMSO and the product was isolated by preparative HPLC. (70 mg, 56%)
  • Y is the observed percent hydrolysis at time t
  • Ymax is the asymptotic maximal % hydrolysis
  • K is the hydrolysis rate constant.
  • Example 5 Evaluating spacing between the maleimide and basic group of the self-stabilizing linker assembly
  • an antibody conjugate would have to be held at pH 8 and 37 °C for approximately 5 hours to achieve complete hydrolysis (about 5 half-lives). Exposure of an antibody or other protein to such conditions for extended periods can potentially result in covalent modifications and misfolding events, and so raaleimides with even faster hydrolysis rates were sought.
  • This phenomenon may arise from a competing reaction such as direct nucleophilic attack of the primary amine on the succinimide ring, or may be due to an isomeric impurity in the maleimide which leads to biphasic hydrolysis kinetics.
  • Example 7 Evaluating spacing between the maleimide and carboxamide group of the self- stabilizing linker assembly
  • Both DPR maleimides possess a basic primary amine which is separated from the maleiraido nitrogen by two carbon atoms. Both also possess an electron withdrawing carboxamide group, however the distance from the maleimido nitrogen the carboxaraide varies from 1 to 2 carbon units (a. and ⁇ , respectively). Finajly, the separation between the basic amine and the carboxamide also varies from 1 to 2 carbon units ( ⁇ and a, respectively).
  • a self-stabilizing maleimido-drug-iinker was prepared.
  • This drug-linker contains the maleimido-DPR group coupled to the cytotoxic agent MMAE via a protease- cleavable val-cit PAB self-imrnolative group (referred to herein as maleimido-DPR-val-cit-PAB- MMAE).
  • a non self-stabilizing drug-linker was used (referred to herein as maleimido-caproyl-val-cit-PAB-MMAE).
  • the maleimide units of these drug-linkers can be prepared using maleic anhydride and mono -protected diaminopropionic acid and aminocaproic acid, respectively
  • DPR diamine roprionyl
  • ADCs were prepared with 8 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyi-val-cit-PAB-MMAE using a fully reduced humanized IgGl .
  • Drug loading was confirmed by reversed-phase HPLC on a polymeric PLRP-S column as described previously (Sun 2005). Complete succinimide hydrolysis of the self-stabilizing linker was also confirmed by electrospray mass spectrometry.
  • the ADC prepared with the self-stabilizing DPR maleimido drug-linker exhibited minimal loss of drug over this tirnecourse (from 8.0 to 7.9 drugs per antibody over 14 days), while the ADC prepared with the caproyl maleimido drug-linker lost approximately half of its drug load (from 8.0 to 3.9 drugs per antibody over 14 days) under these conditions.
  • ADCs were prepared with 8 drugs per antibody of either maleimido-DPR-val-eit-PAB-M AE or raaleimido-caproyl-vai-cit-PAB-MMAE using a fully reduced human IgGl. These ADCs (0.25 mg/mL) were incubated in sterile rat plasma for 7 days at 37 °C.
  • a second assay format was utilized to assess ADC stability in rat and human plasma ex vivo.
  • ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB- MMAE or maleimido-caproyl-val-cit-PAB-MMAE using a human IgGl partially reduced to a level of 4 thiols per antibody (resulting in an ADC with 4 drugs per antibody).
  • These two ADCs were spiked into rat and human plasma and incubated at 37 °C for 7 days. At seven timepoints during this incubation, aliquots were removed and frozen at -80 °C until completion of the timecourse.
  • ADCs were then isolated from each sample and MMAE released proteolytically from the isolated ADCs as described previously (Sanderson 2005), The released MMAE was then quantified by LC-MS/MS and normalized to the initial value for each ADC ( Figure 9).
  • the ADC prepared with a self- stabilizing maleimide lost little or no drug under these conditions, while approximately half of the drug was lost from a ma!eimido- caproyi ADC.
  • the drug:antibody ratio can be measured for ADCs in rat plasma by reversed-phase HPLC analysis following purification with IgSeiect resin. This method was applied to samples derived from an in vivo pharmacokinetic experiment in rats. ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-vai-cit-PAB-MMAE using a humanized IgGl partially reduced to an average of 4 thiols per antibody (resulting in drug : antibody ratio of 4).
  • ADCs were further purified as described previously (Sanderson 2005) by hydrophobic interaction chromatography to isolate the species containing 4 drugs per antibody. These ADCs were dosed intravenously at 10 nig/kg in Sprague-Dawley rats. At five timepoints, three animals from each dosing group were sacrificed and the collected blood was processed to plasma and frozen at. -80 °C. Upon completion of the study, all samples were processed by the IgSeiect resin method described above, except that the sample volume, varied. The drug : antibody ratio at each timepoint in this study are plotted in Figure 10.
  • the ADC prepared with a self-stabilizing maleimide exhibits minimal loss of drug in vivo, dropping from an initial value of 4.3 drugs per antibody to a value of 3.6 drugs per antibody ( 12% reduction) after 7 days.
  • the drug : antibody ratio of an ADC prepared with a maleimido-caproyl linker dropped from an initial value of 3.9 to a value of 1.5 (61 % reduction). This illustrates that the increased stability of a self-stabilizing drug-linker that is observed ex vivo translates into an in vivo setting.
  • Example 12 Pharmacokinetics
  • maleimido-caproyl ADCs are prone to loss of drug through raaleirnide elimination whereas self-stabilizing maleimide ADCs are not, it is reasonable to predict that exposure to antibody-conjugated drug will be greater following equivalent doses of the two ADCs.
  • ADCs were prepared with 4 drugs per antibody of either ma!eimido-DPR-vai-cit-PAB-MMAE or raaleimido-caproyl-val-cit-PAB-MMAE using a human IgG l partially reduced to an average of 4 thiols per antibody (resulting in drug : antibody ratio of 4).
  • ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl- val-cit-PAB-MMAE using a humanized IgGl (which has no known binding to any rat antigen) partially reduced to an average of 4 thiols per antibody (resulting in an average drug : antibody ratio of 4).
  • ADCs w r ere dosed intravenously in female CD®IGS rats (Charles River Laboratories) at 10 mg/kg (6 rats per test article plus 6 rats receiving vehicle only).
  • conjugates were prepared with the anti-CD30 antibody cAC iO using drug-sinkers containing the val-cit-PAB-MMAE cytotoxic pay load linked to the antibody via either a maleimido-caproyl group or a self-stabilizing maleimido-DPR group.
  • ADCs were evaluated in two separate murine xenograft models of CD30+ human malignancies. In the first mode!
  • L428 human Hodgkin Lymphoma cells were implanted subcutaneousiy in female NSG mice and tumors were allowed to grow a volume of approximately 100 mm 3 before dosing at 1 mg kg ever four days for four doses (six mice per dose group).

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Abstract

The present invention provides Ligand-Drug Conjugates, Drug-Linkers, Linkers, and Ligand-Linker Conjugates comprising a self-stabilizing linker assembly component.

Description

SELF-STABILIZING LINKER CONJUGATES
CONTINUITY
[0001 j This application claims the benefit of US Provisional Patent Application No.
61/647,373, filed May 15, 2012, and US Provisional Patent Application No. 61/773,067, filed March 5, 2013, and also claims priority to US Application Ser. No. 13/799,244, filed March 13, 2033, each of which is incorporated herein in its entirety and for ail purposes.
BACKGROUND OF THE INVENTION
[0002] The antibody-drug conjugate (ADC) field has made significant advances with the FDA approval of Brentuximab Vedotin for the treatment of a select group of patients and with the advancement of many other ADCs in the clinic, The linker component of ADCs is one important feature in developing optimized therapeutic agents that are highly active at well tolerated doses. The electrophilic maleimide functional group has proven very useful in the preparation of ADCs due to its high degree of specificity for reacting with thiol groups and the very fast thiol addition kinetics under gentle conditions,
Figure imgf000002_0001
[0003] As has been noted by multiple investigators in the bioconjugate field, the thio- subsiituted product of the reaction between the electrophilic maleimide functional group and free thiol of antibody is subject to slow elimination, thus reversing the above reaction:
Figure imgf000003_0001
o drug-finker
[0004] When this reversible reaction occurs in a purified preparation of the ADC, the reaction is largely undetectable because the maleimide and thiol which are regenerated through the elimination process simply react again, thus reforming the intact conjugate. However, when other thiols are present, the net effect can be the transfer of the maleimide from the antibody of the ADC onto any other available thiol. This process has been documented to occur in plasma, in which the maleimide of an ADC transfers to cysteine 34 of serum albumin (Alley et ai., Bioconjugate Chem. 2008, 19, 759-765). This process has also been reported when an ADC is incubated in the presence of excess cysteine or glutathione (Shen et a!., Nature Biotech, 30(2): 184-9, 2012). The present invention provides, inter alia, biocorijugates t at do not undergo this transfer reaction.
BRIEF SUMMARY OF THE INVENTION
[0005] The invention provides inter alia, Linkers, Drug-Linkers, Ligand-Drug Conjugates, Ligand-Linker Conjugates, Ligand-Functional Agent Conjugates, and Functional Agent-Linkers, and methods of preparing and using them. The Ligand-Drug Conjugates are stable in circulation, yet capable of inflicting cell death once released in the vicinity or within tumor cells.
BRIEF DESCRIPTION OF TOE DRAWINGS j0006] Figure 1 provides a reaction scheme illustrating the reduction of the interchain disulfides of a human IgGl , followed by conjugation of the resulting thiols with a self-stabilizing linker and subsequent hydrolysis of the succinimide ring (Top); and the use of mass spectrometry to monitor the change in the molecular weight of the antibody conjugates due to hydrolysis (Bottom).
Δ [0007J Figure 2 illustrates the timecourse of succinimide ring hydrolysis of a self-stabilizing antibody conjugate by electrospray mass spectrometry. Conjugation of fully reduced cACI O with maieimido-DPR-val-cit-PAB-MMAE was performed at pH 7.2 and 22°C, then samples were subjected to analysis by LC-MS at the indicated times (Top). The resulting data of % hydrolysis was plotted versus time and fit to an exponential equation to determine kinetic parameters (Bottom).
(0008] Figure 3 provides the hydrolysis kinetic profiles for bioconjugates prepared with an IgGl antibody and self-stabilizing linkers with varying spacing between the maleimide and the basic group (a primary amine). Conjugation was performed at pH 8 and 37°C, then hydrolysis of the IgGl light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation.
[0009] Figure 4 provides kinetic profiles of the hydrolysis of bioconjugates prepared with an IgG l antibody and self-stabilizing maleimide linkers with varying spacing between the maleimide and the basic group (a primary amine). Conjugation was performed at pH 8 and 37°C, then hydrolysis of the IgG l light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation.
[0010] Figure 5 provides hydrolysis kinetic profiles for bioconjugates prepared with an IgGl antibody and various N-substituted maieirnides. Conjugation was performed at pH 7.4 and 22°C, then hydrolysis of the IgG l light chain conjugate was immediately monitored by mass spectrometry, plotted as a function of time, and fit to an exponential equation. Hydrolysis of the maleimido-caproyl conjugate (bottom structure) is too slow to produce any detectable hydrolysis in 24 hours under these conditions. The presence of the carboxamide electron withdrawing group (EWG) or the primary amine (BASE) accelerate the hydrolysis, and the combination of the two (top structure) results in a conjugate which hydrolyzes with a half-life of less than 20 minutes under these mild conditions.
[0011] Figure 6 provides hydrolysis kinetic profiles for self-stabilizing maleimido drug- linkers prepared with et-diaminopropionic acid (a-DPR, open circles) and with β- diaminopropionic acid (β-DPR, filled circles). Although isomers of each other, the positioning of the basic amino group and the electron withdrawing carboxamide relative to the succinimide results in a 17-fold difference in the rate of succinimide hydrolysis. [0012] Figure 7 illustrates the change in drug loading over time for an ADC prepared with a self-stabilizing maleimido-DPR drug-linker versus one prepared with a maieimido-caproyl drug linker when incubated in a buffer containing excess thiol. The reversed -phase chromatograms of the two ADCs at lime zero and time 14 days after incubation is shown in the top panel.
Chromatographic peak assignments L0, LI , HO, HI, H2, and H3 correspond to unconjugated light chain, light chain with one drug, unconjugated heavy chain, and heavy chain with 1 , 2, or 3 drugs, respectively. The self-stabilizing maleimido-DPR drug-linker is represented with open circles versus one prepared with a maieimido-caproyl drug linker (open squares). Drug loading remains constant at 8 per antibody for the self-stabilizing drug-linker (open circles), but falls to 4 drugs per antibody over 14 days for the maieimido-caproyl drug linker (open squares), reflecting loss of drug by maleimide elimination.
[0013] Figure 8 illustrates the change in drug loading over time for ADCs prepared with a self-stabilizing maleimido-DPR drug-linker and a maieimido-caproyl drug linker, when incubated in rat plasma at 37 °C (R=val-cit-PAB-MMAE). ADC samples at each timepoint were purified by Ig Select affinity resin and their drug loading evaluated by reversed-phase HPLC analysis of the ADCs.
[0014] Figure 9 provides the stability profile of drugs conjugated to antibodies via a maieimido-caproyl drug- linker (squares) or a self-stabilizing maleimide linker(circles) during incubation in rat (open symbols) or human (filled symbols) plasma (R=vaI-cii-PAB-MMAE). ADCs were captured on Protein A affinity resin at each timepoint and the drug released enzymatically via its protease-cleavable linker. The released drug was then quantified by LC- MS/MS and normalized to the initial value. Each timepoint reflects the percent of the conj ugated drug that was observed at tO.
[0015] Figure 1Θ illustrates the decrease in drug loading in vivo (rats) for ADCs prepared with a self-stabilizing maleimido-DPR drug-linker and a maieimido-caproyl drug linker (R=val-cit- PAB-M AE). ADCs were dosed i.v. and plasma samples from each timepoint were purified by Ig Select affinity resin and their drug loading evaluated by reversed-phase HPLC analysis of the ADCs.
[0016] Figure 11 illustrates the antitumor activity of ADCs in a murine xenograft model of ALCL (Karpas-299 cell line). ADCs were prepared with the anti-CD3G antibody cACl O and drug linkers containing the val-cit-PAB-MMAE cytotoxic payload linked to the antibody via either a maleimido-caproyl group (closed circles) or a self-stabilizing maleimido-DPR group (open circles). Tumors were allowed to reach a volume of approximately 250 mmJ before dosing at 1 mg/kg weekly for three doses (six mice per dose group). The self-stabilizing ADC dose group experienced complete responses (no detectable tumor) in all six animals, with five animals experiencing durable regressions, while the maleimido-caproyl ADC experienced no complete responses.
DETAILED DESCRIPTION
Abbreviations aji Definitions
[0017] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings. When trade names are used herein, the trade name includes the product formulation, the generic drug, and the active pharmaceutical ingredient(s) of the trade name product, unless otherwise indicated by context.
[0018] The term "electron-withdrawing group" refers to a functional group that draws electrons away from a reaction center. Exemplary electron withdrawing groups include, but are not limited to, -C(=0), -CN, -N<¾, -CX3, -X, -COOR, -CQNR2, -COR, -COX, -S02R, -S02OR, -SO2NHR, -S02NR2, -PO3R2, -P(0)(CH3)NHR, NO, ·ΛΚΛ ~C R CR-;. and -C≡CR wherein X is F, Br, CI, or I, and R is, at each occurrence, independently selected from the group consisting of hydrogen and Cj.6 alkyl. Exemplary electron withdrawing groups can also include aryl groups (e.g., phenyl) and certain heteroaryj groups (e.g., pyridine). The term "electron withdrawing groups" includes aryls or heteroaryls further substitued with electron withdrawing groups.
Preferred electron withdrawing groups are -C(=G), -CN, -N02, -CX3, and -X.
(0019] The term "base" refers to a functional group that deprotonates water to produce a hydroxide ion. Exemplary bases are amines and nitrogen containing heterocycles.
Representative bases include -N(R3)(R4) wherein R3 and R4 are independently selected from H or Ci-6 alkyl, preferably H or methyl,
Figure imgf000007_0001
Figure imgf000007_0002
wherein R5, R&, R7 and R8 are, at each occurrence, independently selected from hydrogen or Ci-g alky], preferably H or methyl, and e is 0-4. In some aspects, the base, is a nitrogenous base.
[0020] The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, monospecific antibodies, muitispecific antibodies (e.g., bispecific antibodies), and antibody fragments that exhibit the desired biological activity. An intact antibody has primarily two regions: a variable region and a constant region. The variable region binds to and interacts with a target antigen. The variable region includes a complementary determining region (CDR.) that recognizes and binds to a specific binding site on a particular antigen. The constant region may be recognized by and interact with the immune system (see, e.g., Janeway et al, 2001, Immuno, Biology, 5th Ed., Garland Publishing, New York). An antibody can be of any type (e.g. , IgG, IgE, IgM, IgD, and IgA), class (e.g., IgGl, IgG2, lgG3, IgG4, IgAl and IgA2) or subclass. The antibody can be derived from any suitable species. In some embodiments, the antibody is of human or murine origin, An antibody can be, for example, human, humanized or chimeric,
[0021] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal''' ind icates t he character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
[0022] An "intact antibody" is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CH1 , CH2, Q arid CH , as appropriate for the antibody class. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof.
[0023] An "antibody fragment" comprises a portion of an intact antibody, comprising the antigen-binding or variable region thereof, Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, scFv, scFv-Fc, multispecific antibody fragments formed from antibody fragment(s), a fragments) produced by a Fab expression library, or an epitope-binding fragments of any of the above which immunospecificaily bind to a target antigen (e.g., a cancer ceil antigen, a viral antigen or a microbial antigen).
[0024] An "antigen" is an entity to which an antibody specifically binds.
[0Θ25] The terms "specific binding" and "specifically binds" mean that the antibody or antibody derivative will bind, in a highly selective manner, with its corresponding target antigen and not with the multitude of other antigens. Typically, the antibody or antibody derivative binds with an affinity of at least about IxlO"7 M, and preferably 10"8 M to iO"9 M, 10"10 M, 10~n M, or 10"!2 M and binds to the predetermined antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. [0026] The term "inhibit" or "inhibition of means to a reduce by a measurable amount, or to prevent entirely.
[0027] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer ceils; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may inhibit growth and/or kill existing cancer ceils, it may be cytostatic and/or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and/or determining the response rate (RR).
[0028] The term "substantial" or "substantially" refers to a majority, /', e. >50% of a population, of a mixture or a sample, preferably more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% of a population.
[0029] The terms "intracellulariy cleaved" and "intracellular cleavage" refer to a metabolic process or reaction inside a ceil on a Ligand Drug conjugate (e.g., an Antibody Drug Conjugate (ADC) or the like), whereby the covalent attachment, e.g., the linker, between the Drug moiety (D) and the Ligand unit (e.g., an antibody (Ab)) is broken, resulting in the free Drug, or other metabolite of the conjugate dissociated from the antibody inside the cell. The cleaved moieties of the Drug-Linker-Ligand conjugate are thus intracellular metabolites.
[0030] The term "cytotoxic activity" refers to a cell-killing, a cytostatic or an anti-proliferative effect of a Drug-Linker-Ligand con jugate compound or an intracellular metabolite of a Drug- Linker-Ligand conjugate. Cytotoxic activity may be expressed as the IC50 value, which is the concentration (molar or mass) per unit volume at which half the cells survive.
[0031] The term "cytotoxic agent" as used herein refers to a substance that inhibits or inhibits the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., 211 At, 13iI, 1251, 90Y, f 86Re, 188Re, 1S3Sm, 2i2Bi, 32P, 60C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enz raatically active toxins of bacterial fungal, plant or animal origin, including synthetic analogs and derivatives thereof.
[0032] The terms "cancer" and "cancerous" refer to or describe the physiological condition or disorder in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells.
[0033] An "autoimmune disease" herein is a disease or disorder arising from and directed against an individual's own tissues or proteins.
[0034] Examples of a "patient" include, but are not limited to, a human, rat, mouse, guinea pig, monkey, pig, goat, cow, horse, dog, cat, bird and fowl. In an exemplary embodiment, the patient is a human.
[0035] The terms "treat" or "treatment," unless otherwise indicated by context, refer to therapeutic treatment and prophylactic measures to prevent relapse, wherein the object is to inhibit or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., no worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder. f0036] in the context of cancer, the term "treating" includes any or all of: inhibiting growth of tumor cells, cancer cells, or of a tumor; inhibiting replication of tumor cells or cancer cells, lessening of overall tumor burden or decreasing the number of cancerous cells, and ameliorating one or more symptoms associated with the disease.
[0037] In the context of an autoimmune disease, the term "treating" includes any or all of: inhibiting replication of cells associated with an autoimmune disease state including, but not limited to, cells that produce an autoimmune antibody, lessening the autoimmune-antibody burden and ameliorating one or more symptoms of an autoimmune disease. [0038] As used herein, the term "Detection unit" refers to refers to any molecule which produces, or can be induced to produce, a detectable signal. Detection units having reporter molecules that can be detected by imaging equipment include, but are not limited to, radioactive, paramagnetic, fluorescent or radtoopaque chemical entities. In some embodiments, the
Detection unit will be a radioactive compound, a chemilummescent agent, a fluorescent agent, or a chromogen. In some embodiments, the Detection unit will be a fluorescent molecule such as a fluorophore.
[0039] As used herein, the term "Stability unit" refers to a compound that promotes the stability of the conjugate, e.g., by increasing systemic retention of the Ligand when administered to a patient. A Stability unit can also increase the water solubility of the conjugate. An exemplary Stability unit is polyethylene glycol,
[0040] The phrase "pharmaceutically acceptable salt," as used herein, refers to
pharmaceutically acceptable organic or inorganic salts of a compound (e.g. , a Drug, Drug- Linker, or a Ligand-Drug Conjugate). Th compound can contain at least one amino group, and accordingly acid addition salts can be formed with the amino group. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tarmate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1 ,1 '-methyl ene-bis - (2-hydroxy-3- naphthoate)) salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterfoil may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. "Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
[0041] Unless otherwise indicated, the term "alkyl" by itself or as part of another term refers to a substituted or unsubstituted straight chain or branched, saturated or unsaturated hydrocarbon having the indicated number of carbon atoms (e.g., "-CfCg alkyl" or "-Cf-Cio alkyl refer to an aikyl group having from I to 8 or 1 to 10 carbon atoms, respectively). When the number of carbon atoms is not indicated, the aikyl group has from 1 to 8 carbon atoms. Representative straight chain "-Cj -Cg aikyl" groups include, but are not limited to, -methyl, -ethyl, -n-propyl, - n-butyl, -n-pentyl, -n-hexyl, -n-heptyi and -n-octyl; while branched -Ci-Cg alkyis include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -fert-butyl, -isopent l, and -2-methylbutyl;
unsaturated -C2-C8 alkyis include, but are not limited to, -vinyl, -allyl, -1 -butenyl, -2-butenyl, -isobutyienyl, -1-pentenyl, -2-pentenyl, -3-methyI- 1 -butenyl, -2-methyl-2~butenyl,
-2,3~dimethyl-2-buteny3, -1-hexyl, 2-hexyl, -3-hexyl, -acetylenyl, -propynyl, - -butynyl, -2-butynyl, - 1 -pentynyl, -2-pentyiiyl and -3-methyl-l butvnyl. In some embodiments, an aikyl group is unsubstituted. An aikyl group can be substituted with one or more groups. In some aspects, an aikyl group will be saturated,
[0042] Unless otherwise indicated, "alkylene," by itself of as part of another term, refers to a substituted or unsubstituted saturated, branched or straight chain or cyclic hydrocarbon radical of the stated number of carbon atoms, typically 1-10 carbon atoms, and having two monovalent radical centers derived by the removal of two hydrogen atoms from the same or two different carbon atoms of a parent, alkane. Typical alkylene radicals include, but are not limited to:
methylene (-(¾-), 1 ,2-ethyl (-CH2CH2-), 1 ,3-propyl (-CH2CH2CH2-), 1,4-butyl
(-CH2CH2CH2CH2-), and the like. In preferred aspects, an alkylene is a branched or straight chain hydrocarbon (i.e., it is not a cyclic hydrocarbon).
[0043 J Unless otherwise indicated, "aryl," by itself or as part of another term, means a substituted or unsubstituted monovalent carbocyclic aromatic hydrocarbon radical of 6-20 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Some aryl groups are represented in the exemplary structures as "Ar". Typical aryl groups include, but are not limited to, radicals derived from benzene, substituted benzene, naphthalene, anthracene, biphenyl, and the like. An exemplary aryl group is a phenyl group as follows:
Figure imgf000012_0001
[0044] Unless otherwise indicated, an "arylene," by itself or as part of another term, is an aryl group as defined above which has two covalent bonds (i.e., it is divalent) and can be in the ortho, meta, or para configurations as shown in the following structures, with phenyl as the exemplary group:
Figure imgf000013_0001
|Θ045] Unless otherwise indicated, a C3-CS heterocycle," by itself or as part of another term, refers to a monovalent substituted or unsubstiiuted aromatic or non-aromatic monocyclic or bicyclic ring system having from 3 to 8 carbon atoms (also referred to as ring members) and one to four heteroatom ring members independently selected from N, O, P or S, and derived by removal of one hydrogen atom from a ring atom of a parent ring system. One or more N, C or S atoms in the heterocycle can be oxidized. The ring that includes the heteroatom can be aromatic or nonaromatic. Unless otherwise noted, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. Representative examples of a C Cg heterocycle include, but are not limited to, pyrrolidinyl, azetidinyl, piperidinyl, morpbolinyl, tetrahydrofuranyl, tetrahydropyranyl, benzofuranyl, benzothiophene, inclolyl, benzopyrazolyl, pyrrolyl, thiophenyl (thiophene), furanyl, thiazoiyl, imidazolyl, pyrazoiyl, pyrimidinyl, pyridinyl, pvrazinyl, pyridazinyl. isothiazolyi, and isoxazolyl. A"C3-Cs Iieteroaryi," is an aromatic C3-C8 heterocycle.
[0046] Unless otherwise indicated, "C3-Q heterocyclo," by itself or as part of another term, refers to a C3-Cg heterocycle group defined above wherein one of the heterocycle group's hydrogen atoms is replaced with a bond (i.e., it is divalent). A "C3-Q heteroaryiene," by itself or as part of another term, refers to a C3-C8 heteroaryl group defined above wherein one of the Iieteroaryi group's hydrogen atoms is replaced with a bond (i.e., it is divalent).
[0047] Unless otherwise indicated, a "C3-C3 carbocycle," by itself or as part of another term, is a 3-, 4-, 5-, 6-, 7- or 8-menibered monovalent, substituted or unsubstituted, saturated or unsaturated non-aromatic monocyclic or bicyclic carbocyclic ring derived by the removal of one hydrogen atom from a ring atom of a parent ring system. Representative -C3-C>< carbocycles
32 include, but are not limited to, cyclopropyi, cydobutyl, cyelopentyl, cydopentadienyl, cyelohexyl, cyelohexenyi, 1 ,3-cyclohexadienyl, 1,4-cyclohexadienyl, eyeloheptyl, 1,3- cycioheptadienyl, 1 ,3,5-cydoheptatrieny I, cyciooctyl, and cyclooctadienyl.
IS] Unless otherwise indicated, a "C3-Cg carbocyclo," by i tself or as part of another term, refers to a Q Cs carbocycle group defined above wherein another of the carbocycle groups' hydrogen atoms is replaced with a bond (i.e., it is divalent),
[0049] Unless otherwise indicated, the term "heteroalkyi," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain hydrocarbon, or combinations thereof, fully saturated or containing from 3 to 3 degrees of unsaturation, consisting of the stated number of carbon atoms and from one to ten, preferably one to three, heteroatoms selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N and S may be placed at any interior position of the heteroalkyi group or at the position at which the alky! group is attached to the remainder of the molecule. The heteroatom Si may be placed at any position of the heteroalkyi group, including the position at which the alkyl group is attached to the remainder of the molecule, Examples include -CH2-CH2-0-CH3, -C¾-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, - CH2-CH2-S(0)~CH3, -NH-CH2-CH2" H-C(0)-CI-l2-CIl3, ~CH2-CH2-S(0)2-CH3; -CH=CH-0- CH3, -Si(CH3)3, -CH2-CH=N-0-CH3, and -CH=CH-N(CH3)-CH3, Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-0-Si(CH3)3. in preferred embodiments, a Cj to C4 heteroalkyi or heteroaikylene has 1 to 4 carbon atoms and 1 or 2 heteroatoms and a Q to C3 heteroalkyi or heteroaikylene has 1 to 3 carbon atoms and 2 or 2 heteroatoms. In some aspects, a heteroalkyi or heteroaikylene is saturated.
)| Unless otherwise indicated, the term "heteroaikylene" by itself or as part of another subsiituent means a divalent group derived from heteroalkyi (as discussed above), as exemplified by -CH2-CH2-S-CH2-CH2- and - H2-S-CH2-CH2-NH-CH2-. For heteroaikylene groups, heteroatoms can also occupy either or both of the chain termini. Still further, for alkylene and heteroaikylene linking groups, no orientation of the linking group is implied,
[0051] "Substituted alkyl" and "substituted aryl" mean alkyl and aryl, respectively, in which one or more hydrogen atoms are each independently replaced with a subsiituent. Typical substiiuerrts include, but are not limited to, -X, -R, -O", -OR, -SR, -S", -NR2, -NR3,
=NR, -CX3, ~CN, -OCN, -SCN, -N=C=0, -NCS, -NO, -N02, =N2, -N3, -NRC(=0)R, -C£=0)R, -
C(=0)NR2, -SO3 \ -SO3H, -S(=0)2R; -QS(O)20R, -S(0)2NR, -S(=0)Rs -OP(-0)(OR)2, -
P(=0)(OR)2, -P0' 3, -PO3H2, -As02H2, -C(=0)R, -CH))X, -C(=S)R, -C02R, -C02 "
, -C(=S)OR, -C(=0)SR, -C(-S)SR, -C(=0)NR2, -C(-S)NR2, or -C(=NR)NR2, where each X is independently a halogen: -F, -CI, -Br, or -I; and each R. is independently -H, -C;~C2o alkyl, -
C6-C20 aryl, -C3-C14 heterocycle, a protecting group or a prodrug moiety. Alkylene, carbocycle, carbocycio, arylene, heteroalkyi, heteroalkylene, heterocycle, heterocyclo, heteroaryl, and heteroarvlene groups as described above may also be similarly substituted.
[0052] R.G is a reactive group that contains a reactive site (RS) that is capable of forming a bond with either the components of the Linker unit (i.e., A, W, Y) or the Drug unit D. RS is the reactive site within a Reactive Group (RG). Reactive groups include sulfhydryj groups to form disulfide bonds or thioether bonds, aldehyde, ketone, or hydrazine groups to form hydrazone bonds, carboxylic or amino groups to form peptide bonds, carboxylic or hydroxy groups to form ester bonds, sulfonic acids to form sulfonamide bonds, alcohols to form carbamate bonds, and amines to form sulfonamide bonds or carbamate bonds. The following table is illustrative of Reactive Groups, Reactive Sites, and exemplary functional groups that can form after reaction of the reactive site. The table is not limiting. One of skill in the art will appreciate that the noted IV and R" portions in the table are effectively any organic moiety (e.g.. an alkyl group, aryl group, heteroaryl group, or substituted alkyl, aryl, or heteroaryl, group) which is compatible with the bond formation provided in converting RG to one of the Exemplary Functional Groups, it will also be appreciated that, as applied to the embodiments of the present invention, R' may represent one or more components of the self-stabilizing linker or optional secondary linker, as the case may be, and R" may represent one or more components of the optional secondary linker, Drug unit, Stability unit, or Detection unit, as the ease may be.
Figure imgf000015_0001
2) R'-C(=0)OH -C(=0)- R'-C(=0)NH-R"
3) R'-C(=0)ONHS -C(-O i- R'-C(=0)NH-R"
4) R'S(=0)2-OH -S(=0)2- R'S(=0)2NH-R"
5) R'-CHrX (X is Br, i. CI) -t'i !;··· R'-CH2-S-R"
6) R'-NH2 -N- R'-NHC(=0)R"
it will be understood that, once reacted, the reactive site RS can form a new bond with components of the Linker unit or the Drug unit, as the case may be. The reactive site, RS, once linked to the remainder of the Linker unit has typically lost its reactivity,
[0053] The term "dilactam" as used herein refers to a cyclic amide that forms from a macro- cyclicization reaction with a thio-substituted succinimide and base present on the self-stabilizing linker assembly.
General
[0054] Hydrolysis of a maieimide (or thio-substituted succinimide) represents a nucieophi!ic addition reaction in which water, acting as the nucleophile, attacks one of the electrophi!ic carbonyl carbon atoms of the maieimide ring (or succinimide ring). The rate of this reaction is influenced by eleciroph iicity of the carbonyls, which can vary with the substitution of electron- donating or electron- withdrawing groups present on the nitrogen of the imide group. The rate of the hydrolysis reaction is also influenced by the pH of an aqueous solvent, which effectively increases the nucleopliilicity of water with increasing pH. It has been disco vered by the present inventors that the placement of a basic group on an N-substituted maieimide also increases the rate of the hydrolysis. By careful engineering of an N-substituent group on the maieimide, the combination of its electron withdrawing influence on the maieimide ring (thus increasing its elecirophiiicity) and localized basicity (increasing the effective nucleopliilicity of nearby water) can be used to tune the rate of hydrolysis of either the parent maieimide or its thio-substituted succinimide derivative. The present invention provides, inter alia, N-substituted maleimides with hydrolysis rates that fall within a useful range wherein their reaction with thiols occurs mors quickly than their hydrolysis to the maieie acid derivative, but which yield thio-substituted succinimides with hydrolysis rates that are sufficiently rapid to achieve complete hydrolysis under gentle conditions that are very suitable for the manufacture of protein-based bioconjugates.
[0055] The present invention is based, in part, on the discovery that a basic functional group proximal to a maleimide will catalyze the hydrolysis of a thio-substituted succinimide which is formed upon conjugation of the maleimide and a protein thiol leading to a stable bioeonjugate. By further combining a proximal basic group with an electron withdrawing group, the rate of thio-substituted succinimide ring hydrolysis can be tuned to a desirable level. Design parameters that affect the rate of hydrolysis include the p a of the basic group, the strength of the electron withdrawing group when present, and the proximity of both groups to the maleimide carbonyl carbons. Design parameters that affect the percentage hydrolysis include the nature and proximity of the base to the maleimide carbonyl carbons.
[00S6| Conceptual ly, without limiting the invention, a Linker unit comprising a self-stabilizing linker assembly is referred to herein as a Self-Stabilizing Linker or Self-Stabilizing Linker unit. The Self-Stabilizing Linker prior to conjugation with the Ligand unit comprises a maleimide group. The Self-Stabilizing Linker is self-stabilizing by virtue of the proximity of the maleimide group to a base within the linker unit which catalyzes the hydrolysis of its own thio-substituted succinimide after conjugation to the Ligand unit. This is represented schematically below:
Ugand
Figure imgf000017_0001
[0057] It will be understood that the term Self-Stabilizing Linker refers to the Linker unit both prior to and post stabilization,
[0058] In view of the above, the present invention provides in one group of embodiments, a Ligand-Functional Agent Conjugate comprising a Ligand unit and at least one Functional Agent selected from a. Drug unit, a Detection Unit, or a Stabilizing Unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide ring directly conjugated to the Ligand unit via a thioether linkage; and a base and an electron withdrawing group (conjugated to the Ligand unit via the succinimide) operably linked to stabilize the conjugate in plasma relative to a ligand drug conjugate lacking the self-stabilizing linker assembly (i.e. by increasing the rate of succinimide ring hydrolysis). In some aspects, the electron withdrawing group is positioned to increase the electrophilicity of the succinimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide ring (e.g., by an intramolecular base cataly sis mechanism). In some aspects, in place of the succinimide ring is a dilactam formed when the base reacts with the succinimide ring. In another group of embodiments, Functional Agent- Linker units are provided wherein the Linker portion comprises a self-stabilizing linker assembly, in another group of embodiments, Ligand-Linker conjugates are provided, wherein the Linker portion comprises a self-stabilizing linker assembly. In some embodiments, the Linker portion further comprises an optional secondary linker assembly (L°).
(0059] in some aspects, the Ligand-Functional Agent Conjugate is a Ligand-Drug Conjugate. Accordingly, the present invention provides in one group of embodiments, a Ligand-Drug Conjugate comprising a Ligand unit and at least one Drug unit, wherein the Ligand unit and each of the Drug unit(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide ring directly conjugated to the Ligand unit via a thioether linkage; and a base and an electron withdrawing group (conjugated to the Ligand unit via the succinimide ring) operably linked to stabilize the conjugate in plasma relative to a ligand drug conjugate lacking the self-stabilizing linker assembly (i.e. by increasing the rate of succinimide ring hydrolysis), in some aspects, the electron withdrawing group is positioned to increase the electrophilicity of the succinimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide ring (e.g., by an intramolecular base catalysis mechanism), in some aspects, in place of the succinimide ring is a dilactam formed when the base reacts with the succinimide ring. In another group of embodiments, Drug-Linker units are provided wherein the Linker portion comprises a self-stabilizing linker assembly. In another group of embodiments, Ligand-Linker conjugates are provided, wherein the Linker portion comprises a self-stabilizing linker assembly, In some embodiments, the Linker portion further comprises an optional secondary linker assem bly (L°). In some embodiments, the secondary linker assembly is a releasabie linker assembly (L* ) which comprises a Cleavable unit and optionally one or more of a Stretcher and a Spacer unit. In some other embodiments, the secondary linker assembly is a non-releasable linker assembly (LN) which comprises one or more of a Stretcher unit and a Spacer unit. In still other embodiments, the invention provides methods of treating cancer, immune disease, infectious diseases and other diseases and disorders using a Ligand-Drug Conjugate comprising a self-stabilizing linker assembly,
[0060] The Linker unit of the Ligand-Functional Agent Conjugate (or Ligand-Drug Conjugate) can further comprise, in addition to a self-stabilizing linker assembly, an optional secondary linker assembly (L°) which joins each Functional Agent (or Drug unit) to the self-stabilizing linker assembly. The secondary linker assembly can be a releasabie linker assembly or a non- releasable linker assembly.
[0061] The term Linker unit can be used herein to refer to the linker portion of the Ligand- Functional Agent Conjugate (or Ligand-Drug Conjugate) comprising the self-stabilizing linker assembly and optional secondary linker assembly.
The Self-Stabilizing Linker Assembly
|0062] The Self-Stabilizing Linkers are designed such that the rate of the post-conjugation hydrolysis of the succinimide ring will be controllable and fall within a desired range. The limits of this range are typically dictated by issues which arise in the manufacture of ligand-drug conjugates. On the one hand, hydrolysis which is too slow would require unacceptable delays in the manufacturing process or aggressive conditions of pH and temperature which may induce damage to the protein backbone, Conversely, a maleimide which is too reactive with water may be hydrolyzed to the corresponding maleic acid derivative before it can react with available protein thiols (see undesired pathway):
Figure imgf000019_0001
hydrolyzed maieimide maleimide thio-substituted succinimide hydrolyzed succinimide
(maleic acid derivative) (2 possible positional isomers) Such maleic acid derivatives are not reactive with thiols, and thus this reaction pathway does not result in a bioconjugate. Therefore, maleimides which undergo hydrolysis faster than thiol addition under applicable conditions are not useful reagents, in general, structural features which increase the hydrolysis rate of a thio-substituted succinimide will also increase the hydrolysis rate of the parent maleimide.
[0063] in designing the Self-Stabilizing Linkers of the present invention, it will be understood that the p a of the basic group, the strength of the electron withdrawing group(s), and the proximity of both groups to the maleimide are inter-related variables and will affect the hydrolysis rate of both the maleimide and corresponding thio-substituted succinimide product. Accordingly, positioning of the electron withdrawing group and base will be dependent upon the pKa of the base and the strength of the electron withdrawing group(s). The skilled artisan will understand that for particularly strong electron withdrawing groups such as fluoro,
trifluoromethyi, and nitro, the group can be further from the maleimide. In some embodiments, the hydrolysis reaction may compete with a macro-cyclization reaction such that the resultant conjugates comprise a heterogenous mixture of hydrolyzed thio-substituted succinimide conjugates and cyclized thio-substituted dilactam conjugates. In preferred embodiments, a dilactam will not be formed.
Selected Embodiments of the Invention
[0064] in some embodiments, the Ligand-Functionai Agent Conjugate is represented by Formula 1:
Figure imgf000020_0001
L' ss
(I)
or a salt thereof (e.g., pharmaceutically acceptable salt thereof;!;
wherein L is a Ligand unit;
D' is a Drug unit, a Detection unit, or a Stability unit:
L° is the optional secondary linker assembly; and
Lss is the self-stabilizing linker assembly, wherein
M! is a succinimide ring or a hydroiyzed succiniraide or together with BIJ forms a dilactam;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci-8 alkylene, C1-8 heteroalkylene, Ce-io arylene, or Quo heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or C4_;0 heteroarylene;
the subscript a and b are each 0 or 1 , and the sum of a+b is 1 ; and
the subscript p ranges from 1 to 20,
[0065] In some aspects, when r is 1, HE does not comprise a carbonyl group,(i.e., C(=0))
[0066] in some aspects, r is zero, in some aspects, a is 1 and b is zero. In other aspects, a is zero and b is 1.
[0067] In some aspects m + q + r is 0. in such aspects, the scaffold is a C6-io arylene or C4.10 heteroarylene and acts as the electron withdrawing group. Exemplary aryls and heteroaryls include phenyl and pyridinyl,
[0068] In some aspects m + q + r is 1 or 2.
[0069] In some aspects, the Conjugate is represented by Formula I or a sal t thereof wherein a is 1 and r is zero.
[0070] In some aspects, the Conjugate is represented by Formula I or a salt thereof wherein L,° is present and is a releasable linker assembly, the circle represents a scaffold that is Cj.g alkylene or Cj.g heteroalkylene (preferably C1-4 alkylene or C1.4 heteroalkylene), a is 1, r is zero, and the sum of m+q is 1. In some such aspects, the scaffold is Ci-3 alkylene or d-3 heteroalkylene. In some such aspects, the alkylene is straight chain or branched. [0071 J in some aspects, the Conjugate is represented by Formula I or a salt thereof wherein V" is present and is a releasable linker assembly, the circle represents a scaffold that is C1-8 alkylene or Ci-8 heteroalkylene (preferably C].4 alkylene or C heteroalkylene), a is 1 , and m and r are zero. In some such aspects, the scaffold is C1 -3 alkylene or C1.3 heteroalkylene. in some such aspects, the alkylene is straight chain or branched.
[0072] in some aspects, the Conjugate is represented by Formula I or a salt thereof wherein L° is present and is a releasable linker assembly, the circle represents a scaffold that is Ci , C2, C3 or C4 straight or branched chain alkylene, a is i, r is zero, and the sum of m+q is 1.
[0073] In some aspects, the Conjugate is represented by Formula 1 or a salt thereof wherein L° is present and is a releasable linker assembly, the circle represents a scaffold that is Q, C2, C3 or Q straight or branched chain alkylene, a is 1 , and m and r are zero.
[0074] in some aspects, there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succininiide (hydroiyzed or non- hydrolyzed) or dilactam and there are no more than 5 atoms, no more than 4 atoms, no more than 3 atoms, or no more than 2 intervening atoms between the electron withdrawing group and the nitrogen atom of the succininiide ring (hydroiyzed or non-hydrolyzed) or dilactam.
[0075] In each of these embodiments, the alkylene or heteroalkylene chain can be straight or branched, n some aspects, the alkylene or heteroalkylene chain will be a straight chain. In other aspects, it will be branched.
»] In each of these embodiments, p can range from 1 to 20, preferably 1 to 12, even more preferably i to 10, or 1 to 8.
[0077] In each of these embodiments, M1 is preferably a succinimide ring (i.e., non- hydrolyzed) or a hydroiyzed succinimide ring (also referred to herein as hydroiyzed
succinimide).
[0078] in each of these embodiments, D' can be a Drug unit, D, and the Ligand-Functional Agent Conjugate can be a Ligand-Drug conjugate.
In some aspects wherein the scaffold itself is directly linked to the optional secondary assembly or D', (for example, in select embodiments when q is zero or when q is zero and r is zero), th scaffold will comprise a reactive site suitable for attachment to the optional secondary linker assembly or D'.
[0080] in some embodiments, the self-stabilizing linker assembly (L¾ ) is represented by Formula Π:
or a salt thereof (e.g., pharmaceutically acceptable salt) wherein the wavy lines indicates points for attachment of the optional secondary linker assembly to D' or D, and wherein // indicates the point of attachment to a Ligand Unit. In the self-stabilizing linker assembly above, M 1 represents a succinimide ring or a hydroiyzed succiniraide ring or a diiactam formed when the base reacts with the succinimide ring, BU is a Basic unit, HE is a hydrolysis enhancer comprising an electron withdrawing group, and the circle represents a scaffold that can be Ci.g alkylene, Cj.s heteroalkyiene, C& io arylene, or C4-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly, D!, or D; and the subscripts m, q and r are each 0 or J , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-;o arylene or C4.10 heteroarylene.
[0081] In some aspects, when r is 1 , HE does not comprise a carbonyl group,(i.e., C(=0))
[0082] in some aspects, the self-stabilizing linker assembly is represented by Formula 11 wherein r is zero.
[0083] In some aspects m + q + r is 0. In such aspects, the Cg-jo arylene or C4.]o heteroarylene act as the electron withdrawing group. Exemplary aryls and heteroaryis include phenyl and pyridinyl.
[0084] In some aspects m + q + r is 1 or 2.
77 [0085] In some aspects, the self-stabilizing linker assembly is represented by Formula 11 or a salt thereof wherein the circle represents a scaffold that is Cf.g aikylene or Cj.g heteroalkylene (preferably C aikylene or heteroalkylene), r is zero, and the sum of m+q is i . In some such aspects, the scaffold is C1.3 aikylene or Cj.3 heteroalkylene. In some such aspects, the aikylene is a straight chain or branched aikylene.
[0086] in some aspects, the self-stabilizing linker assembly is represented by Formula II or a sait thereof wherein, the circle represents a scaffold that is C1-8 aikylene or C]- heteroalkylene (preferably C aikylene or heteroalkylene) and m and r are zero. In some such aspects, the scaffold is C1.3 aikylene or Cj.3 heteroalkylene. In some such aspects, the aikylene is a straight chain or branched aikylene.
[0087J In som aspects, the self-stabilizing linker assembly is represented by Formula II or a salt thereof wherein the circle represents a scaffold that is Ci , C2, C3. or C4 straight or branched chain aikylene, r is zero, and the sum of m+q is I .
[00881 in some aspects, the self-stabilizing linker assembly is represented by Formula II or a salt thereof wherein the circle represents a scaffold that is C C2, C3, or C4 straight or branched chain aikylene, and m and r are zero.
[0089] In some aspects, there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succininiide (hvdroiyzed or non- hydroiyzed) or dilactam and there are no more than 5 atoms, no more than 4 atoms, no more than 3 atoms, or no more than 2 intervening atoms between the electron withdrawing group and the nitrogen atom of the succinimide ring (hydrolyzed or non-hydrolyzed) or dilactam.
[0090] In each of these embodiments, the aikylene or heteroalkylene chain will preferably be a straight or branched chain. In some aspects, the aikylene or heteroalkylene chain will be a straight chain. In other aspects, it will be a branched chain.
[0091 in each of these embodiments, M1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
[0092] In each of these embodiments, D' is preferably D, a Drug unit. [0093 j Returning to the embodiments of the invention wherein the Ligand-Fursctional Agent Conjugate has the Formula (I);
Figure imgf000025_0001
or a salt thereof, wherein each of the scaffold, L, M!, HE, BU, L°, D', and the subscripts p, m, q and r have the meanings provided above, selected embodiments include those wherein
1) m is 1, and q and r are 0;
2) q is 1 , and m and r are 0;
3) r is 1 , and m and q are 0;
4) m i I , q and r are 0, and a is ί ;
5) q is 1 , m and r are 0, and a is 1 ;
6) r is 1, m and q are 0, and a is 1 ;
7) m is 1 , q and r are 0, and D' is a Drug unit, D;
8) q is 1. m and r are 0, and D' is a Drug unit, D;
9) r is 1, m and q are 0, and D' is a i ug unit, D;
10) m is 3 , q and r are 0, a is 1, and D: is a Drug unit, D:
11) q is 1, m and r are 0, a is 1, and D' is a Drug unit, D; or
12) r is i , m and q are 0, a is I , and D' is a Drug unit, D. [0094] In other selected embodiments, including those based on each of the selected embodiments of J ), 2) 3), 4), 5), 6), 7), 8), 9), 10), 1 1 ), and 12) above , the Basic unit (BU) comprises a primary, a secondary amine, or a tertiary amine. In still other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 1 1 ), and 12) above, the Basic unit is selected from the group consisting of-(C(R )( R!° ))XNH2, - (C(R9)( R10 ))xNHRa, and -~(C(R9)( R30 ))xNRa 2, wherein x is an integer of from 0-4 (or from 1 to 4) and each Ra is independently selected from the group consisting of Ci-ό alky! and Cj-6 haloaikyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each R' and Rw are independently selected from H or Ci.j alkyl. In still other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 1 1), and 12) above, the Basic unit is selected from the group consisting of -(CH2 )XNH2, -(CH2 )xNHRa, and -(CH2 )xNRa 2, wherein x is an integer of from 0 to 6 (preferably 0 to 4, or 1 to 4) provided that if x is zero there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each Ra is independently selected from the group consisting of C1-6 alkyl and C1-6 haloaikyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group. I n yet other selected embodiments, x is an integer of from 1 to 4. In even other selected embodiments, including those based on each of the selected embodiments of 1), 2) 3), 4), 5), 6). 7), 8), 9), 10), 1 1), and 12) above, the Basic unit is Ni k -CH2NH2, -CH2CH2NH2, - CH2CH2CH2 NH2, or -- ¾CH2CH2CH2NH2 provided that if the Basic unit is -NH2, there are no less than 2 intervening atoms between the base and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam.
[0095] in still other selected embodiments, including those based on the selected embodiments of 2), 5), 8), and 1 1) above and including those based on the embodiments of the preceding paragraph, HE preferably comprises a carbonyl, sulfonyl or phosphoryl moiety.
[0096] In yet other selected embodiments, including those based on each of the selected embodiments above (e.g., each of the selected embodiments of 1), 2) 3), 4), 5 ), 6), 7), 8), 9), 10), 1 1), and 12) above and including those based on the embodiments of the preceding paragraphs), there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydro iyzed.) or dilactam and there are no more than 5 atoms, no more than 4 atoms, no more than 3 atoms, or no more than 2 intervening atoms between the electron withdrawing group and the nitrogen atom of the succinimide ring (hydrolyzed or non-hydrolyzed) or dilactam.
[0097] In yet other selected embodiments, including those based on each of the selected embodiments above (e.g., each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 1 1), and 12) above and including those based on the embodiments of the preceding paragraphs, 1 is a succinimide ring or hydrolyzed succinimide.
[0098 ] In yet other selected embodiments, including those based on each of the selected embodiments above (e.g., each of the selected embodiments of 1), 2) 3), 4), 5), 6), 7), 8), 9), 10), 11), and 12) above and including those based on the embodiments of the preceding paragraphs, the circle represents a scaffold that is Cj-8 alkylene or Ci..g heteroalkylene (preferably Cj.4 alkylene or C heteroalkylene). In some such aspects the alkylene is a straight or branched chain alkylene,
[0099] In yet other selected embodiments, the Ligand-Functional Agent Conjugates have the formula;
Figure imgf000027_0001
1 O or a pharmaceutically acceptable salt thereof, wherein each of the scaffold, L, M , HE, BIJ, L , D', and the subscript p has the meaning provided above, selected embodiments include those wherein: 1) the Basic unit (BU) comprises a primary, a secondary amine, or a tertiary amine, and D' is preferably a Drug unit D.
2) the Basic unit is selected from the group consisting of -(C(R9)( R.1 ))XNH2) -(C(R9)( R10 ))xNHRa, and -(C(R9)( R 10 ))xNRa 2, wherein x is an integer of from 0-4 (or 1-4) and each Ra is independently selected from the group consisting of C1-6 alk l and Q.6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that if x is zero, there are no less than 2 intervening- atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and R9 and RlU are independently selected from H or C1.3 alkyl, and D' is preferably a Drug unit D.
3) the Basic unit is selected from the group consisting of-(CH2 )XNH2, ~(CH2 )XNHR\ and -~(CH2 )xNRa 2, wherein x is an integer of from 0 to 6 (preferably 0 to 4 or 1 to 4) provided that if x is zero, there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam, and each Ra is independently selected from the group consisting of C e alkyl and .6 haloalkyl. or two R8 groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, and D' is preferably a Drug unit D. In yet other selected embodiments, X is an integer of from 1 to 4.
4) the Basic unit is -N¾, -CH2N¾ ~CH2CH2NH25 -C¾CH2CH2 NH2, or
-CH2CH2CH2CH2 N¾ provided that if the Basic unit is -NH2, there are no less than 2 intervening atoms between the base and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam; and D' is preferably a Drug unit D.
[0100] In still other selected embodiments, including those based on each of the selected embodiments above, HE comprises a carbonyl, sulfonyl or phosphoryl moiety, and D' is preferably a Drug unit D.
[0101] in yet other selected embodiments, including those based on each of the selected embodiments above, there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed) or dilactam and there are no more than 5 atoms, no more than 4 atoms, no more than 3 atoms, or no more than 2 intervening atoms between the electron withdrawing group and the nitrogen atom of the succinimide ring (hydrolyzed or non-hydrolyzed) or dilactarn, and D! is preferably a Drug unit (D).
[0102] In yet other selected embodiments, including those based on each of the selected embodiments above, M1 is a succinimide ring or hydrolyzed succinimide, and D' is preferably a Drug unit (D).
[0103] in yet other selected embodiments, including those based on each of the selected embodiments above, the circle represents a scaffold that is Q-g alkylene or C-.-g heteroalkylene (preferably CM alkylene or C heteroalkylene), and D' is preferably a Drug unit (D). In some- such aspects, the alkylene is a straight chain or branched chain alkylene.
[0104] In still other selected embodiments, including those based on each of the selected embodiments above, HE is a carbonyl, and D' is preferably a Drug unit (D).
[0105] In still other selected embodiments, including those based on each of the selected embodiments above, H E is a carbonyl and the circle represents a scaffold that is a straight chain Ci-8 alkylene or C heteroalkylene (preferably CM alkylene or C heteroalkylene), and D' is preferably a Drug unit (D).
In still other selected embodiments, including those based on each of the selected embodiments above, H E is a carbonyl and the circle represents a scaffold that is a branched chain C i.g alkylene or Ci-8 heteroalkylene (preferably CM alkylene or CM heteroalkylene), and D' is preferably a Drug unit (D).
I In yet other selected embodiments, Ligand-Drug Conj ugates have the formula:
Figure imgf000029_0001
or a pharmaceutically acceptable salt thereof, wherein the Ligand portion is an antibody (Ah), the subscript p ranges from 1 to 20 (preferably 1 to 12), and M1, BU, L° are as described in any of the embodiments provided herein, and D is a Drug unit. For example, in some aspects, L° is a releasable linker assembly, and BU is (CI ! ;■ )vNf -(CH2 )xNHRa, and ΐί"! h ),\'R:' 2, wherein x is an integer of from 1-4 and each Ra is independently selected from the group consisting of Q. 6 alkyl and C-i-6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, In yet other aspects, L° is a releasable linker assembly, and BU is C! !-N! k -CH2CH2NH2, -CH2CH2CH2 H2, or - CH2CH2CH2CH2 NH2. in some aspects, the Ab can be replaced by a non-antibody protein. M1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
[010S] in yet other selected embodiments, the Ligand-Drug Conjugates have the formula:
Figure imgf000030_0001
or a pharmaceutically acceptable salt thereof wherein the Ligand portion is an antibody (Ab) and the subscript p ranges from I to 20 (preferably i to 12) and M1, BU, and L° are as described in any of the embodiments provided herein, and D is a Drug unit. For example, in some aspects, L° is a releasable linker assembly, and BU is -(OL )xNH2, -(CH2 ) NHRa, and -(Cl¾ )xNRa 2, wherein x is an integer of from 1-4 and each Ra is independently selected from the group consisting of Cj.6 alkyl and C;.6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group. In yet other aspects, L° is a releasable linker assembly, and BU is -CH2NH2, -CH2CH2NH2, --CH2CH2CH2 NH2, or -CH2CH2CH2CH2 H2. In some aspects, the Ab can be replaced by a non-antibody protein. M1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
[0109] in yet other selected embodiments, the Ligand-Drug Conjugates have the formula:
Figure imgf000030_0002
or a pharmaceutically acceptable salt thereof wherein the Ligand portion is an antibody (Ab), the subscript p ranges from 1 to 20 (preferably 1 to 12), and M1, BU, and L° are as described in any of the embodiments provided herein and D is a Drug unit. For example, in some aspects, L° is a releasable linker assembly, and BU is -(CH2 )XNH2, -(CH2 )xNHRa, and ~(CH2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Cj. 6 alkyl and Ci-6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group. In yet other aspects, L° is a releasable linker assembly, and BU is -NH2, -CH2NH2, -CH2CH2NH2, (Ί LCM K i h L. or -CH2CH2CH2CH2 NH2. In some aspects, the Ab can be replaced by a non-antibody protein. M1 is preferably a succinimide ring or a hydrolyzed succinimide ring.
[0110] Having described a variety of Ligand-Functional Agent Conjugates and Ligand-Drug
Conjugates provided by the present disclosure, one of skill in the art will appreciate that component assemblies are also useful. Accordingly, the present invention provides Fuctional Agent-Linker Conjugates (e.g., Drug-Linker Conjugates), Linkers, and Ligand-Linker assemblies.
Functional Agent-Linker Conjugates ,
[0111] In another embodiment, the present invention provides Functional Agent-Linker Conjugates (e.g., Drug-Linker Conjugates) having the formula:
Figure imgf000031_0001
or a salt thereof (e.g., pharmaceutically acceptable salt) wherein,
D' is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly: and
Lss is the self-stabilizing linker assembly, wherein BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci_g alkylene, Cj.g heteroalkylene, C6-io arylene, or C,i..]0 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
the subscripts rn, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or C4.10 heteroarylene, and the subscript a and b are each 0 or 1 , and the sum of a+b is 1.
[0112] Irs certain selected embodiments, the Functional Agent-Linker Conjugate is represented by the formula:
Figure imgf000032_0001
or a salt thereof, while in other selected embodiments, the Drug-Linker Conjugate is represented by the formulae:
Figure imgf000032_0002
Figure imgf000033_0001
Figure imgf000033_0002
or a saii thereof, wherein the circle, HE, BU, 1/ and D' have the meanings provided herein for Formula 5 and D is a Drug unit. Additionally, each of the specifically recited selected embodiments for the circle, HE, BU, L° and D' (for Formula 1 or any of the conjugates provided herein) are equally applicable to these Drug-Linker Conjugates, In preferred aspects D' is a Drug unit, D,
Linkers
[0113} Also provided herein are Linkers having the formula:
Figure imgf000033_0003
or a. salt thereof (e.g., pharmaceutically acceptable salt), wherein RG is a reactive group (comprising a reactive site) at the terminus of L°, suitable for attaching a Drug unit;
L° is an optional secondary linker assembly that is present: and
Lss is the self-stabilizing linker assembly, wherein
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci-s alkyiene, C\.% heteroalkyiene, C6-i0 arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit:
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C&io arylene or Gno heteroarylene: and the subscript a and b are each 0 or 1, and the sum of a+b is 1 .
(0114] in some aspects wherein the Linker is attached to a Detection unit or a Stability unit, RG is a reactive group that contains a reactive site that is capable of forming a bond with a Detection unit or a Stability unit instead of a Drug unit.
[0115] In certain selected embodiments, the Linker is represented by the formula:
Figure imgf000034_0001
or a salt thereof, while in other selected embodiments, the Linker is represented by the formulae:
Figure imgf000035_0001
Figure imgf000035_0002
Figure imgf000035_0003
or a salt thereof (e.g., pharmaceutically acceptable salt) wherein the circle, HE, BU, L° and RG have the meanings provided above. Additionally, each of the specifically recited selected embodiments for BU, L° and RG (for any of the conjugates provided herein) are equally applicable to these Linkers,
Ligand-Linker Conjugates [0116] Also provided herein are Ligand-Linker Conjugates, having the formula:
Figure imgf000036_0001
or a salt thereof (e.g., pharmaceutically acceptable salt) wherein
L is a Ligand unit;
the subscript p ranges from 1 to 20;
RG is a reactive group (comprising a reactive site) at the terminus of L° which is suitable for attaching a Drug unit;
Lu is an optional secondary linker assembly that is present; and
Lss is a self-stabilizing linker assembly, wherein
M: is a succinimide ring or hydrolyzed succinimkle;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be C1-8 a!kylene, C].g heteroalkyiene, C6-io arylene, or C4.10 heteroaryiene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit;
the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6.io arylene or C -io heteroaryiene; and
the subscript a and b are each 0 or 1, and the sum of a b is 1.
[0117] In some aspects wherein the Ligand-Linker Conjugate is attached to a Detection unit or a Stability unit instead of a Drug unit, RG is a reactive group that contains a reactive site that is capable of forming a bond with a Detection unit or a Stability unit instead of a Drug unit.
[0118] In certain selected embodiments, the Ligand-Linker Conjugate is represented fay the formula:
Figure imgf000037_0001
ss or a salt thereof while in other selected embodiments, the Ligand-Linker Conjugate is represented by the formula:
Figure imgf000037_0002
Figure imgf000037_0003
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein L is an antibody (Ab). and the circle, HE, M\ BU, L° and RG have the meanings provided above. Additionally, each of the specifically recited selected embodiments for Ab, M ', BU, L° and RG (for any of the conjugates provided herein) are equally applicable to these Ligand-Linker conjugates. [0119] In some embodiments of the invention, the self-stabilizing linker assembly instead of being represented by the structure for Lss is represented by L' 1 and has Formula (III):
Figure imgf000038_0001
or a pharmaceutically acceptable salt thereof, wherein the wavy line indicates points of attachment of the optional secondary linker assembly or Drug unit and wherein // indicates the point of attachment to a Ligand unit;
wherein M1 is a non-hydro lyzed or hydrolyzed succinimide or M1 forms a dilactam with B (e.g., a dilactam is formed when B reacts with the succinimide ring), wherein the succinimide or dilactam is conjugated to the Ligand unit via a thioether linkage;
V, Q, T, and G are independently selected from -(C(R9)( R10))-;
R1 is H or C| -3 alk l;
R9 and R'°are, in each occurrence, independently selected from H or C1.3 alkyl;
F is C(E')( E ) wherein E1 and E2 are independently selected from hydrogen, an electron withdrawing group, or E1 and E2 together are (=0);
RS is a reactive site for conjugation to a component of the optional secondary linker assembly or Drug unit;
g is 0 to 5;
m is 0 to 5;
n is 0 to 5;
d is 0 or 1 ;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
and B is a base.
[0120] In some aspects, there are from 1 to 20 drug-linkers attached to each Ligand unit. [0121] In selected embodiments, Ligand-Drug Conjugates have the formula:
Figure imgf000039_0001
or a pharmaceutically acceptable salt thereof.
[0122] In selected embodiments, Drug-linkers have the formula:
Figure imgf000039_0002
a pharmaceutically acceptable salt thereof.
[0124] In selected embodiments, Ligand -Linker Conjugates have the formula:
Figure imgf000040_0001
or a pharmaceutically acceptable salt thereof in formulas ilia, 1Mb, IIIc, and Hid or pharmaceutically acceptable salts thereof:
L, if present, is a Ligand unit;
L° is an optional secondary linker assembly;
RG, if present, is a reactive group (comprising a reactive site) at the terminus of L° which is suitable for attaching a Drug unit;
M1, if present, is a non-hydrolyzed or hydrolyzed succinimide or 1 forms a diiactam with B (e.g., a diiactam is formed when the base reacts with the succinimide ring), wherein the succinimide or diiactam is conjugated to the Ligand unit via a thioether linkage;
V, Q, T, and G are independently selected from -<C(R9)( R10))-;
R' is H or Ci.3 alkyl;
R9 and R!° are, in each occurrence, independently selected from H or Cj.3 aikyl; F is C(E')(E') wherein El and E2 are independently selected from hydrogen, an electron withdrawing group, or E1 and E2 together are (=0);
RS is a reactive site for conjugation to a component of the optional secondary linker assembly or Drug unit;
g is O to 5;
m is 0 to 5;
n is 0 to 5; d is 0 or ] ;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
p, if present, ranges from 1 to 20, preferably 1 to 12;
and B is a base.
[0125] it will be understood that for Formula ί 11 (including Ilia, illb, ilic, and II Id) and . pharmaceutically acceptable salts thereof, the electron withdrawing group will either be represented by F (e.g., E1, E~ or E: and Ez") or by the reactive site RS. For example, when d is zero, or when E' and E2 are hydrogen, the reactive site will act as an electron withdrawing group. In some aspects, when d is zero, RS is -C(=0)-. In some aspects, n, d, and g are zero or m, n, d, and g are zero and RS is -C(=0)-.
[0126] Exemplary embodiments wherein the Ligand-Drug Conjugates, Drug-Linkers, Linkers, or Ligand-Linker conjugates are represented by Formula III (or formulae I l ia, illb, Hie, or IIId, as the case may be) or pharmaceutically acceptable salts thereof, include those wherein m is zero; m is zero and n is zero, one, two, or three; x is I ; x is zero and n is zero, one, two, or three; and m is zero, n is zero, and x is 1. Exemplary embodiments include those described herein wherein R and R1 are hydrogen. Exemplary embodiments include those wherein E* and Ez are independently selected from H, -CN, -N02, -CX3 wherin X is halogen or E! and Έ/' together are (=0). The remainder of the substituents are as defined.
[0127] Exemplary embodiments wherein the Ligand-Drug Conjugates. Drug-Linkers, Linkers, or Ligand-Linker conjugates are represented by Formula III (or formulae Ilia, Illb, Ilic, or IIId, as the case may be) or pharmaceutically acceptable salts thereof include those wherein;
(i) E' and E" are independently selected from hydrogen, -CN, -N02, -CX , and -X wherein is halogen or E! and E:2 together are (-O);
(ii) m is zero and n is zero, one two or three;
(iii) x is 1 ;
(iv) x is 4;
(v) x is zero, and n is zero, one , two or three; (vi) ra is zero, n is zero, and x is 3 :
(vii) d is 1 and g is 1 to 5;
(viii) d is 1 and g is 2 to 5;
(ix) n, d, and g are zero;
(x) rn, n, d, and g are zero;
(xi) RS is -€(=€) ;
(xii) E' and E are together (=0);
ixiii) B is
Figure imgf000042_0001
Figure imgf000042_0002
Figure imgf000042_0003
, or -N(R )(R',)5 wherein R3, R4, R5, R6. R7 and R8 are endently selected from H or Cj-6 alkyl and e is 0 to 4; (xiv) B is■■ N(R3)(R4), wherein R3 and R4 are independently selected from H or Ci-g aikyl;
(xv) B is as in (xiii) or (xiv) and R3, R4, R Rb, R7 and R8 are independently selected from H or Ci-3 alkyl;
(xvi) B is as in (xiii) or (xiv) and R3, R4, R5, RB, R and R8 are independently selected from 1 1 or methyl;
(xvii) B is as in (xiii) or (xiv) or (xvi) and R3 and R4 are hydrogen;
(xviii) B is as in (xiii) or (xiv) or (xvi) and at least one of RJ and R4 are hydrogen;
(xix) B is as in (xiii) or (xiv) or (xvi) and at least one of R3 and R4 is not hydrogen;
(xx) R!, RQ, and R '° are independently selected from H or methyl;
(xxi) R! , R9, and RL U are hydrogen;
(xxii) R\ RY, and R!° are independently selected from H or methyl;
(xxiii) The cleavable unit is present;
(xxiv) The cleavable unit is present and has the formula
-(AA-AA)!.6-, w},erejn js at each occurrence independently selected from an amino acid;
(xxv) The cleavable unit is present and conjugated directly to the Drug unit;
(xxvi) The cleavable unit is present and conjugated directly to the Drug unit via a cleavable peptide, disulfide, or hydrazone bond;
(xxvii) The cleavable unit is present and the Spacer and Stretcher unit are absent:
(xxviii) The Drug is an auristatin;
(xxix) M1 is a hydrolyzed or non-hydrolyzed succinimide;
(xxx) p is about 4;
(xxxi) p is about 8; (xxxii) the t i/2 of hydrolysis of the thio-substituted succinimide of the Self-Stabilizing Linker unit is from about 10 minutes to about 2.5 hours at pH 7.4 and 22 °C;
(xxxiii) the ti/2 of hydrolysis of the thio-substituted succinimide of the Self-Stabilizing Liinker unit is from about 10 minutes to about 1 hour at pH 7.4 and 22 °C;
(xxxiv) the tl/2 of hydrolysis of the thio-substituted succinimide of the Self-Stabilizing Linker unit is from about 10 minutes to about 30 minutes at pH 7.4 and 22 °C;
(xxxv) the Ligand unit is an antibody;
(xxxvi) the Ligand unit is an antibody and is attached to the Linker unit though a cysteine residue of an interchain disulfide;
(xxxvii) the Ligand unit is a monoclonal antibody; and any combinations or subcombinations of (i) througli (xxxvii) provided that the combinations or subcombinations do not conflict with each other (e.g., xxx and xxxi conflict because p cannot be both about 4 and about 8). For example, in selected embodiments, m is zero, and n is zero, one, two, or three. In other selected embodiments, m is zero, n is zero, and x is one. in any of these selected embodiments, d can be one and g can be from 1 to 5 or d can be one and g can be from 2 to 5. In any of these embodiments, one or more of (i), (iii) or (xi)-(xxxvi) can apply.
[0128] In each of the selected embodiments wherein the Ligand -Drug Conjugates, Drug- Linkers, Linkers, or Ligand-Linker conjugates are represented by Formul III (or formulae Ilia, lilb, Hie, or Hid, as the case may be) or pharmaceutically acceptable salts thereof, the optional secondary linker assembly can be represented by the following formula:
Figure imgf000044_0001
wherein -A- is an optional Stretcher unit, the subscript a' is 0 or I ;
-W- is an optional Cleavable unit, the subscript v' is 0 or 1 ; and -Ύ- is an optional Spacer unit, and the subscript y' is 0 or 1.
[0129] Also included are selected embodiments wherein the Linker has the formula:
Figure imgf000045_0001
or a pharmaceatically acceptable salt thereof wherein V, T, B, I , Q, F, G, m, x, n, d, and g are as defined for formula III and RG is a reactive group comprising a reactive site, RS, for conjugation to the Drug unit D when the secondary linker assembly is absent or to a component of the secondary linker assembly wherein the secondary linker assembly secondary linker has the following formula:
Figure imgf000045_0002
wherein -A- is an optional Stretcher unit, the subscript a' is 0 or 1 :
-W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or I .
Further Embodiments of the invention
[0130] Exemplary self-stabilizing linker assemblies prior to conjugation with a Ligand and following conjugation and hydrolysis of the thio-substituted sueccinimide which is formed upon conjugation are as follows:
Figure imgf000046_0001

Figure imgf000047_0001
Figure imgf000047_0002
or pharmaceutically acceptable salts thereof wherein V, Q, m. n, x, and B, are as defined above for Formula III or any other selected embodiment, c is from 1 to 4, and R' ' and R » 1!2 are, at each occurrence, independently selected from H or Ci-C6 alkyl, in an exemplary embodiment c is 1 or 4, The "S" of the hydrolyzed thio-succinimide represents a sulfur atom of the Ligand (e.g., antibody). The wavy line indicates linkage to the secondary linker assembly or Drug unit. In an exemplary embodiment, the wavy line indicates linkage to the following secondary linker assembly
I- wherein -A- is an optional Stretcher unit, the subscript a' is 0 or 1 ; -W- is an optional Cleavable unit, the subscript w! is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or 1 . It will be understood that more than one (e.g., 1 to 20) drug-linkers can be attached to each Ligand.
[0131] In some aspects of the present invention, a self-stabilizing linker assembly may undergo macro-cyclization to form a dilactam as follows wherein R represents the remainder of the conjugate:
Figure imgf000048_0001
Seeondai Linker Assembly
[0132] The optional secondary linker can comprise a variety of linking groups. In each of the embodiments provided herein, including the specifically recited embodiments, L° can be present and have the formula:
Figure imgf000048_0002
wherein
-A- is an optional Stretcher unit, the subscript a' is 0 or 1 ;
-W- is an optional Cleavable unit, the subscript v' is 0 or 1 ; and
-Y- is an optional Spacer unit, and the subscript y' is 0 or 1 ;
[0133] The optional secondary linker assembly can be a releaseabie linker assembly, LR. In those embodiments, w is I . In some other aspects, the optional secondary linker assembly is a non-reieasable linker assembly. In those embodiments w is 0 and release of drug is via a total protein degradation pathway (i.e., non-cleavabie pathway). The Ligand Unit
[0134] In some embodiments of the invention, a Ligand Unit is present. The Ligand unit (L-) is a targeting agent that specifically binds to a target moiety. The Ligand can specifically bind to a cell component (a Cel l Binding Agent) or to other target molecules of interest. In some aspects, the Ligand unit acts to deliver the Drug unit to the particular target cell population with which the Ligand unit interacts. Ligands include, but are not limited to, proteins, polypeptides and peptides. Suitable Ligand units include, for example, antibodies, e.g., full-length antibodies and antigen binding fragments thereof, interferons, lymphokines, hormones, growth factors and colony-stimulating factors, vitamins, nutrient-transport molecules (such as, but not limited to, transferrin), or any other cell binding molecule or substance. In some aspects, the ligand is a non- antibody protein targeting agent. In some aspects, a Ltgand-Functional Agent is provided wherein D* is a Detection Unit or Stability unit and the Ligand unit is a protein (e.g., a non- antibody protein).
[0135] In some aspects, a Ligand unit forms a bond with the maleirnide of the Self-Stabilizing Basic unit via a sulfhydryl group of the Ligand to form a thio-substituied succinimide. The su!fhydryi group can be present on the Ligand in the Ligand's natural state, for example a naturally-occurring residue, or can be introduced into the Ligand via chemical modification.
[0136] It has been observed for bioconjugates that the site of drug conj ugation can affect a number of parameters including ease of conjugation, drug-linker stability, effects on biophysical properties of the resulting bioconjugates, and in-vitro cytotoxicity. Willi respect to drug-linker stability, the site of conj ugation of a drug-linker to a ligand can affect the ability of the conjugated drug-linker to undergo an elimination reaction and for the drug linker to be transferred from the ligand of a bioconjugate to an aiternative reactive thiol present in the milieu of the bioconjugate, such as, for example, a reactive thiol in albumin, free cysteine, or glutathione when in plasma. Use of the Self-Stabilizing Linkers of the present invention is particularly beneficial when conjugated to thiol residues at sites that are susceptible to the elimination reaction and subsequent transfer of drug-linker if non-self-stabilizing alkyl maleimides are used (e.g., maleimido-eaproyi drug linker). Such sites include, for example, the interchain disulfides as well as select cysteine engineered sites, Use of the Self-Stabilizing Linkers of the present invention provides a stable linkage and ability to attach multiple drugs to each Ligand unit.
[0137] In one aspect, the Ligand unit has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryi groups. The reagents that can be used to modify lysines include, but ar not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2- Iminothiolane hydrochloride (Traut's Reagent).
[0138] In another embodiment, the Ligand unit can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryi groups.
[0139] In another embodiment, the Ligand is an antibody and the sulfhydryi group is generated by reduction of an interchain disulfide. Accordingly, in some embodiments, the Linker unit is conjugated to a cysteine residue of the reduced interchain disulfides.
[0140] In another embodiment, the sulfhydryi group is chemically introduced into tire antibody, for example by introduction of a cysteine residue. Accordingly, in some embodiments, the Linker unit is conjugated to an introduced cysteine residue.
[0141 j Useful non-immunoreactive protein, polypeptide, or peptide Ligands include, but are not limited to. transferrin, epidermal growth factors ("EGF"), bombesin, gastrin, gastrin- releasing peptide, platelet-derived growth factor, IL-2, IL-6, transforming growth factors ("TGF"), such as TGF-a and TGF-β, vaccinia growth factor ("VGF"), insulin and insuiin-like growth factors I and II, somatostatin, lectins and apoprotein from low density lipoprotein,
[0142] Particularly preferred ligands are antibodies. Useful polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of immunized animals. Useful monoclonal antibodies are homogeneous populations of antibodies to a particular antigenic determinant (e.g. , a cancer cell antigen, a viral antigen, a microbial antigen, a protein, a peptide, a carbohydrate, a chemical, nucleic acid, or fragments thereof). A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art which provides for the production of antibody molecules by continuous cell lines in culture.
[0143] Useful monoclonal antibodies include, but are not limited to, human monoclonal antibodies, humanized monoclonal antibodies, or chimeric human-mouse (or other species) monoclonal antibodies. The antibodies include full-length antibodies and antigen binding fragments thereof. Human monoclonal antibodies may be made by any of numerous techniques known in the art (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA. 80:7308-7312: Kozbor et al, 1983, Immunology Today 4:72-79; and Olsson et al, 1982, Meth, Enzymol 92:3-16).
[0144] The antibody can be a functionally active fragment, derivative or analog of an antibody that immunospecifically binds to target ceils (e.g., cancer cell antigens, viral antigens, or microbial antigens) or other antibodies bound to tumor cells or matrix. In this regard,
"functionally active" means that the fragment, derivative or analog is able to elicit anti-anii- idiotype antibodies that recognize the same antigen that the antibody from which the fragment, derivative or analog is derived. Specifically, in an exemplary embodiment the antigenicity of the idiotype of the immunoglobulin molecule can be enhanced by deletion of framework and CDR sequences that are C-terminal to the CDR sequence thai specifically recognizes the antigen. To determine which CDR sequences bind the antigen, synthetic peptides containing the CDR sequences can be used in binding assays with the antigen by any binding assay method known in the art (e.g., the BIA core assay) (See, e.g., Kabat et al., 1991, Sequences of Proteins of
Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md; Kabat E et al, 1980, J. Immunology 125(3):961 -969).
[0145] Other useful antibodies include fragments of antibodies such as, but not limited to, F(ab')2 fragments, Fab fragments, Fvs, single chain antibodies, diabodies, tribodies, tetrabodies, scFv, scFv-FV, or any other molecule with the same specificity as the antibody.
[0146] Additionally, recombinant antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as for example, those having a variable region derived from a murine monoclonal and human immunoglobulin constant regions. (See, e.g., U.S. Patent No, 4,816,567; and U.S. Patent No. 4,816,397, which are incorporated herein by reference in their entirety.) Humanized antibodies are antibody molecules from non- human species having one or more complementarity determining regions (CDRs) from the non- human species and a framework region from a human immunoglobulin molecule. (See, e.g., U.S. Patent No. 5,585,089, which is incorporated herein by reference in its entirety.) Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in International Publication No. WO 87/02671 ; European Patent Publication No. 0 184 187; European Patent Publication No.
0 171 496; European Patent Publication No. 0 173 494; International Publication No. WO 86/01533; U.S. Patent No. 4,816,567; European Patent Publication No.012 023; Berter et al, 1988, Science 240: 1041-1043; Liu et al, 1987, Proc. Natl Acad Sci. USA 84:3439-3443; Liu et al, 1987, J. Immunol. 139:3521-3526; Sun et ., 1987, P oa Natl. Acad. Sci. USA 84:214-218; Nishimura e/ £?L 1987, Cancer. Res. 47:999-1005; Wood et al, 1985, Nature 314:446-449; and Shaw et al, 1988, J. Natl. Cancer Inst. 80: 1553-1559; Morrison, 1985, Science 229:1202-1207;
01 et al., 1986, BioTechniques 4:214; U.S. Patent No. 5,225,539; Jones et al. , 1986, Nature 321 :552-525; Verhoeyan et al, 1988, Science 239: 1534; and Beidier et al, 1988, J. Immunol 141 :4053-4060: each of which is incorporated herein by reference in its entirety.
[0147] Completely human antibodies are particularly desirable and can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes.
[0148] Antibodies include analogs and derivatives that are either modified, i.e., by the covalent attachment of any type of molecule as long as such covalent attachment permits the antibody to retain its antigen binding immunospecificity. For example, but not by way of limitation, derivatives and analogs of the antibodies include those that have been further modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular antibody unit or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, forrnylation, metabolic synthesis in the presence of tunicamycin, etc. Additionally, the analog or derivative can contain one or more unnatural amino acids.
[0149] Antibodies can have modifications (e.g., substitutions, deletions or additions) in amino acid residues that interact with Fc receptors. In particular, antibodies can have modifications in amino acid residues identified as involved in the interaction between the anti-Fc domain and the FcRn receptor (see, e.g.. International Publication No. WO 97/34631, which Is incorporated herein by reference In its entirety ). [0150] Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., chemical synthesis or recombinant expression techniques. The nucleotide sequence encoding antibodies
immunospecific for a cancer cell antigen can be obtained, e.g., from the GenEiank database or a database like it, the literature publications, or by routine cloning and sequencing.
[0151] in a specific embodiment, known antibodies for the treatment of cancer can be used. Antibodies immunospecific for a cancer cell antigen can be obtained commercially or produced by any method known to one of skill in the art such as, e.g., recombinant expression techniques. The nucleotide sequence encoding antibodies immunospecific for a cancer ceil antigen can be obtained, e.g., from the GenBank database or a database like it, the literature publications, or by routine cloning and sequencing.
[0152] In another specific embodiment, antibodies for the treatment of an autoimmune disease are used in accordance with the compositions and methods of the invention. Antibodies immunospecific for an antigen of a cell that is responsible for producing autoimmune antibodies can be obtained from any organization {e.g.. a university scientist or a company) or produced by any method known to one of skil l in the art such as, e.g., chemical synthesis or recombinant expression techniques, in another embodiment, useful antibodies are immunospecific for the treatment of autoimmune diseases incl ude, but are not limited to, anti-nuclear antibody; anti-ds DNA; Anti-ss DNA, anti-cardiolipin antibody igM, IgG; anti-phospholipid antibody IgM, IgG; anti-SM antibody; anti-mitochondrial antibody; thyroid antibody; microsomal antibody;
thyroglobulin antibody; anti-SCL-70 antibody; anti-Jo antibody; anti-U;RNP antibody;
anti-La/SSB antibody; anti-SSA; anti-SSB antibody; anti-perital ceils antibody; anti-histones antibody; anti-RNP antibody; C-ANCA antibody; P-ANCA antibody; anti-centromere antibody; Anti-Fibriilarin antibody and anti-GBM antibody.
[0153] in certain embodiments, useful antibodies can bind to a receptor or a receptor complex expressed on an activated lymphocyte. The receptor or receptor complex can comprise an immunoglobulin gene superfamily member, a TNF receptor superfamily member, an integrin, a cytokine receptor, a chemokine receptor, a major histocompatibility protein, a lectin, or a complement control protein. Non-limiting examples of suitable immunoglobulin superfamily members are CD 2, CDS, CD4, CDS, CD 19, CD20, CD22, CD28, CD30, CD70, CD79, CD90, CD 152/CTLA-4, PD-1 , and 1COS. Non-limiting examples of suitable TNF receptor superfamily members are CD27, CD40, CD95/Fas, CD134/OX40, CD 137/4-1 BB, T F-R 1 , T F -2, RANK, TACI, BCMA, osteoprotegerin, Apo2/TRAIL-Rl, TRAIL-R2, TRAIL-R3, TRAIL-R4, and APO-3. Non-limiting examples of suitable integrins are CD1 l a, CD i lb, CD i lc, CD 18, CD29, CD41 , CD49a, CD49b, CD49c, CD49d, CD49e, C! )49f. ( 1 ) 1 03, and CD 104. Non- limiting examples of suitable lectins are C-type, S-type, and I-type lectin.
The Drag Unit, D
[0154] The drug unit (D) can be any cytotoxic, cytostatic or immunosuppressive drug also referred to herein as a cytotoxic, cytostatic or immunosuppressive agent. The Drug unit has an atom that can form a bond with the Linker Unit, in some embodiments, the Drug unit D has a nitrogen atom that can form a bond with the Linker unit. In other embodiments, the Drug unit D has a carboxylic acid that can form a bond with the Linker unit. In other embodiments, the Drug unit D has a su!fhydryl group that can form a bond with the Linker unit . In other embodiments, the Drug unit D has a hydroxy! group or ketone that can form a bond with the Linker unit.
[0155] Useful classes of cytotoxic or immunosuppressive agents include, for example, antitubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g. , platinum complexes such as cis-platin, mono(p!atinum), bis(platinum) and tri-nuelear platinum complexes and carbopiatin), anthracyclin.es, antibiotics, antifolates-, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinois, pre-formirsg compounds, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisornerase inhibitors, vinca alkaloids, or the like. Particularly examples of useful classes of cytotoxic agents include, for example, DNA minor groove binders, DNA alkylating agents, and tubulin inhibitors. Exemplary cytotoxic agents include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM! and DM4), taxanes, benzodiazepines (e.g.,
pyrrolo [1 ,4] benzodiazepines (PBDs). indolinobenzodiazepines, and
oxazolidinobenzodiazepines) and vinca alkaloids. Select benzodiazepine containing drugs are described in WO 2010/091 150, WO 2012/1 12708, WO 2007/085930, and WO 201 1/023883.
[0156] Individual cytotoxic or immunosuppressive agents include, for example, an androgen, antbramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin, busuJfan, buthionine sulfoximine, calicheamicin, camptothecin, carbopiatin, carmustine (BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide, cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine, dactinomycin (formerly actinomycin). daunorubicin, decarbazine, docetaxel, doxorubicin, etoposide, an estrogen, 5-fluordeoxyuridine, 5~fluorouracil, gemcitabine, gramicidin D, hydroxyurea, idarubicin, ifosfamide, irinoteean, iomustine (CCNU), mayiansine, mechlorethainine, melphalan, 6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxei, paly toxin, plicamycin, procarbizine, rhizoxin, streptozotocin, tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine, vincristine, vinorelbine, VP- 16 and VM-26.
[0157] In some typical embodiments, suitable cytotoxic agents include, for example, DNA minor groove binders (e.g., enediynes and {exitropsins, a CBI compound; see also U.S. Patent No. 6,130,237), duocarmycras (see U.S. Publication No. 20060024317), taxanes (e.g., paclitaxei and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan, morpholino- doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, echinomvcin, combretastatin, netropsin, epothilone A and B, estramustine, cryptophysins, cemadotin, maytansinoids, discodermoiide, eleutherobin, and mitoxantrone.
[0158] in some embodiments, the Drug unit is an anti-tubulin agent. Examples of anti-tubulin agents include, but are not limited to, taxanes (e.g., Taxol® (paclitaxei), Taxotere© (docetaxel)), T67 (Tularik) and vinca alkyloids (e.g., vincristine, vinblastine, vindesme, and vinorelbine). Other antitubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colcimid, estramustine, cryptophysins, cemadotin, maytansinoids, combretastatins, discodermoiide, and eleutherobin.
[0159] ! certain embodiments, the cytotoxic agent is maytansine or a maytansinoid, another group of anti-tubulin agents. (ImmunoGen, Inc.: see also Chari et ah, 1992, Cancer Res. 52:127- 131 and U.S. Patent No. 8,163,888).
[0160] in some embodiments, the Drug unit is an auristatin. Auristatins include, but are not limited to, AE, AFP, AEB, AEVB, MMAF, and MMAE. The synthesis and structure of auristatins are described in U.S. Patent Application Publication Nos. 2003-0083263, 2005- 0238649 2005-0009751 , 2009-01 1 1756, and 201 1 -0020343: International Patent Publication No. WO 04/010957, international Patent Publication No. WO 02/088172, and U.S. Patent Nos. 7,659,241 and 8,343,928; each of which is incorporated by reference in its entirety and for all purposes. Exemplary auristatins of the present invention bind tubulin and exert a cytotoxic or cytostatic effect on the desired cell line.
[0161 ] Exemplary auristatin Drug units have the following formula or a pharmaceutically acceptable sal t thereof wherein the wavy line indicates site of attachment to the linker unit:
Figure imgf000056_0001
[0162] In some embodiments, the Drug is a benzodiazepine (including benzodiazepine containing drugs e.g., pyrrolo[l ,4]benzodiazepiries (PBDs), indolinobenzodiazepines, and oxazo!idino benzodiazepines) .
[0163] PBDs are of the general structure:
Figure imgf000057_0001
but can differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N= ), a carbinolamine(NH-CH(OH))> or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C1 1 position, which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have a (S)-configuration at the chiral CI la position which provides them vvitli a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a. snug fit at the binding site. The ability of PBDs to form an adduct in the minor groove enables them to interfere with DNA processing, hence their use as antitumour agents. The biological activity of these molecules can be potentiated by, for example, joining two PBD units together through their C8/C -hydroxy! functionalities via a flexible alkylene linker. The PBD dimers are thought to form sequence-selective DNA lesions such as the palindromic 5'-Pu-GATC-Py-3' interstrand cross-link which is thought to be mainly responsible for their biological activity.
[0J 4] There are a number of different assays that can be used for determining whether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effect on a cell line. In one example for determining whether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effect on a ceil line, a thymidine incorporation assay is used. For example, cells at a density of 5,000 ceils/well of a 96-weli plated is cultured for a 72-hour period and exposed to 0,5 μθ of Ή-thyrmdine during the final 8 hours of the 72-hour period, and the incorporation of JH-thyrmdine into cells of the culture is measured in the presence and absence of Ligand-Drug Conjugate. The Ligand- Drug Conj ugate has a cytostatic or cytotoxic effect on the cell line if the cells of the culture have reduced Ή-thymidine incorporation compared to cells of the same cell hne cultured under the same conditions but not contacted with the Ligand-Drug Conjugate.
[0165] in another example, for determining whether a Ligand-Drug Conjugate exerts a cytostatic or cytotoxic effect on a ceil line, cell viability is measured by determining in a cell the uptake of a dye such as neutral red, trypan blue, or ALAMAR.™ blue {see, e.g.. Page et l.t 1993, Intl. J. of Oncology 3:473-476). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cy toxicity (Skehan et al., 1990, J. Nat'l Cancer Inst. 82:1107-12). Preferred Ligand-Drug Conjugates include those with an IC50 value (defined as the mAB concentration that gives 50% cell kill) of less than 1000 ng/ml, preferably less than 500 ng/ml, more preferably less than 100 ng ml, even most preferably less than 50 or even less than 10 ng/rnl on the cell line,
[1)166] General procedures for linking a drug to linkers are known in the art. See, for example, U.S. Patent Nos. 8,163,888, 7,659,241, 7,498,298, U.S. Publication No. US20110256157 and International Application Nos. WO201 1023883, and WO2005112919.
M1 - The succinimide
[0167] A non-hydrolyzed succinimide (also referred to herein as a succinimide ring) conjugated to the Ligand unit via a thioether linkage can be represented as follows wherein R represents the remainder of the Linker unit optionally conjugated to a Drug unit, Detection unit or Stability unit:
Ligand
Figure imgf000058_0001
8] A hydrolyzed succinimide (also referred to herein as a hydrolyzed succinimide ring) conjugated to the Ligand unit via a thioether linkage can be represented as one of its two positional isomers as follows wherein R represents the remainder of the Linker unit optionally conjugated to a Drug unit, Detection unit or Stability unit:
Figure imgf000058_0002
[0169] It will be understood for the non-hydrolyzed succinimides and hydrolyzed succinimide representations, there can be from 1 to 20, preferably 1 to 12, 1 to 10 or 1 to 8 self-stabilizing linkers conjugated to each Ligand. In some aspects, there are from I to 20, preferably 1 to 12, 1 to 10 or 1 to 8 drug-linkers conjugated to each Ligand. Additionally, for the conjugates described herein where a Ligand is not attached, the succinimide is in an unsaturated form as a maieimide (capable of reactive with a thiol or the Ligand).
Basic units
[0170] in Formula I, as well as the other formulae comprising a self-stabilizing linker (T_r ), the Basic unit (BU) can be essentially any base capable of facilitating a hydroxide ion (or vvaier) attack to hydrolyze a nearby succinimide group. Accordingly, BU represents any "base" but is typically a group comprising a tethered amine or nitrogen containing heterocycle; the amine or nitrogen containing heterocycle acting as the base of the Basic unit. R epresentative amines include ~N(R3)(R4) wherein R3 and R4 are independently selected from H or Ci^ alkyl, preferably H or methyl.
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000060_0001
wherein R5, R6, R7 and R8 are, at each occurrence, independently selected from hydrogen or Ci^ alkyl, preferably H or methyl, and e is 0-4. in the formulae above, the wavy line indicates the point of attachment to a tethering group (typically an aikylene linker -~(C(R )( R1,J )) ~ wherein the subscript x is an integer of from 0 to 6 (or 1 to 6 ) provided that if x is 0 there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydroiyzed) or diiactam and R.y and R'° are independently selected from H or C: .3 a!kyi. In some aspects, the aikylene linker is -(CH2 )x- wherein the subscript x is an integer of from 0 to 6 (or 1 to 6) provided that if x is 0 there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non- hydroiyzed) or diiactam. The subscript x is preferably 0 to 4, 1 to 4, or from 1 to 3, or from 2 to
3, or from 2 to 4, but can also be 0, 1, 2, 3 or 4. Accordingly, the Basic unit, will in some embodiments, be selected from the group consisting of -(€¾ )XNH2, -( ¾ )xNHRa, and --(€¾ )XNR" 2, wherein x i s an integer of from 0 to 4, 1 to 4 , or from 1 to 3, or from 2 to 3 , or from 2 to
4, but can also be 0, 1 , 2, 3 or 4, and each Ra is independently seiected from the group consisting of Cue alkyl and Q.6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group. In preferred aspects, the base of the Basic unit will be a nitrogenous base.
Hydrolysis Enhasieers (HE) and Electron-withdrawing groups
[0171] The hydrolysis enhancers (HE) of Formula I, as well as the other formulae comprising a self-stabilizing linker (LSs>), can be essentially any electron-withdrawing group capable of facilitating the hydrolysis of a nearby succinimide group, The hydrolysis is further facilitated by the Basic unit (BU) assisting a hydroxide ion (or water) attack to hydrolyze a nearby succinimide group; or to render the nearby succinimide group more susceptible to hydrolysis. Accordingly, HE can include a functional group that draws electrons away from a reaction center. Exemplary electron withdrawing groups include, but are not limited to, -C(::::0), ( ()}. -C , -NO?, -CX3, - X, -COOR, -CONR2, -COR, -COX, -SO2R, -SO2OR, -SO2NHR, -SO2NR2, -PO3R2, - P(0)(CH3)NHR, NO, - R f . -CR=CR2, and -C≡CR wherein X is F, Br, CI or Ϊ, and R is, at each occurrence, independently selected from the group consisting of hydrogen and Ch alky!. Exemplary electron withdrawing groups can also include aryl groups (e.g., phenyl) and certain heteroaryl groups (e.g., pyridine). The term "electron withdrawing groups" includes aryls or heteroaryls further substitued with electron withdrawing groups.
[0172] in some embodiments, HE comprises a carbony], sulfonyl or phosphoryl moiety. In some embodiments, the hydrolysis enhancer (HE)
is -CH2C(0)-, -C(O)-, -C(0)CH2-, -CH2CH2C(0)- , or -CH2C(0)NH-.
[0173] In some embodiments wherein HE is directly linked to the secondary Iinlcer assembly or Drug unit or Stability unit or Detection unit, HE will comprise a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit. In some aspects, the electron withdrawing group will itself act as both the electron withdrawing group and a reactive site for attachment to the optional secondary linker assembly or Drug unit (e.g., -C(=0)-).
The Optional Secondary Linker Assembly (L°)
[0174] As noted above, the optional secondary linker assembly can be represented by the formula:
Figure imgf000061_0001
wherein - A- is an optional Stretcher unit, the subscrip a' is 0 or 1 ; -W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; and ~Y- Is an optional Spacer unit, and the subscript y' is 0 or 1. Hie wavy line adjacent to the optional Stretcher unit indicates the site of attachment to the self- stabilizing linker assembly and the wavy line adjacent, to the optional Spacer unit indicates the site of attachment to the Drue unit.
[0175] Genera] methods of linking a Drug unit, a Detection unit, or a Stability unit to a Ligand unit are known in the art. and linkers known in the art can be adapted for use with a self- stabilizing linker assembly or modified to include a basic component and/or electron withdrawing group using the teachings described herein. For example, auristatin and maytansine ADCs are currently in clinical development for the treatment of cancer,
Monomethyi auristatin E is conjugated tbrough a protease cleavable peptide linker to an antibody, monomethyi auristatin F is conjugated directly to an antibody through
raaleimidocaproic acid, DM1 is conjugated through a disulfide or directly through the heterobifunctional SMCC linker, and DM4 is conjugated through a disulfide linker. These linker systems can be adapted for use with a self-stabilizing linker assembly or modified to include a basic component and/or electron withdrawing group using the teachings described herein and provide release of drug by a cleavable or non-cleavable system depending on the linker system used. Disulfide, thioether, peptide, hydrazine, ester, or carbamate bonds are all examples of bonds that can be used to connect a Drug Unit to a Linker Unit. Stretcher units, Cleavable units, and Spacer units are described in more detail below.
[0176] Also contemplated within the present invention are branched linkers. Accordingly, in one aspect, the Stretcher unit is designed in such a way to allo branching within the Linker unit, e.g., the attachment of more than one Drug unit or Detection unit or Stability unit to each self-stabilizing linker assembly, as represented by the following formula:
Figure imgf000062_0001
wherein the wavy line indicates the site of attachment to the self-stabilizing linker assembly, -W- is an optional Cleavable unit, the subscript vv' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , u is from 2 to 20 (preferably from 2 to 10); A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; and a' is 0 or 1 . Each A', W, Y, and D' can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit. In some aspects, D' is a Drug unit D.
[0177] Exemplary Ligand-Functional Agent Conjugates or Ligand-Drug Conjugates having either branched or non-branched linkers have the following formulae:
Figure imgf000063_0001
Figure imgf000063_0002
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of L, M !, HE, BU, D', and the subscripts p, a, b, m. q, and r have the meanings provided for Formula I and any of the selected embodiments for Formula I, D is a Drug unit, the circle represents a scaffold that can be C!-8 alkylene, Ci.g heteroalkylene, C6-io arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D' (or D as the case may be); -W- is an optional Cleavable unit, the subscript W is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or 1, A is a. Stretcher unit, A' is an optional Stretcher unit component at the terminus of A: a' is 0 or I ; and u is from i to 20 (preferably from I to 10) wherein when u is from 2 to 20 A is present and when u is 1, A can be present or absent. Each A', W, Y, and D' (or D as the case may be) can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit. In some aspects, w' is 1. In some aspects, w' is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1 and a' is 0, In other aspects, wherein the linker is branched, u is from 2 to 20 (preferably from 2 to 10), In each of these selected embodiments, the circle can represent a scaffold that is Ci.g alkyiene or Q-g heteroalkyiene (preferably C alkyiene or C heteroalkyiene) or C-..3 alkyiene or C1.3 heteroalkyiene. In some such aspects, the alkyiene is straight chain or branched,
[0178] Ligand-Functional Agent Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000064_0001
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of L, Ml, HE, BU, D', and the subscript p have the meanings provided for Formula I and any of the selected embodiments for Formula I , the circle represents a scaffold that can be C;.g alkyiene, C1-8 heteroalkylene, C6-1o aryiene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D', -W- is an optional Cieavable unit, the subscript w' is 0 or 1; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and 11 is from 1 to 20 (preferably from 1 to 10), wherein when u is from 2 to 20 A is present and when u is 1 , A can be present or absent. Each A', W, Y, and D' can be the same or different. Each Cieavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, ' is 1. In some aspects, w' is I and a' is 0. In aspects wherein the linker isn't branched u is 1 , a' is 0, and A can be present or absent. In other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10). In some preferred embodiments, BU is selected from the group consisting of -(CH2 )XN!¾, -iC¾ )xNHRa, and
-(CH2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Q-6 alkyl and C1-6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidiny) group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrol zed). In some such aspects X is 0-4 and each Ra is Ci.6 alkyl.
[0179] Ligand-Drug Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000065_0001
Figure imgf000066_0001
Figure imgf000066_0002
Figure imgf000066_0003
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of L, M , HE, BU, and the subscript p have the meanings provided for Formula 1 and any of the selected embodiments for Formula I, D is a Drug unit, the circle represents a scaffold that can be Ct-g alkylene, C1-8 heteroalkylene, C io arylene, or C4.10 heteroaryiene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D, -W- is an optional Cleavable unit, the subscript W is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 3 ; and u is from 3 to 20 (preferably from 1 to 10), wherein when u is from 2 to 20 A is present and when u is 1 , A can be present or absent. Each A', W. Y, and D can be th same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit. In some aspects, w' is 1. In some aspects, w! is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1 , a' is 0, and A can be present or absent. In other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10). In some preferred embodiments, BU is selected from the group consisting of --(CHb )ΧΝΗ2. -{CH2 )xNHRa, and -(CH-2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Cj..6 alky! and haloalkyl, or two Ra groups are combined with the nitrogen to which they are aitached to form an azetidinyi, pyrrol idinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydro !yzed or non-hydrolyzed). In some such aspects X is 0-4 and each Ra is C1-6 alkyl.
[0180] Functional Agent-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000067_0001
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of HE, BU, D!, and the subscripts p, a, b, m, q, and r have the meanings provided for Formula I and any of the selected embodiments for Formula I, the circle represents a scaffold that can be Ci-g alkyiene, C1-8 heteroalkylene, C6-JO arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D'; W- is an optional Cleavable unit, the subscript w* is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y! is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20 (preferably from 1 to 10, wherein when u is from 2 to 20, A is present and when u is 1, A can he present or absent. Each A', W, Y, and D! can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 1 . in some aspects, w' is i and a' is 0. In aspects wherein the linker isn't branched u is 1, a' is 0, and A can be present or absent. In other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10), In each of these selected embodiments, the circle can represent a scaffold that is Q.g alkylene or Q.g heteroalkylene (preferably CM alkylene or C heteroalkylene) or Q.3 alkylene or C1..3 heteroalkylene. In some such aspects, the alkylene is straight chain or branched. In each of these selected embodiments, D' can he D.
IJ In some aspects
Figure imgf000068_0001
is represented by
Figure imgf000068_0002
some such aspects, D' is D. in some preferred embodiments, BU is selected from the group consisting of -(CH2 )XNH2, ---(CH2 )xNHR , and -(CH2 )xNRa 2, wherein x is an integer of from 0- 4 and each Ra is independently selected from the group consisting of Cj-e alkyl and Ci-g haloa!kyl, or two R3 groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non-hydrolyzed). In some such aspects X is 0-4 and each Ra is C1-6 alkyl.
[0182] Ligand-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000069_0001
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of L, M1 , HE. BU, and the subscripts p, a, b, m. q, and r have the meanings provided for Formula I and any of the selected embodiments for Formula Ϊ, the circle represents a scaffold that can be C1-8 alkylene. Cs.g heteroalkylene, C5.10 arylene, or€ .10 heteroarylene, and optionally comprises a reactive site suitable for attachment to A, W, Y or D; RG is a reactive group (comprising a reactive site) at
~~S~A Aa.— - Ww.— Yy - - the terminus of > Q which is suitable for attaching a Drug unit (or alternatively a Detection unit or a Stability unit) , W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y* is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2 to 20, A is present and when u is 1, A can be present or absent. Each A', W, Y, and D can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit. In some aspects, W is 1 . In some aspects, w' is 1 and a' is 0. In aspects, wherein the linker isn't branched u is I , a! is 0, and A can be present or absent. In other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10). In each of these selected embodiments, the circl can represnet a scaffold that is C .$ alkylene or Ci„g heteroalkylene (preferably C|. alkylene or C1.4 heteroalkylene) or Q.3 alkylene or C1.3 heteroalkylene. in some such aspects, the alkylene is straight chain or branched.
[0183] In some aspects
Figure imgf000070_0001
represented by:
Figure imgf000070_0002
in some preferred embodiments, BU is selected from the group consisting of -{€¾ )XNH2, - (CH2 )XNHR\ and -(CH2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is
independently selected from the group consisting of Ci-6 alkyl and C1-6 haloalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyi group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydrolyzed or non- bydrolyzed). in some such aspects X is 0-4 and each RR is Ci-6 alkyl.
[0184] Branched or non-branched Linkers can be represented by the following formulas:
Figure imgf000070_0003
Figure imgf000071_0001
or a salt thereof (e.g., pharmaceutically acceptable salt), wherein each of the scaffold, HE, BU, and the subscripts a, b, m, q, and r have the meanings provided for Formula Ϊ and any of the selected embodiments for Formula I, the circle represents a scaffold that can be d.« alkylene, Ci-8 heteroalkylene, C6-i0 arylene, or C4.10 heteroarylene, and optionally comprises a reactive site- suitable for attachment to A, W, Y or D; RG is a reactive group (comprising a reactive site) at
-¾-A— -— Aa Ww. Yy-¾- the terminus of J ς which is suitable for attaching a Drug umt (or aiiernatively a Detection unit or a Stability unit), W- is an optional Cleavable unit, the subscript w' is 0 or 1 : - Y- is an optional Spacer unit, the subscript y' is 0 or 1 , A is a Stretcher unit, A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 3 to 20
(preferably from 1 to 10), wherein when u is from 2 to 20, A is present and when u is 1, A can be present or absent. Each A', W, Y, and D can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 3. In some aspects, w' is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1 , a' is 0, and A can be present or absent, h other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10). In each of these selected embodiments, the circle can represent a scaffold that is Cj.8 alkylene or Ci.g
heteroalkylene (preferably C alkylene or Q.4 heteroalkylene) or C1.3 alkylene or C1.3
heteroalkylene. In some such aspects, the alkylene is straight chain or branched. 1.85] In some aspects
Figure imgf000072_0001
js represented
Figure imgf000072_0002
In some preferred embodiments, BU is selected from the group consisting of --(C¾ )XNH2, - (CH2 )xNHRa, and (CI 12 )x 'NRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Cf.g alkyl and Q-e haioalkyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrroiidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the suceinimide (hydrolyzed or non- hydrolyzed), In some such aspects X is 0-4 and each Ra is Ci-6 alkyl.
[0186] In some other aspects, exemplary Ligand-Drug Conjugates having either branched or non-branched linkers have the following formulae:
Figure imgf000072_0003
or a pharmaceutically acceptable salt thereof wherein each of L, M1, V, R1, T, B, Q, F, G, and RS and the subscripts p, m, x, n, d, and g ha ve the meanings provided for Formula 111 and any of the selected embodiments for Formula III, L is a Ligand unit, W- is an optional Cleavable unit, the subscript w' is 0 or 1; -Y- is an optional Spacer unit, the subscript y' is 0 or 1 , A is a Stretcher unit. A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1; and u is from 1 to 20 (preferably from 1 to 10) wherein when u is from 2 to 20, A is present and when u is 1 , A can be present or absent. Each A', W, Y. and D can be the same or different. Each Cleavable unit can be attached to the Stretcher unit (either A or A') through the same or different functional group on the Stretcher unit, in some aspects, w' is 1. in some aspects, w' is 1 and a' is 0. In aspects wherein the linker isn't branched u is 1, a' is 0, and A can be present or absent, in other aspects, wherein the linker is branched u is from 2 to 20 (preferably from 2 to 10).
[0187] Stretcher units, Cleavable units, and Spacer units are described in more detail below.
The Stretcher Unit
[0188] The Stretcher unit (-A-), when present, extends the framework of the Linker unit to provide more distance between the self-stabilizing linker assembly and the Drug unit. A Stretcher unit is capable of linking the self-stabilizing linker assembly to the Cleavable unit when the Cleavable unit is present, the self-stabilizing linker assembly to the Spacer unit when the Cleavable unit is absent but the Spacer unit is present and the self-stabilizing linker assembly to the Drug unit when both the Cleavable unit and the Spacer unit are absent. As described, a Stretcher unit is capable of attaching to more than one Cleavable unit, Spacer unit, and/or Drug unit.
[0189] The Stretcher unit can also act to alter the physiochemical properties of the Drug- Linker depending on components of the Stretcher unit, in some aspects, the Stretcher unit will be added in order to increase the solubility of the Drug-Linker and will comprise one or multiple solubility-enhancing groups such as ionic groups or water-soluble polymers. Water-soluble typically includes any segment or polymer that is soluble in water at room temperature and includes poly(ethylene)g!yeol groups as well as other polymers such as polyethyleneimines.
[0190] A Stretcher unit can comprise one or multiple stretcher groups. Exemplary stretcher groups include, for example, -NH-C|-C10 alkylene-, -NH~Ci-Cio alkylene-NH-C(0)-Ci-Cio alkylene-, -NH-C Ci0 alkylene-C(O)-NH-Ci-C)0 alkylene-, -NH~(C ¾(¾<¾-, -NI-I- (C! M . i L .-O 1;:-, -NH-i('[ :i LNS !}„-(( ! L)<.
-NH-(CH2CH2NH)S-(CH2)S-MH-C(0)-(CH2)S) -NH-(C3-C8 carbocycio)-, -NH-(arylene-)-, and -NH-(C3-C8 heteroeyclo-)-, wherein each s is independently 1-10. A representative stretcher group having a carbonyl group for linkage to the remainder of the Linker unit or the Drug unit is as follow:
Figure imgf000074_0001
wherein R! j is -Ci-Cio alkylene-, -C3-Cgcarbocyclo-, -aryiene-, -Ci-Cjoheteroalkylene-, -C3- Cgheterocyclo-, -Cj -Chalky lene-arylene-. -arylene-Ci-Cioaikylene-, -Ci-Cioaikyiene-(C3- Cgcarbocyclo)-, -(C3-C8carbocyclo)-C i -Cj oalkylene-, -C ί -C i oalkylene-(C3-C8 heterocyclo}-, -(C3~ C8 heterocyclo)-CrCio alkylene-, -(CH2CH20)i,1o(-CH2)i.3-J or -(CH2CH2NH)!-io(-CH2)1-3-. In some embodiments, R13 is -Ci-Cio alkylene- or -C j -C3oheteroalkylene-. in some
embodiments, R13 is -Ci~Ci0 alkylene-, -(CH2CH2O)1-10(-CH2)i-3-, or
-(CH2CH2NH)i.1o(-CH2)f-3-. In some embodiments, R is -Ci-Cio alkylene- polyethyleneglycol, or polyethyjeneimine.
[0191 j Non-cleavable drug release systems are known in the art and can be adapted for use with the self-stabilizing linker assemblies of the present invention as Stretcher units and/or Spacer units. A non-cleavab!e linker in capable of Unking a Drug unit to a Ligand in a generally- stable and covalent manner and is substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase- or esterase-induced cleavage, and disulfide bond cleavage. Drug is released from Ligand Drug Conjugates containing non-cieavable linkers via alternative mechanisms, such as proteolytic ligand degradation.
[0192] Cross-linking reagents that form non-cleavable linkers between maytansinoid drugs and ligands are well known in the art and can adapted for use herein. Exemplary cross-linking reagants that form non-cleavable linkers between the maytansinoid drugs and ligands comprise a maleimido or haloacetyl-based moiety. They include N-suceinimidyl 4- (maieimidomethyi)cyclohexaneearboxyiate (SMCC), N-succiniraidyl-4-(N-maleimidomethyl)- cyclohexane- 1 -carboxy~(6-amidocaproate), which is a "long chain" analog of SMCC (LC- SMCC), -maleimidoundecanoic acid N-succinimidyl ester (KMUA), γ -maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), ra-raaleirnidobenzoyl-N-hydroxysuccinimide ester(MBS), N-(.alp a.-maleirnidoacetoxy)- succinimide ester [AMAS], succinimidyl-6-( -maleimidopropionamido)hexanoate (SMPH), N- succinimidyl 4-(p-maleimidophenyJ)-butyrate (SMPB), and N-(p-nialeimidophenyl)isocyanate (PMPI), N-succinimidyl-4-(iodoacetyl)-aniinobenzoate (SLAB), N-succinimidyl iodoacetate (SI A), N-suceinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP). Additional Stretcher units for use in combination with the self-stabilizing linker assembly of the present invention can be found, for example, in U.S. Patent No. 8,142,784, incorporated herein by reference in its entirety and for all purposes.
The Cleavable Unit
[0193] The Cleavable unit (- W-), when present, is capable of linking the self-stabilizing linker assembly to the Spacer unit when the Spacer unit is present or the self-stabilizing linker assembly to the Drug unit when the Spacer unit is absent. The linkage from the self-stabilizing linker assembly to the Spacer unit or to the Drug unit can be directly from the self-stabilizing linker assembly when the Stretcher unit is absent or via the Stretcher unit if the Stretcher unit is present.
[0194] in some embodiment, the Cleavable unit will be directly conjugated to the self- stabilizing linker assembly on one end and to the Drug unit on the other end. in other embodiments, the Cleavable unit will be directly conjugated to the Stretcher unit on one end and to the Drug unit on the other end. In yet other embodiments, the Cleavable unit will be directl conjugated to the Stretcher unit on one end and to the Spacer unit on the other end. in even yet other embodiments, the Cleavable unit will be directly conjugated to the self-stabilizing linker assembly on one end and to the Spacer unit on the other end. Any of specifically described self- stabilizing linker assemblies described herein can be used in these embodiments.
[0J95] The Cleavable unit is capable of forming a cleavable bond with a Drug unit or a Spacer unit. Reactive groups for forming cleavable bonds can include, for example, sulfhydryi groups to form disulfide bonds, aldehyde, ketone, or hydrazine groups to form hydrazone bonds, carboxylic or amino groups to form peptide bonds, and carboxylic or hydroxy groups to form ester bonds.
[0196] The nature of the Cleavable unit can vary widely. For example, cleavable linkers include disulfide containing linkers that are cleavable through disulfide exchange, acid-labile linkers that are cleavable at acidic pH, and linkers that are cleavable by hydrolases, peptidases, esterases, and glucuronidases.
[0197] In some aspects, the structure and sequence of the Cleavable unit is such that the unit is cleaved by the action of enzymes present at the target site. In other aspects, the Cleavable unit can be cleavable by other mechanisms. The Cleavable unit can comprise one or multiple cleavage sites.
[0198] In some embodiments, the Cleavable unit will comprise one amino acid or one or more sequences of amino acids. The Cleavable unit can comprise, for example, a monopeptide, a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit.
[0199 j Each amino acid can be natural or unnatural and/or a D- or L-isomer pro vided of course that there is a cleavable bond, in some embodiments, the Cleavable unit will comprise only natural amino acids. In some aspects, the Cleavable unit will comprise 1 to 12 amino acids in contiguous sequence.
[020Θ] In some embodiments, each amino acid is Independently selected from the group consisting of alanine, arginine, asparlic acid, asparagine, histidme, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, selenocysteine, ornithine, penicillamine, β-alanine, aminoalkanoic acid, aminoalkynoic acid, aminoalkanedioic acid, arainobenzoic acid, amino- heterocyclo-alkanoic acid, heterocycio-carboxyiic acid, citrulline, statine, diaminoalkanoic acid, and derivatives thereof. In some embodiments, each amino acid is independently selected from the group consisting of alanine, arginine. asparlic acid, asparagine, hisiidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, valine, cysteine, methionine, and selenocysteine. In some embodiments, each amino acid is independently selected from the group consisting of alanine, arginine, aspartic acid, asparagine, histidme, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, proline, tryptophan, and valine, in some embodiments, each amino acid is selected from the proteinogenic or the non-proteinogenic amino acids. [0201] In another embodiment, each amino acid is independently selected from the group consisting of the following L-(natural) amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan and valine.
[0202] in another embodiment, each amino acid is independently selected from the group consisting of the following D-isomers of these natural amino acids: alanine, arginine, aspartic acid, asparagine, histidine, glycine, glutamic acid, glutamine, phenylalanine, lysine, leucine, serine, tyrosine, threonine, isoleucine, tryptophan and valine.
[0203] In some embodiments, the bond between the Cleavable unit and the Drug unit can be enzymaticaliy cleaved b one or more enzymes, including a tumor-associated protease, to liberate the Drug unit (-D), which in one embodiment is protonated in vivo upon release to provide a Drug (D).
[0204] Useful Cleavable units can be designed and optimized in their selectivity for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease. In one embodiment, a linkage (or bond) between the Cleavable unit and the Drug unit or Spacer unit is that which cleavage is catalyzed by cathepsin B, C and D, or a plasmin protease.
[0205] In certain embodiments, the Cleavable unit can comprise only natural amino acids. In other embodiments, the Cleavable unit can comprise only non-natural amino acids. In some embodiments, the Cleavable unit can comprise a natural amino acid linked to a non-natural amino acid. In some embodiments, the Cleavable unit can comprise a natural amino acid linked to a D~isomer of a natural amino acid.
[0206] An exemplary Cleavable unit is the dipeptide -Val-Cit-, -Phe-Lys- or -Val-Ala.
[0207] in some embodiments, the Cleavable unit will comprises a peptide and will comprise from 1 to 22 amino acids. In some such embodiments, the peptide will be conjugated directly to the Drug unit and the Spacer unit will be absent. In some such embodiments, the Stretcher unit and Spacer unit will be absent. In one aspect, the peptide will be a dipeptide.
[0208] In some embodiments, the Cleavable unit -™WW- will be represented by -(-ΑΑ~)ι.. ΐ 2-, or (-ΑΑ~ΑΑ~)ι..ό wherein AA is at each occurrence independently selected from natural or non- natural amino acids, in one aspect, AA is at each occurrence independently selected from natural amino acids. One of skill in the art would appreciate that amino acids are typically linked to the Drug unit or Spacer unit through functional units present in the amino acid, e.g., its earboxylie acid or amino termini.
[0209] In some such aspects, the Ligand Drug Conjugates and Drug-Linkers are represented by the following formulae or salts thereof wherein L, LbS, L1 r A, a', AA, Y, y', D, and p are as defined in any of the embodiments described herein and f is an integer from 1 to 12:
Figure imgf000078_0001
Figure imgf000079_0001
Aa.— AA? 0
Aa>— AAf D _
AAT-D , or
ss
AAf D
10210] It will be understood that although not reflected in the above formulae, such formulaes can be modified as taught herein to include branched linkers, i.e., multiple Drug units can be attached to each self-stabilizing linker assembly,
[0211] In other aspects, the Cieavable unit will comprise a glucoronide unit, preferably 1 or 2 glucoronide units. In some such embodiments, the Glucuronide unit comprises a sugar moiety (Su) linked via a glycoside bond (-()'-) to a self-immolative Spacer:
-[Su-O'-Y]-
[0212] The glycosidic bond (~0'~) is typically a β-glucuronidase-cleavage site, such as a bond cieavable by human, lysosomal β-glucuronidase,
[0213] In some aspects, -[Su-O'-Y]- is represented by the following formula:
Figure imgf000079_0002
wherein Su is a Sugar moiety,_-0'- represents a glycosidic bond; each R is independently hydrogen, a halogen, -CN, or -N02; wherein the wavy bond adjacent to the nitrogen atom indicates covalent attachment to the Stretcher unit or to the Ligand and the wavy bond adjacent to the oxygen indicates covalent attachment to the Spacer unit or to the Drug unit. An exemplary Linker unit comprising a glucoronide prior to conjugation to an antibody and post conjugation is as follows wherein the wavy line indicates attachment to a Drug unit or Spacer unit and Ab represents an antibody and S is a sulfur atom of the antibody, It will be understood that more than one self-stabilizing assembly can be attached to each antibody:
Figure imgf000080_0001
Figure imgf000080_0002
[0214] In some embodiments, the Cleavable unit itself will comprise a sulfur atom that is capable of forming a bond with a sulfur atom of a Spacer unit or Drug unit to form a disulfide or hindered disulfide. Cleavage occurs between the two sulfur atoms of the disulfide. In some such embodiments, one of the sulfur atoms is cleaved from the Drug unit and, provided there is no further release mechanism, the other sulfur atom remains attached to the Drug unit. A Linker unit comprising a Cleavable unit having a sulfur atom is capable of forming a bond with a sulfur atom of a Spacer unit or Drug unit to form a disulfide or hindered disulfide
[0215] Exemplary linkers include, for example, the following Drug-Linker wherein the wavy line indicates the site of attachment to the remainder of the Linker unit, D is a mavtansinoid drug, and Ra and ¾ are independently selected from H or meth l.
Figure imgf000081_0001
[0216] A variety of disulfide linkers are known in the art and can adapted for use in the present invention, including, for example, those that can be formed using SATA (N-succirdraidyl-S- aeetyithioacetaie), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate)> SPDB (N- succinimidyl-3-(2~pyridyldithio)butyrate), SMPT (N-succiniraidyl-oxycarbonyl-alpha-methyl- alpha-(2-pyridyl-dithio)toluene), and SPP (N-succinimidyl 4-(2-pyridyIdithio)pentanoate). (See, e.g., Thorpe ei al, 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al, In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C, W. Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935.)
[0217] In some embodiments, the cleavable linker is pH-sensitive and will comprise, for example, an acid-labile linker that is hydrolyzable in the iysosome (e.g., a hydrazone, seraicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetai, or ketal group) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville ei al,, 1989, Biol. Che n. 264: 14653-14661.) Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the Iysosome.
[0218] In some embodiments, the Cleavable unit will be conjugated directly to the Drug unit and the Spacer unit will be absent and the Cleavable unit will be linked to the Drug unit via a cleavable peptide, disulfide, or hydrazone bond.
The Spacer Unit
[0219] The Spacer unit (-Y-), when present, links a Cleavable unit to the Drug unit or a Stretcher unit to the Drug unit or a self-stabilizing linker assembly to a Drug unit. Like the Stretcher unit, the Spacer unit, when present can act to extend the framework of the Linker unit. The Spacer unit can comprise multiple self-immolative or non-self immolative groups. In some embodiments, the Spacer unit comprises one or more self-immolative groups, in this context, the term "self-immolative group" refers to a Afunctional chemical moiety that is capable of covalentiy linking together two spaced chemical moieties into a normally stable tripartite molecule. It will spontaneously separate from the second chemical moiety if its bond to the first moiety is cleaved. In other embodiments, the Spacer unit is not self-imraolative. In these embodiments, part, or all of the Spacer unit remains attached to the Drug unit.
[0220] In some embodiments, -Y - is a self-immolative group and is linked to a Cleavable unit via the methylene carbon atom of the self-immolative group, and linked connected directly to the Drug unit via a carbonate, carbamate or eiher group.
[Θ221] In. some embodiments, -Yy- is a p-aminobenzyl alcohol (PAB) unit whose phenylene portion is optionally substituted with -C]-Cg alkyl, -0-(Ct~Cg alkyl), -halogen,- nitro or -cyano. In another embodiment, -Yy- can be a carbonate group. An unsubstituted PAB unit is as follows:
Figure imgf000082_0001
[0222] Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PAB group such as 2-arainoitnidazol-5-methanol derivatives (see, e.g., Hay et ah, 1999, Bioorg. Med. Chem, Lett. 9:2237) and ortho or para- arninobenzyiaeetals. Spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (see, e.g., Rodrigues et ah, 1995, Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (s e, e.g.. Storm et ah, 1972, J. Amer. Chem. Soc. 94:5815) and 2-arninophenylpropionic acid amides (see, e.g., Amsberry et ah, 1990, J. Org. Chem. 55:5867). Elimination of amine- containing drugs that are substituted at the a-position of glycine (see, e.g., Kingsbury et ah, 1984, J. Med. Chem. 27: 1447) are also examples of self-immolative groups.
[0223] Other suitable Spacer units are disclosed in Published U.S. Patent Application No. 2005-0238649, the disclosure of which is incorporated by reference herein.
[0224] Exemplary Stretcher units, Cleavable units, and Spacer units that can be used with the present compositions and methods are described in WO 2004010957, WO 2007/038658, WO 2005/1 12919, U.S. Patent No. 6,214,345, 7,659,241 , 7,498,298, 7,968,687, 8, 163,888, and U.S. Publication No. 2009-01 1 1 756, 2009-0018086, 2009-0274713, each of which is incorporated herein by reference in its entirety and for all purposes.
[0225] In embodiments wherein the Conjugates are conjugated to a Stability unit or a
Detection unit in lieu of a Drug unit, the optional Secondary Linker Assembly will typically be absent. In embodiments where the Secondary Linker Assembly is present, the Stretcher unit will generally be present but the Cleavable unit and the Spacer unit will be absent. The Stretcher unit will extend the framework of the Linker unit to provide more distance between the self- stabilizing assembly and the Detection unit or Stability unit, in such aspects, the Stretcher unit is capable of linking the self-stabilizing linker assembly to the Detection unit or the Stability unit.
DRUG LOADING
[0226] The number of self-stabilizing linkers per Ligand is represented by p. In embodiments wherein the linkers are not branched, p represents the number of drug-linker molecules (or detection-linker or stability-linker molecules) per Ligand molecule (e.g., antibody). Depending on the context, p can represent the average number of self-stabilizing linkers per Ligand (or in embodiments where the linkers are not branched, the average number of drug-linker molecules (or detection-linker or stability-linker molecules) per Ligand (e.g., antibody)). The variable p ranges from 1 to 20, typically 1 to 12, 1 to 10 and is preferably from 1 to 8. In some preferred embodiments, when p represents the average, number of self-stabilizing linkers per antibody, p ranges from about 2 to about 5. in some embodiments, p is about 2, about 4, or about 8. in some preferred embodiments, when p represents the average number of drug-linker molecules per antibody, p ranges from about 2 to about 5. In some embodiments, p is about 2, about 4, or about 8. The number of D' per self-stabilizing linkers is represented by u. u ranges from 1 to 10.
[0227] The average number of Drugs units per Ligand unit in a preparation from a conjugation reaction may be characterized by conventional means such as mass spectroscopy, ELISA assay, HiC and HPLC. The quantitative distribution of Drug-Linker-Ligand conjugates in terms of p may also be determined. In some instances, separation, purification, and characterization of homogeneous Ligand-Drug Conjugates, where p is a certain value from Ligand -Drug Conj gate with other drug loadings may be achieved by means such as reverse phase HPLC or
electrophoresis.
Self-Stabilizing Linker Assem ly (Lss or L{ f) and Rates of Hydrolysis
[0228| The Self-Stabilizing linker assembly links the Ligand unit to a Stretcher unit if the Stretcher unit is present, links the Ligand unit to a Cleavabie unit if the Stretcher unit is absent and a Cleavabie unit is present, links the Ligand unit to a Spacer unit if the Stretcher unit and the Cleavabie unit are absent and the Spacer unit is present, or links the Ligand Unit to D' (e.g., a Drug Unit) if the Stretcher unit. Cleavabie unit and Spacer unit are absent. In some
embodiments, the Stretcher unit, Cleavabie unit, and Spacer unit will be absent and the self- stabilizing linker assembly will be conjugated directly to D' (e.g., a Drug Unit). In other embodiments, one or more of the Stretcher unit, Cleavabie unit, and Spacer unit will be present.
[0229] The rate at which the thio-substituted succinimide of the Self-Stabilizing Linker when part of a Ligand-Drug Conjugate undergoes hydrolysis can be quantified using the tl/2 of hydrolysis, tl/2 of hydrolysis refers to the time taken for half of the compound of interest to hydrolyze, i.e., undergo a ring opening, under stated conditions (e.g., pH 7.4 and 22°C). in some embodiments of the present invention, the tl/2 of hydrolysis of the thio-substituted succinimide of the Self-Stabilizing Linker unit is less than 4 hours, preferably less than 3 hours, even more preferably, less than 2 hours, less than 1 hour, less than 45 minutes, less than 30 minutes, less than 15 minutes using the following assay and sta ed conditions.
[0230] The hydrolysis reaction rates of maleimido drug linkers following conjugation to antibody cysteines can be determined by mass spectrometry, as the hydrolyzed product has a molecular weight 18 daltons greater than the unhydrolyzed conj ugate. Reduction of the interchain disulfides of a human IgGl creates a single reduced cysteine on the light chain and three reduced cysteines on the heavy chain. The self-stabilizing raaleimide drug-linker can then be conjugated to the reduced antibody at pH 7,4 and 22°C and introduced to a high-resolution electrospray mass spectrometer via a reversed-phase HPLC column which separates the conjugated light and heavy chains. The masses of the conjugated light and heavy chains can thus be measured, and the peak intensities determined by standard mass spectrometry data processing software (e.g., MassLynx). By performing a series of injections over time, the disappearance of the peak corresponding to the mass of the original, unhydrolyzed conjugate and the appearance of the peak corresponding to the mass of the hydrolyzed conjugate can be monitored, the intensities of the peaks determined, and the percentage of hydrolyzed conjugate calculated at each timepoint. By plotting the hydrolysis percentage versus time, a curve is generated (e.g., using PRISM) which can be fit to a standard equation for exponential phenomena which includes a parameter for tl/2.
[0231] In some aspects, the Self-Stabilizing Linker will be designed such that the maleimide component of the Self-Stabilizing Linker does not substantially undergo hydrolysis prior to conjugation to the Ligand unit,
[0232] In some embodiments of the present invention, the tl 12 of hydrolysis of the thio- substituted succinimide of the Self-Stabilizing Linker is from about 5 or about 10 minutes to about 24 hours, preferably from about 5 or about 10 minutes to about 12 hours, more preferably from about 5 or about 10 minutes to about 5 hours, more preferably from about 5 or about 10 minutes to about 2.5 hours, even more preferably from about 5 or about 10 minutes to about 1 hour, even more preferably from about 5 or about 10 minutes to about 30 minutes, even more preferably from about 5 or about 10 minutes to about 20 minutes, and even more preferably from about 10 minutes to about 15 minutes at a ρϊ of about 7 to about 7.5 (e.g., 7.4) and a temperature of about 22°C,
[0233] In some such embodiments wherein the tl/2 of hydrolysis is as stated above, the hydrolysis goes to completion. Complete hydrolysis is considered to be achieved if 90% of the thio-substiiuted succinimide hydroiyzes. Preferably, 95% or greater, 96%. 97%, 98%, 99% or 100% hydrolysis will be achieved. In some embodiments, the hydrolysis reaction will compete with a dilactam formation and will not achieve completion. In some such embodiments, at least 90% of the reaction product will be a combination of either a hydrolyzed thio-substituted succinimide Ligand-Drug Conjugate or a thio-substituted dilactam Ligand-Drug Conjugate. Preferably at leas t 95% or greater, 96%, 97%, 98%, 99% or 100% of the reaction product will be a combination of either a hydrolyzed thio-substituted Ligand-Drug Conjugate or a thio- substituted dilactam Ligand-Drug Conjugate. The percentage of hydrolysis can be calculated from the mass spectrometrie data of the conjugate at the final timepoint by determining the intensity of the peak corresponding to the mass of the original, unhydrolyzed conjugate and the intensity of the peak corresponding to the mass of the hydrolyzed conjugate, and using the sum of the peak intensities to determine the percentage hydrolyzed and percentage unhydrolyzed,
[0234] In addition to characterizing the Ligand-Drug Conjugate by its tl/2 of hydrolysis and/or the efficiency of the hydrolysis reaction, the stability of the Ligand-Drug Conjugate can he characterized by the ability of the Ligand-Drug Conjugate to undergo an elimination reaction and for the Drug-Linker to be transferred from the Ligand unit to a alternative reactive thiol present in the milieu of the Ligand-Drug Conjugate, in some embodiments, the Drug-Linker will exhibit no or substantially no disassociation from the Ligand under the following assay and stated conditions. The phrase "substantially no disassociation from the Ligand" is considered to be achieved if less than 40%, preferably less than 20%, even more preferably less than 10%, or even more preferably less than 5% or less than 2% of the Drug-Linker in a sample disassociates from the L gand.
[0235] The elimination of a drug-linker containing an enzyme-cleavable linker from an antibody can be measured in ex vivo plasma by the following method. The conjugate is placed in sterile plasma and incubated at 37°C. At the beginning of the incubation and at varying timepoints from 1 hour to 1 week or longer, an aliquot is removed at fro/en at -80°C. Upon completion of the timepoints, the samples are passed over a protein A affinity resin to capture the antibody, the resin is washed with buffer, and then drug is released from the captured antibody by treatment with an appropriate enzyme (e.g. papain or proteinase K for peptide-based cleavable linkers). The released drug can then be quantified by standard LC-MS methodology, and the quantity of drug measured at each timepoint divided by the q uantity of drug measured for the pre-incubation aliquot to determine the percentage of drug remaining conjugated to the antibody at each timepoint. The precision of this assay can be improved by including an internal standard antibody-drug conjugate which is prepared using an isotopically labeled version of the same drug-linker, such that the drug which is released from it can be detected independently in the LC-MS assay from the drug released from the test drug-linker by virtue of its mass difference. This isotopically labeled internal standard antibody-drug conjugate is added to each sample in equal amounts immediately prior to the protein A capture step. The quantitation of the drug released from the test ADC is then performed ratiometrically to the signal from the internal standard by conventional LC-MS techniques. | 0236] An alternative method for evaluating the elimination of a maieimide drug-linker from an antibody (or other ligand) is to incubate the conjugate in buffer (e.g., phosphate-buffered saline) at slightly elevated pH (e.g., pH 8.0) in the presence of a large excess of a small-molecule thiol (e.g., N-acetyl cysteine, NAC) which will react with any maleimide that eliminates from the parent conjugate. LC-MS assays can be performed to detect and quantify the drag-linker conjugated to NAC, or the parent !igand-eonjugate. In the latter case, the ratio of the ligatid- conjugate to unconjugated ligand can be measured and will remain constant over time if the ligand-conjugate is stable. Additional methods are provided in the examples section.
Treatmesii of Cancer
[0237] The Ligand-Drug Conjugates are useful for inhibiting the multiplication of a tumor ceil or cancer cell, causing apoptosis in a tumor or cancer ceil, or for treating cancer in a patient. The Ligand-Drug Conjugates can be used accordingly in a variety of settings for the treatment of cancers. The Ligand-Drug Conjugates can be used to deliver a drug to a tumor cell or cancer cell. Without being bound by theory, in one embodiment, the Ligand unit of a Ligand-Drug Conjugate binds to or associates with a cancer-ceil or a tumor-cell-associated antigen, and the Ligand-Drug Conjugate can be taken up (internalized) inside a tumor cell or cancer cell through receptor-mediated endocytosis or other internalization mechanism. The antigen can be attached to a tumor cell or cancer cell or can be an extracellular matrix protein associated with the tumor cell or cancer cell. Once inside the cell, via a cleavable or non-cleavable mechanism, depending upon the components of the linker system, the drug is released within the cell, in an alternative embodiment, the Drug or Drug unit is cleaved from the Ligand-Drug Conjugate outside the tumor ceil or cancer cell, and the Drug or Drug unit subsequently penetrates the cell,
[Θ238] The Ligand-Drug Conjugates can provide conjugation-specific tumor or cancer drug targeting, thus reducing genera! toxicity of the drug. In some embodiments, the Linker units stabilize the Ligand-Drug Conjugates in blood, yet are capable of liberating drug once inside the cell.
[0239] In one embodiment, the Ligand unit binds to the tumor cell or cancer cell. [0240] In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is on the surface of the tumor eel! or cancer cell.
[0241] In another embodiment, the Ligand unit binds to a tumor cell or cancer cell antigen which is an extracellular matrix protein associated with the tumor cell or cancer cell.
[0242] The specificity of the Ligand unit for a particular tumor cell or cancer cell can be important for determining those tumors or cancers that are most effectively treated. For example, a ligand drug conjugate haying a BR96 Ligand unit can be useful for treating antigen positive carcinomas including those of the lung, breast colon, ovaries, and pancreas. Ligand- Drug Conjugates having an anti-CD30 or an anti-CD70 binding Ligand unit can be useful for treating hematologic malignancies,
[0243] Other particular types of cancers that can be treated with a ligand drug conjugates include, but are not limited to, those disclosed in Table 1 :
Table 1
[0244] Solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangtosarcoma, lymphangioendotheliosareoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophogeal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papil lary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medullobfastoma, craniopharyngioma, ependymoma, pineal orna, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, retinoblastoma blood-borne cancers, including but not limited to:
acute lymphoblastic leukemia "ALL", acute lymphoblastic B-eell leukemia, acute lymphoblastic T-ceil leukemia, acute myeloblasts leukemia "AML", ac promyelocyte leukemia "APL", acute monoblastic leukemia, acute erythroleukeraic leukemia, acute megakaryoblastic leukemia, acute myeiomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia "CML", chronic lymphocytic leukemia "CLL", hairy ceil leukemia, multiple myeloma acute and chronic leukemias:
lymphoblastic, myelogenous, lyraphocytic, myelocytic leukemias Lymphomas:
I lodgkin's disease, non-Hodgkin's Lymphoma, Multiple myeloma, Waldenstrom's macroglobulinemia, Heavy chain disease, Polycythemia vera
Multi-Modality Therapy for Cancer
[0245] Cancers, including, but not limited to, a tumor, metastasis, or other disease or disorder characterized by uncontrolled cell growth, can be treated or inhibited by administration of a Ligand-Drug Conjugate.
[0246] In other embodiments, methods for treating cancer are provided, including
administering to a patient in need thereof an effective amount of a Ligand-Drug Conjugate and a. chemotherapeutic agent. In one embodiment the chemotherapeutic agent is that with which treatment of the cancer has not been found to be refractory, In another embodiment, the chemotherapeutic agent is that with which the treatment of cancer has been found to be refractory. The Ligand-Drug Conjugates can be administered to a patient that has also undergone surgery as treatment for the cancer. [0247] in some embodiments, the patient also receives an additional treatment, such as radiation therapy. In a specific embodiment, the Ligand-Drug Conjugate is administered concurrently with the chemotherapeutic agent or with radiation therapy, In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of a ligand drug conjugate,
[0248] A chemotherapeutic agent can be administered over a series of sessions. Any one or a combination of the chemotherapeutic agents, such a standard of care chemotherapeutic agent(s), can be administered,
[0249] Additionally, methods of treatment of cancer with a Ligand-Drug Conj ugate are provided as an alternative to chemotherapy or radiation therapy where the chemotherapy or the radiation therapy has proven or can prove too toxic, e.g., results in unacceptable or unbearable side effects, for the subject being treated. The patient being treated can, optionally, be treated with another cancer treatment such as surgery, radiation therapy or chemotherapy, depending on which treatment is found to be acceptable or bearable.
Treatment of Autoimmune Diseases
[0250] The Ligand-Drug Conjugates are useful for killing or inhibiting the replication of a cell that produces an autoimmune disease or for treating an autoimmune disease. The Ligand-Drug Conjugates can be used accordingly in a variety of settings for the tTeatment of an autoimmune disease in a patient. The Ligand-Drug Conjugates can be used to deliver a drug to a target ceil. Without being bound by theory, in one embodiment, the Ligand-Drug Conjugate associates with an antigen on the surface of a target cell, and the ligand drug conjugate is then taken up inside a target-cell through receptor-mediated endocytosis. Once inside the cell, one or more specific peptide sequences within the Linker unit are cleaved, resulting in release of the Drug or Drug unit. The released Drug or Drug unit is then free to migrate in the cytosol and induce cytotoxic or cytostatic activities, in an alternative embodiment, the Drug is cleaved from the Ligand- Drug Conjugate outside the target cell, and the Drug or Drug unit subsequently penetrates the cell. [0251 j lii one embodiment, the Ligand unit binds to an autoimmune antigen. In one aspect, the antigen is on the surface of a ceSi involved in an autoimmune condition.
[0252] In another embodiment, the Ligand unit binds to an autoimmune antigen which is on the surface of a cell.
[0253] in one embodiment, the Ligand unit binds to activated lymphocytes that are associated with the autoimmune disease state.
[0254] In a further embodiment, the Ligand-Drug Conjugate kills or inhibit the multiplication of cells that produce an autoimmune antibody associated with a particular autoimmune disease.
[0255] Particular types of autoimmune diseases that can be treated with the ligand drug conjugates include, but are not limited to, Th2 lymphocyte related disorders (e.g., atopic dermatitis, atopic asthma, rhinocorijunctivitis, allergic rhinitis, Omenn's syndrome, systemic sclerosis, and graft versus host disease); Thl lymphocyte-related disorders (e.g., rheumatoid arthritis, multiple sclerosis, psoriasis, Sjorgren's syndrome, Hashimoto's thyroiditis, Grave 's disease, primary biliary cirrhosis, Wegener's granulomatosis, and tuberculosis): activated B lymphocyte-related disorders (e.g., systemic lupus erythematosus, Goodpasture's syndrome, rheumatoid arthritis, and type 1 diabetes); and those disclosed in Table 2.
Table 2
Active Chronic Hepatitis, Addison's Disease, Allergic Alveolitis, Allergic Reaction, Allergic Rhinitis, Alport's Syndrome, Anaphlaxis, Ankylosing
Spondylitis, Anti-phosholipid Syndrome, Arthritis, Ascariasis, Aspergillosis, Atopic Allergy, Atropic Dermatitis, Atropic Rhinitis, Behcet's Disease, Bird- Fancier's Lung, Bronchial Asthma, Caplan's Syndrome, Cardiomyopathy, Celiac Disease, Chagas' Disease, Chronic Glomerulonephritis, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Rubella Infection, CREST Syndrome, Crohn's Disease, Cryoglobulinemia. Gushing 's Syndrome, Dermatomyositis, Discoid Lupus, Dressier' s Syndrome, Eaton-Lambert Syndrome, Echovirus Infection, Encephalomyelitis, Endocrine opthalmopathy, Epstein-Barr Virus Infection, Equine Heaves, Erythematosus, Evan's Syndrome, Felty's Syndrome, Fibromyalgia, Fuch's Cyclitis, Gastric Atrophy. Gastrointestinal Allergy, Giant Cell Arteritis, Glomerulonephritis, Goodpasture's Syndrome, Graft v. Host Disease, Graves' Disease, Gui!iain-Barre Disease, Hashimoto's Thyroiditis, Hemolytic Anemia, Henoch-Schonlein Purpura, Idiopathic Adrenal Atrophy, idiopathic Pulmonary Fibritis, IgA Nephropathy, Inflammatory Bowel Diseases, insulin-dependent Diabetes Mellitus, Juvenile Arthritis, Juvenile Diabetes Mellitus (Type 1), Lambert-Eaton Syndrome, Laminitis, Lichen Planus, Lupoid Hepatitis, Lupus, Lymphopenia, Meniere's Disease, Mixed Connective Tissue Disease, Multiple Sclerosis, Myasthenia Gravis, Pernicious Anemia,
Polyglandular Syndromes, Presenile Dementia, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Psoriatic Arthritis, Raynauds Phenomenon, Recurrent Abortion, Reiter's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sampter's Syndrome, Schistosomiasis, Schmidt's Syndrome, Scleroderma, Shulman's Syndrome, Sjorgen's Syndrome, Stiff-Man Syndrome, Sympathetic Ophthalmia, Systemic Lupus Erythematosus, Takayasu's Arteritis, Temporal Arteritis, Thyroiditis, Thrombocytopenia, Thyrotoxicosis, Toxic Epidermal Necrolysis, Type B insulin Resistance, Type I Diabetes Mellitus, Ulcerative Colitis, Uveitis, Vitiligo, Waldenstrom's Macroglobulemia, Wegener's
Granulomatosis
Multi-Drug Therapy of Autoimmune Diseases
[0256] Methods for treating an autoimmune disease are also disclosed including administering to a patient in need thereof an effective amount of a Ligand-Drug Conjugate and another therapeutic agent known for the treatment of an autoimmune disease.
Treatment of Infectious Diseases
[0257] The Ligand-Drug Conjugates are useful for killing or inhibiting the multiplication of a cell that produces an infectious disease or for treating an infectious disease. The Ligand-Drug Conjugates can be used accordingly in a variety of settings for the treatment of an infectious disease in a patient. The Ligand-Drug Conjugates can be used to deliver a drug to a target cell. In one embodiment, the Ligand unit binds to the infectious disease cell. [0258] In one embodiment, the conjugates kill or inhibit the multiplication of cells that produce a particular infectious disease.
[0259] Particular types of infectious diseases that can be treated with the Ligand-Drug Conjugates include, but are not limited to, those disclosed in Table 3.
Table 3
Bacterial Diseases:
Diphtheria, Pertussis, Occult Bacteremia, Urinary Tract infection, Gastroenteritis, Cellulitis, Epiglottitis, Tracheitis, Adenoid Hypertrophy, Retropharyngeal Abcess, impetigo, Ecthyma, Pneumonia, Endocarditis, Septic Arthritis, Pneumococca, Peritonitis, Bactermia, Meningitis. Acute Purulent Meningitis, Urethritis, Cervicitis, Proctitis, Pharyngitis, Salpingitis, Epididymitis, Gonorrhea, Syphilis, Listeriosis, Anthrax, Nocardiosis, Salmonella, Typhoid Fever, Dysentery, Conjunctivitis, Sinusitis, Brucellosis, Tuliaremia, Cholera, Bubonic Plague, Tetanus, Necrotizing Enteritis, Actinomycosis, Mixed
Anaerobic Infections, Syphilis, Relapsing Fever, Leptospirosis, Lyme Disease, Rat Bite Fever, Tuberculosis, Lymphadenitis, Leprosy, Chlamydia, Chlamydial Pneumonia, Trachoma, Inclusion Conjunctivitis
Systemic Fungal Diseases:
Histoplarnosis, Coccidiodomyeosis, Blastomycosis, Sporotrichosis, Cryptococcsis, Systemic Candidiasis, Aspergillosis, Mucormycosis, Mycetoma, Chromomycosis
Rickettsial Diseases:
Typhus, Rocky Mountain Spotted Fever, Ehrlichiosis, Eastern Tick-Borne Rickettsioses, Rickettsialpox, Q Fever, Bartonellosis
Parasitic Diseases:
Malaria, Babesiosis, African Sleeping Sickness, Chagas' Disease, Leishmaniasis, Dum-Dum Fever, Toxoplasmosis, Meningoencephalitis, Keratitis, Entamebiasis, Giardiasis, Cryptosporidiasis, Isosporiasis, Cyc!osporiasis, Microsporidiosis, Ascariasis, Whipworm Infection, Hookworm Infection, Threadworm Infection, Ocular Larva Migrans, Trichinosis, Guinea Worm Disease, Lymphatic Fiiariasis, Loiasis, River Blindness, Canine Heartworm Infection, Schistosomiasis, Swimmer's Itch, Oriental Lung Fluke, Oriental Liver Fluke, Fascioliasis, Fasciolopsiasis, Opisthorchiasis, Tapeworm Infections, Hydatid Disease, Alveolar Hydatid Disease
Viral Diseases:
Measles, Subacute sclerosing panencephalitis. Common Cold, Mumps, Rubella, Roseola, Fifth Disease, Chickenpox, Respiratory syncytial virus infection, Croup, Bronchiolitis, infectious Mononucleosis, Poliomyelitis, Herpangina, Hand-Foot- and-Mouth Disease, Bornholm Disease, Genital Herpes, Genital Warts, Aseptic Meningitis, Myocarditis, Pericarditis. Gastroenteritis, Acquired
immunodeficiency Syndrome (AIDS), Human Immunodeficiency Virus (HIV), Reye's Syndrome, Kawasaki Syndrome, influenza, Bronchitis, Viral "Walking" Pneumonia, Acute Febrile Respiratory Disease, Acute pharyngoconj uncti val fever, Epidemic keratoconjunctivitis, Herpes Simplex Virus i (HSV-1), Herpes Simplex Virus 2 (HSV-2), Shingles, Cytomegalic Inclusion Disease, Rabies, Progressive Multifocal Leukoencephalopathy, Kuru, Fatal Familial Insomnia, Creutzfeldt- Jakob Disease, Gerstraann-Straussler-Scheinker Disease, Tropical Spastic Paraparesis, Western Equine Encephalitis, California Encephalitis, St. Louis Encephalitis, Yellow Fever, Dengue, Lymphocytic choriomeningitis, Lassa Fever, Hemorrhagic Fever, Hantvirus Pulmonary Syndrome, Marburg Virus Infections, Ebola Virus Infections, Smallpox
Multi-Drug Therapy of Infectious Diseases
[0260] Methods for treating an Infectious disease are disclosed including administering to a patient in need thereof a Ligand-Drug Conjugate and another therapeutic agent that is an anti- infectious disease agent. COMPOSITIONS AND METHODS OF ADMINISTRATION
[0261 ] The present invention provides pharmaceutical compositions comprising the Ligand- Drug Conjugates described herein and a pharmaceutically acceptable carrier. The Ligand-Drug Conjugates can be in any form that allows for the compound to be administered to a patient for treatment of a disorder associated with expression of the antigen to which the Ligand unit binds. For example, the conjugates can be in the form of a liquid or solid. The preferred route of administration is parenteral. Parenteral administration includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In one aspect, the compositions are administered parenterally. In one aspect, the compounds are administered intravenously.
[0262] The present invention also provides pharmaceutical compositions comprising the Ligand-Functional Agent Conjugates described herein and a pharmaceutically acceptable carrier. The Ligand-Drug Conjugates can be in any form that allows for the compound to be
administered to a patient for treatment of a disorder or for diagnostic purposes
[0263] Pharmaceutical compositions can be formulated so as to allow a compound to be bioavailable upon administration of the compositi on to a patient. Compositions can take the form of one or more dosage units, where for example, a tablet can be a single dosage unit.
[0264] Materials used in preparing the pharmaceutical compositions can be non-toxic in the amounts used. It will be evident to those of ordinary skill in the art that the optimal dosage of the active ingredient(s) in the pharmaceutical composition will depend on a variety of factors. Relevant factors include, without limitation, the type of animal (e.g., human), the particular form of the compound, the manner of administration, and the composition employed.
[0265] The composition can be, for example, in the form of a liquid. The liquid can be useful for delivery by injection, in a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. |0266] The liquid compositions, whether they are solutions, suspensions or other like form, can also include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digyleerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as amino acids, acetates, citrates or phosphates; detergents, such as nonionic surfactants, polyols; and agents for the adjustment of tonicity such as sodium chloride or dextrose. A parenteral composition can be enclosed in ampoule, a disposable syringe or a mul tiple-dose vial made of glass, plastic or other material. Physiological saline is an exemplary adjuvant. An injectable composition is preferably sterile.
[0267] The amount of the conjugate that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques, in addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges, The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances.
[0268] The compositions comprise an effective amount of a compound such that a suitable dosage will be obtained. Typically, this amount is at least about 0.01% of a compound by weight of the composition.
[0269] For intravenous administration, the composition can comprise from about 0.01 to about 100 mg of a Ligand-Drug Conjugate per kg of the animal's body weight, in one aspect, the composition can include from about I to about 100 mg of a Ligand-Drug Conjugate per kg of the animal's body weight, in another aspect, the amount administered will be in the range from about 0.1 to about 25 mg/kg of body weight of a compound.
[0270] Generally, the dosage of a compound administered to a patient is typically about 0.01 mg/kg to about 100 mg/kg of the subject's body weight. In some embodiments, the dosage administered to a patient is between about 0.01 mg/kg to about 35 mg/kg of the subject's body- weight. In some embodiments, the dosage administered to a patient is between about 0.1 mg/kg and about 15 mg/kg of the subject's body weight. In some embodiments, the dosage
administered to a patient is between about 0.1 mg/kg and about 20 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 0.1 mg/kg io about 5 mg/kg or about 0.1 mg/kg to about 10 mg/kg of the subject's body weight. In some
embodiments, the dosage administered is between about 1 mg/kg to about 15 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 1 mg/kg to about 10 mg/kg of the subject's body weight. In some embodiments, the dosage administered is between about 0.1 to 4 mg/kg, even more preferably 0.1 to 3.2 mg/kg, or even more preferably 0.1 to 2.7 mg/kg of the subject's body weight over a treatment cycle.
(02711 The Ligand-Functional Agent Conjugates e.g., Ligand-Drug Conjugates) can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa). Administration can be systemic or local. Various delivery systems are known, e.g.,
encapsulation in liposomes, microparticles, microcapsules, capsules, and can be used to administer a compound. In. certain embodiments, more than one compounds or composition is administered to a patient,
[0272] The term "carrier" refers to a diluent, adjuvant or excipient, with which a compound is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea,. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a patient, the compound or compositions and pharmaceutically acceptable carriers are sterile. Water is an exemplary carrier when the compounds are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanoi. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. [0273] in an embodiment, the conjugates are formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to animals, particularly human beings. Typically, the carriers or vehicles for intravenous administration are sterile isotonic aqueous buffer solutions. Where necessary, the compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally comprise a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where a conjugate is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceuticaf grade water or saline. Where the conjugate is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.
[0274] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
[0275 ] Pharmaceutical compositions of the present invention comprise the Ligand Drug Conjugates of the present invention and a pharmaceutically acceptable carrier. In some preferred embodiments, all, or substantially ail. or more than 50% of the Ligand Drug Conjugates present in the pharmaceutical composition comprises a hydrolyzed thio-substituted succinimide. In some preferred embodiments, more than 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99% of the Ligand Drug Conjugates present in the pharmaceutical composition comprises a hydrolyzed thio-substituted succinimide.
Methods for preparing Ligasid-Prog Conjugates
[0276] in another aspect, the present invention provides methods of preparing Ligand- Drug Conjugates or Ligand-Functional Agent Conjugates comprising a Self-Stabilizing Linker.
[0277] In some embodiments, methods of the present invention comprise the steps of providing a Drug-Linker or Linker unit as described herein, conj gating said Drug-Linker or Linker unit to a sulfhydryl group of a Ligand unit to form a conjugate, allowing the resultant conjugate to undergo a hydrolysis reaction to form a Ligand-Drug conjugate comprising a thio- substituted hydrolyzed succinirnide.
[0278] The rate of the ihio-substitued succinirnide hydrolysis can be manipulated by adjusting the reaction conditions following conjugation of the Drug-Linker to the Ligand, e.g., by adjusting the pH or temperature. In some embodiments of the present invention, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95% of the thio- substituted succinirnide is hydrolyzed without manipulation of the reaction conditions, i.e., the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction. In some embodiments, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90%. or even 95% of the thio-substituted succinirnide is hydrolyzed from 20 minutes, to 4 hours following conjugation, preferably from 20 minutes to 2 hours following conj ugation, in exemplary embodiments, the conjugation conditions are pH of about 7.4 and a temperature of about 22° C,
[Θ279] In some embodiments, methods for preparing a Ligand-Drug Conjugate comprises the steps of providing a Drug-Linker or Linker unit comprising a Self-Stabilizing Linker;
conjugating said Drug-Linker or Linker unit to a sulfhydryl group of a Ligand to form a Ligand- Drug Conjugate conjugate comprising a non-hydrolyzed thio-substituted succinirnide; al lowing the non-hydrolyzed thio-substituted succinirnide to undergo a hydrolysis reaction, wherein all, substantially all, or at least 50%, 60%, 70%, 80% or even 85% of the succinirnide is hydrolyzed from 10 minutes to 4 hour's following conjugation. In some embodiments, all, substantially all, or at least 50%, 60%, 70%, 80%, 85%, 90% or even 95 % of the succinirnide is hydrolyzed by 10 minutes, by 20 minutes, 40 minutes 60 minutes, 90 minutes or 120 minutes following conjugation. In some embodiments, the hydrolysis reaction occurs under the same reaction conditions as the conjugation reaction. In exemplary embodiments, the conjugation conditions are pH of about 7.4 and a temperature of about 22° C.
Methods for Synthesizing Self-Stabilizing Linkers
[0280] The present invention provides, inter alia, Self-Stabilizing Linkers. Methods of preparing Self-Stabilizing Linker units are encompassed within the scope of the present invention. [0281 ] Maleimide compounds are typically prepared from corresponding amines by reaction of the primary amine with maleic anhydride followed by cyclodehydration of the maleamic acid. The overall scheme for the preparation of maleimde compounds is shown in the scheme below.
Figure imgf000100_0001
[0282] For preparation of maleimides containing basic groups in the side chain of the starting amine, such basic groups should be protected, if necessary. The appropriate protecting groups should be stable under conditions of maleimide preparation, yet should be removable later in the presence of maleimide. Suitable protecting groups consist, but are not limited to, acid labile protecting groups. "Boc" protecting group is one of the preferred protecting group.
[0283] The first step of maleimide preparation, the formation of the maleamic acid is very facile and can be usually accomplished in good yield by slow addition of the amine to a suspension containing a stoichiometric excess of the maleic anhydride.
[0284] The second step, cyclodehydration of the maleamic acid, can be accomplished in a number of ways known to skilled in the art. For example, the use of chemical dehydrating agents has been a well established method for accomplishing this step. Carbodiimides in combination with isomerizing alcohols, for example: DCC/HOBt, have been used to effect, cyclodehydration of amic acids to maleimides.
[0285] Thermal cyclodehydration with use of azeotropic distillation in the presence of acid catalyst is another well known method to generate maleimides. The use of an azeotropic solvent permits the efficient removal of the water co-product as it forms, thereby driving reaction to maleimide. Suitable azeotropic solvents include cyclohexane, benzene, toluene, ethylbensene, mesitylene, and the like. Toluene is considered to be the most desirable since it boils at 1 10 °C at atmospheric pressure. Boiling temperatures below 200 °C are preferable to minimize possible thermal isomerization of maleamic acid to the more thermodynamically stable trans (fumaramic acid) structure. [0286] The use of polar aprotic co-solvents can be beneficial for overall yield improvement as well as reducing time of cyciodehydration. Several polar aprotic solvents including
dimethy!formamide, dimethylacetamide, acetonitrile, N-methylpyrrolidone, dimethylsulfoxide, arid sulfonate have been claimed to be useful. The most useful polar aprotic solvent is dimethylformamide.
[0287] Incorporation of certain amine salts instead of aprotic solvents can be further beneficial for maleimide formation according to Patent US 5,973,166.
[0288] One step microwave assisted maleimide synthesis has been also reported starting from maleie anhydride and an appropriate amine using no solvent (H. N. Borah, et aL J. Chem. Research (S), 1 98, 272-272).
[0289] Example using water as a solvent for maleimide formation has been reported in ARKIVOC, 2001 (v) 60-67 by V. Ondrus, et al.
[0290] Alternatively, maleimide compounds can be generated from maleimide and appropriate alcohol using, for example, Mitsunobu reaction conditions as shown in the scheme below (M.A. Walker, Tetrahedron Letters, 1994, v. 35, n 5, pp. 665-668),
Figure imgf000101_0001
[0291] The self-stabilizing linker assembly of the present invention are linked to the Stretcher unit, Cleavable unit, Spacer unit, or Drug unit using the teachings described herein in
combination with methods known in the art. The Linkers and Drug-Linkers are conj gated to Ligand units using teachings described herein in combination with methods known in the art. For example, for conjugation to interchain disulfides, an antibody ca be treated with a reducing agent, such as dithiothreitol (DTT) to reduce some or all of the interchain disulfide cysteine residues to form highly nucleophilic cysteine thiol groups. The full reduced antibody or partially reduced antibody can be subsequently conjugated to the maleimide of the Linker Unit. In exemplary embodiments, conjugation conditions are gentle ones, pH of about 7 and a temperature of about 22 °C.
Irster mediates
[0292] The present invention provides intermediates for use in making Self-Stabilizirig Linkers. Intermediates include the following wherein T, c, R31 and R12 are as previously described.
Figure imgf000102_0001
Mono-thio-substituted or Di-thio-substituted Maleimide or Succinimide SeSf- Stabilizing Linkers
[0293] In addition to designing self-stabilizing linkers for increasing the hydrolysis rates of mono thio-substituted succinimides, self-stabilizing linkers can also be used to increase the hydrolysis rate of mono-thio-substituted maleimides, di-thio-substituted maleimides, or di-thio- substituted succinimides.
[0294] in view of the above, the present invention provides in one group of embodiments, a Ligand-Functionai Agent Conjugate comprising a Ligand unit and at least one Functional Agent selected from a Drug unit, a Detection unit, or a Stability unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring wherein the succinimide ring, maleimide ring, hydrolyzed succinimide ring or hydrolyzed maleimide ring is directly conjugated to the Ligand unit via one or two thioether linkages; and a base and an electron withdrawing group operabiy linked to stabilize the conjugate in plasma relative to a Ligand-Functionai Agent Conjugate lacking the self-stabilizing Sinker assembly (i.e. by increasing the rate of succinimide or maleimide ring hydrolysis). In some aspects, the electron withdrawing group is positioned to increase the electrophilicity of the succinimide or maleimide rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide or maleimide ring (e.g., by an intramolecular base catalysis mechanism),
[0295] In some embodiments, the Ligand-Functionai Agent Conjugates are represented by- Formula IV or IVa:
Figure imgf000103_0001
Figure imgf000104_0001
or a salt thereof (e.g., pharmaceutically acceptable salt thereof); wherein
L is a Ligand unit
LL is a Ligand unit that can be present or absent, wherein L and LL can be the same or different Ligand units;
D! is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly;
* is a maleimide ring, a hydrolyzed maieimide, a succinimide ring, or a hydrolyzed succinimide conjugated to at least one of L or L^ via a thioether linkage; and
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
The circle represents a scaffold that can be C;-8 alkylene, Cj.3 heteroalkylene,
Figure imgf000104_0002
aryiene, or C4-1o heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0. the scaffold is a Ce-ιο aryiene or C4.10 heteroarylene;
the subscript p ranges from 1 to 20;
-W- is an optional Cieavable unit,
the subscript w' is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 ,
-A- is an optional Stretcher unit, -A'- is an optional Stretcher unit component at the terminus of A;
a' is 0 or i ; and
u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present, and when u is L A can be present or absent.
[0296] L, HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, L, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates. L and LL can be different Ligand units or the same Ligand unit, In embodiments, wherein L and Lt, are the same Ligand unit, the succinimide or maleimide can be conjugated to the Ligand unit on the same or different polypeptide chains of the Ligand unit.
[0297] in some aspects, when r is 1, HE does not comprise a carbonyl group,(i.e., C(=0)) [0298] In some aspects, m + q + r is 0, 1 or 2 [0299] in some aspects, r is zero.
[0300] in some aspects, the Ligand-Funciional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein r and m are zero and q is one.
[0301] In some aspects, the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is C1-g alkylene or Cj-g heteroalkylene (preferably C1.4 alkylene or C heteroalkylene), r is zero, and the sum ofm+q is 1. in some such aspects, the scaffold is Q.3 alkylene or Q.3 heteroalkylene. in some such aspects, the alkylene is straight chain or branched.
[0302] in some aspects, the Ligand-Functional Agent Conjugate is represented by Formula IV or I Va or a salt thereof wherein the circle represents a scaffold that is C1-8 alkylene or Ci-s heteroalkylene (preferably C alkylene or C]..4 heteroalkylene), and m and r are zero. In some such aspects, the scaffold is C1.3 alkylene or C1.3 heteroalkylene. In some such aspects, the alkylene is straight chain or branched.
[0303] in some aspects, the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is Q, C2, C3 or C4 straight or branched chain alkylene, r is zero, and the sum of m+q is 1. [Θ3Θ4] In some aspects, the Ligand-Functional Agent Conjugate is represented by Formula IV or IVa or a salt thereof wherein the circle represents a scaffold that is Ci , C2s C3 or straight or branched chain alkyiene, and m and r are zero.
In some aspects, m and r are zero and
Figure imgf000106_0001
is represented by
-
Figure imgf000106_0002
[0306] In some aspects, there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the maleimide or succinimide (hydrolyzed or non-hydro lyzed) and there are no more than 5 atoms, no more than 4 atoms, no more than 3 atoms, or no more than 2 intervening atoms between the electron withdrawing group and the nitrogen atom of the maleimide or succinimide (hydrolyzed or non~hydrolyzed).
In some aspects, M" is a succinimide ring or hydrolyzed succinimide and Lj_, is present. In some aspects, M2 is a succinimide ring or hydrolyzed succinimide and LL is absent. In some aspects, M" is a maleimide ring or hydrolyzed maleimide and LL is present. In some aspects, M is a maleimide ring or hydrolyzed maleimide and LL is absent. In some aspects, when M2 is a maleimide ring or hydrolyzed maleimide, LL is present or absent and when M2 is a succinimide ring or hydrolyzed succinimide LL is present. In each of these embodiments when L and LL are present, L and LL can be the same Ligand unit or different Ligand units. In some aspects wherein L and LL are present and are the same Ligand unit, the maleimide or succinimide can be conjugated to the ligand unit on the same or different polypeptide chains of the Ligand unit.
[0308] in each of these embodiments, the alkyiene or heteroaikylene chain can be straight or branched, in some aspects, the alkyiene or heteroaikylene chain will be a straight chain. In other aspects, it will be branched.
[0309] In each of these embodiments, p can range from 1 to 20, preferably 1 to 12, even more 1 to 10 or 1 to 8. |Ό310] in some aspects wherein the scaffold itself is directly linked to the optional secondary linker assembly or D1, (for example, in select embodiments when q is zero or when q is zero and r is zero), the scaffold will comprise a reactive site suitable for attachment to the optional secondary linker assembly or D'
[0311] In some aspects wherein the scaffold itself is directly linked to the optional secondary linker assembly or D', (for example, in select embodiments when q is zero or when q is zero and r is zero), the scaffold will comprise a reactive site suitable for attachment to A or D'.
[0312] The maleimide ring can be conjugated to the Ligand unit via one or two thioether linkages as illustrated below both in non-hydrolyzed and hydrolyzed form and the succinimi.de ring can be conjugated to the Ligand unit via two thioether linkages as illustrated below in both non-hydrolyzed and hydrolyzed form wherein the wavy line indicates the point of attachment to the remainder of the linker conj ugate or linker-functional agent, conjugate:
Figure imgf000107_0001
Figure imgf000108_0001
Figure imgf000108_0002
Figure imgf000108_0003
[0313] In embodiments wherein the nialeiraide ring, hydrolyzed maleimide, succinimide ring, or hydrolyzed succinimide is conjugated to the Ligand via two tliioether linkages, p typically ranges from 3 to 30, or 1 to 8, or 1 to 4 and the maleimide or succinimide can be conjugated to the same or different polypeptide chains of the Ligand. In some aspects, the Ligand is an antibody. In other aspects, the Ligand is a non-antibody protein.
|0314J Functional Agent-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000109_0001
Figure imgf000109_0002
or a salt thereof (e.g., pharmaceutically acceptable salt thereof); wherein
D' is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly;
Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen:
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci.g alkylene, Ci.g heteroalkylene, C$.10 arylene, or C4-S 0 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C-6-10 arylene or C4.10 heteroarylene;
W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; -Y- is an optional Spacer unit, the subscript y' is 0 or i. ,
A is an optional Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1 , A can be present or absent
[0315] in some aspects, the halogen is bromine.
[0316] HE and BU, L°, A, W, and Y have the meanings provided for the Drag-Linker Conjugates. Additionally each of the specifically recited selected embodiments for the circle, HE, BU, L°. A, W, and Y are equally applicable to these Conjugates.
[0317] In some aspects, m and r are zero and
Figure imgf000110_0001
is represented by
B|U O
O ii
-CH C- — CH-CH2- , or -CH2-CH—— C-
[0318] Ligand-Linker Conjugates having either branched or non-branched linkers can be represented by the following formulas:
Figure imgf000111_0001
Figure imgf000111_0002
or a salt thereof (e.g., pharmaceutically acceptable salt thereof); wherein
L is a Ligand unit;
LL is a Ligand unit that can be present or absent, wherein L and L|.. can be the same or different Ligand units;
RG is a reactive group (comprising a reactive site) at the terminus of L° or
Figure imgf000111_0003
is suitable for attaching a Drug unit, Detection unit or Stability unit;
L° is an optional secondary linker assembly that is present;
M1 is a maleimide ring, a hydrolyzed ma!eirmde, a succinimide ring, or a hydrolyzed succinimide conjugated to at least one of L or LL via a thioether linkage; and
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group; the circle represents a scaffold that can be Cj-8 alkylene, Ci. heteroalkylene, C6-jo aryiene, or C4.10 heteroarvlene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or FA;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a Q. I G aryiene or C4.10 heteroarylene;
the subscript p ranges from 1 to 20.
W- is an optional Cleavable unit, the subscript w! is 0 or 1 ;
-Y- is an optional Spacer unit, the subscript y' is 0 or 1 ,
A is an optional Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 3 , A can be present or absent.
10319) L, HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, L, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates.
[0320] In some aspects , r and m are zero and
Figure imgf000112_0001
is represented by:
BU BU BU o
O
_|_iH— c—S--- — CH-CH2— C— |— , or ~ j>— CH2-CH—-C- —
[0321] Branched or non-branched Linkers can be represented by the following formulas:
Figure imgf000113_0001
Figure imgf000113_0002
or a salt thereof (e.g., pharmaceutically acceptable salt thereof); wherein
RG is a reactive group (comprising a reactive site) at the terminus of L
~\- A Aa-— Ww. Yy. - - which is suitable for attaching a Drug unit, Detection unit or Stability unit;
L is an optional secondary linker assembly that is present:
Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci„g alkyfene, C;_g heteroalkyiene, C6-io arylene, or C4-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or RG;
the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a -io arylene or C4.10 heteroarylene;
W~ is an optional Cleavable unit, the subscript w' is 0 or i ;
-Y- is an optional Spacer unit. the subscript y' is 0 or 1 ,
A is an optional Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A; a' is 0 or 1 ; and u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1 , A can be present or absent.
[0322] HE and BU, L°, A, W, and Y have the meanings provided for the Ligand-Drug Conjugates. Additionally each of the specifically recited selected embodiments for the circle, HE, BU, L°, A, W, and Y are equally applicable to these Conjugates.
)323] In sortie aspects , r and m are zero and
Figure imgf000114_0001
is represented by:
Figure imgf000114_0002
[0324] Methods of preparing mono- or di-thio-subsiituted halomaleimides as well as di- thiosubstituted succinimides are known in the art as are methods of conjugating them to ligands, see for example, Ryan et al., Chem. Commun., 201 1, 47, 5452-5454 and Smith et al, J, Am. Chem. Soc. 2010, 132(6), 1960-1965.
EXAMPLES
Example 1 - Synthesis of representative Self-Stabilizing Components Ma!eyS-Lysine{boc}OH
Figure imgf000115_0001
Chemical Formula: C15H24 2O7 hemical Formula: C-5iH22N204
Molecular Weight: 344.38
Molecular Weight: 246.30
[0325] In a 50 ml round bottom flask H-Lys(boc)-OH (246mg, 1 mmol) and maleic anhydride (98 mg, 1 mmol) were dissolved in 3 ml (4 vol.) acetic acid and the solution was stirred at room temperature for 3 hours, The reaction mixture was concentrated to an oil on the rotovap, and the product was precipitated by adding ~ 10 ml diehioromethane . The precipitate was collected by vacuum filtration, washed with diehioromethane, and dried overnight in the vacuum oven. 270 mg of product was recovered as a white powder (85% yield)
IViaieoyf-Lysine(boc}-OH
Figure imgf000115_0002
Chemical Formula: C15H 4 2O7
Chemical Formul
Molecular Weight: 344.36
Molecular We
Figure imgf000115_0003
[Θ326] Maleyl-Lys(boc)-OH (100 mg, 0.29 mmol) was suspended in Toluene (3 ml) and txiethylamine (224 uL) over molecular sieves in a 50 ml round bottom flask equipped with a condenser. DMA (~150 uL) was added to aid solubility. The solution was heated to 125 °C and refluxed for 4 hours after which the reaction was shown to be complete by LCMS. The reaction mixture was concentrated to dryness on the rotovap, redissolved in DMSO and purified by- preparative HPLC. 56 mg of product was isolated as a white powder. (60% yield) aieyi-DPR(boc)OH
Figure imgf000116_0001
Chemical Formula: CsH!6N204 Chemical Formula: C12H18N207
Molecular Weight: 204.22 Molecular Weight: 302.28
[0327] Maleyl-DPR(boc)-OH was prepared in the same manner as MaIeyl-Lysine(boe)OH (503 mg, 67%)
1aleoyl-DP (boc)-OH
Figure imgf000116_0002
Chemical Formula: C 2Hi8N207 Chemical Formula: C 2H 6N2Os
Molecular Weight: 302.28 Molecular Weight: 284.27
[0328] Maleoyi-DPR(boc)-OH was prepared in the same manner as MaleoyhLysihoe). (340 mg, 71%)
^aieyl-Dimeihy!lysirse
D
Figure imgf000116_0003
[0329] Maleyl-dimethyllysine was prepared in the same manner as Maleyl-lys(boc)-OH with the exception that the product did not precipitate after addition of dichloromethane. instead the oil was eo-evapaorated with 1 : 1 dichloromethane/ hexane until a white foam was obtained and dried under high vacuum overnight. (109 mg, 99%) a!eoyi-dimetbyHysirse
Ch
Figure imgf000117_0001
[0330] In a 10 ml round bottom flask ,Maleyl-dinieihyllysine (100 mg) was dissolved in acetic acid (1 ml) and refluxed for 4 hours. After 4 hours the reaction mixture was concentrated to dryness on the rotovap and dried to a white foam under high vacuum. NMR of crude material shows --80% conversion based on ratio of the singlet at 6.9 ppm and the olefmic protons from the starting material.
Example 2 - Synthesis of mDPR-Vai-Cit-PAB-MMAE
[0331 j mDPR-Val-Cit-PAB- MAE was prepared by coupling Boc-protected mDPR to Val- Cit-PAB-MMAE using standard methods for peptide coupling. The Boe group was removed in the final step.
Scheme:
Figure imgf000117_0002
Figure imgf000117_0003
Figure imgf000118_0001
Figure imgf000118_0002
Preparation of Fmoc-Val-Cit-PAB-MMAE
[0332] MMAE (5.34g, 6,94 mmol), Fmoc-Val-Cit-PAB-OCO-pNP (5.0g, 6.94 mmol) and HOBi (1.4 mmol) were charged to a 250 mi round bottom flask purged with N2 and dissolved in 15 ml of DMA. DIPEA (2.44 ml, 14 mmol) was then added, and the solution was stirred overnight at room temperature under inert atmosphere. The product was isolated by preparative HPLC, using a linear gradient from 30% MeCN (0.05%TFA) to 100% MeCN (0.05% TFA) over 40 min. Fractions containing product were concentrated on the rotovap to a white powder, affording 3.2 g (34%)
Preparation of Val-Cit-PAB-MMAE
[0333J A solution of 3.2 g of Fmoc- Val-Cit-PAB-MMAE in 7 ml DMF and 7 ml diethyiamine was stirred for 3 hours at room temperature. The reaction mixture was then concentrated to a thick oil on the rotovap. The product was precipitated in diethyl ether ( 100 ml) and filtered affording 2.0 g of product as an off white powder which was used without further purification.
Preparation ofmDPR (hoc) Val-Cit-PAB-MMAE 10334] In a 50 nil round bottom flask mDPR(boc)-OH (25 mg, 0,089 mmol), Vai-Cit-PAB-
MMAE (100 mg, 0.089 mmol), and HATU (41 mg, 0.107 mmol) were dissolved in 2 ml DMF. DIPEA (34 uL) was added and the solution was stirred for 1 hr at rt. The reaction mixture was diluted with 1 ml DMSO and the product was isolated by preparative HPLC. (70 mg, 56%)
Preparation ofmDPR - Val-Cit-PAB-MMAE
[0335] The above material was dissolved in 2 ml 10% TFA/dichloromethane and stirred for 1 h at rt. The reaction mixture was concentrated to dryness, reconstituted in 1 ml DMSO, and purified by preparative HPLC. (56 mg, 86%)
Example 3 - Monitoring thiosuccinimide hydrolysis
[0336] Thiosuccinimide hydrolysis of a self-stabilizing bioconjugate can be monitored by electrospray mass spectrometry, since the addition of water to the conjugate results in an increase of 18 Daltons to the observable molecular weight of the conjugate. When a conjugate is prepared by fully reducing the interchain disulfides of a human IgGl antibody and conjugating the maleimide to the resulting cysteines, each light chain of the antibody will contain a single maleimide modification and each heavy chain will contain three maleimide modifications (see Figure 1 , top). Upon complete hydroly sis of the resulting thiosuccinimides, the mass of the light chain will therefore increase by 18 Daltons, while the mass of the heavy chain will increase by 54 Daltons, This is illustrated in Figure 1 (bottom), with the conjugation and subsequent hydrolysis of a self-stabilizing maleimide drug-linker of the present invention (mDPR- Val-Cit- PAB-MMAE, molecular weight 1289 Da) to the fully reduced anti-CD30 antibody cACl O. The presence of the single N-linked glycosylation site on the heavy chain, results in the heterogeneity of masses observed in the unconjugated antibody.
Example 4 - Monitoring 11/2 hydrolysis
[0337] By monitoring the intensities of the non-hydrolyzed and hydrolyzed peaks in the mass spectrum of a self-stabilizing bioconjugate over time (mDPR- Val-Cit-PAB-MMAE), the hydrolysis kinetics can be evaluated. This is done by plotting the percent of the total population which has hydrolyzed at each timepoint versus time (Figure 2, top). These data are then fit to the exponential equation
where Y is the observed percent hydrolysis at time t, Ymax is the asymptotic maximal % hydrolysis, and K is the hydrolysis rate constant. The half-life for the hydrolysis reaction is defined as
Figure imgf000120_0001
[0338] When this procedure is performed on the light chain of a reduced hlgGl antibody, the analysis is quite straightforward as there is only one conjugation site per light chain and the reaction is a simple progression from the unhydrolyzed species to the hydrolyzed species with a mass change of 18 Daltons. Performing this analysis on the heavy chain is complicated by the fact that there are a total of three conjugation sites, resulting in a series of peaks of + 18, +36, and +54 Daltons as the conjugate undergoes hydrolysis. The analysis of the heavy chain is further complicated by the presence of multiple glycoforms. The analysis presented in Figure 2 was performed by only evaluating the peaks arising from the most abundant glycoform (the transition from 54195 Da to 54250 Da) and assuming that these peaks are a reasonable surrogate for the whole population of heavy chain glycoforms. As is evident in Figure 2, the observed kinetic profiles for ligh t and heavy chains are very similar. For this reason, and because of the added complexities of quantifying hydrolysis rates on the heavy chain noted above, most of the data to characterize hydrolysis rates of self-stabilizing maleimides conjugated to antibodies was determined from evaluation of the light chain hydrolysis.
[0339] One limitation of this methodology is that the eiectrospray ionization process tends to produce a small proportion of sodium adducts in the observed peaks (approximately 10% under the conditions used to generate the data in Figure 2), which have an observed mass 22 Daltons greater than the parent mass. Many mass spectrometers do not have sufficiently high resolution to resolve this +22 mass from the +18 mass thai results from hydrolysis on a protein with a total molecular mass in excess of 25,000 Daltons. Consequently, at the early timepoints when the degree of hydrolysis is low, the appearance of a peak at approximately +20 Daltons is a combination of these two effects which cannot be easily separated experimentally. As a result, the estimate of the percent of hydro!yzed product at the earliest timepoints is probably an overestimate, but the magnitude of this effect diminishes as the reaction proceeds toward completion
Example 5 - Evaluating spacing between the maleimide and basic group of the self-stabilizing linker assembly
[0340] it was hypothesized that the presence of a basic amino group adjacent to maleimide would accelerate the hydrolysis of thiosuccinimides prepared with those maleimides and thus result in stable bioconjugates. The distance between the maleimide and the basic amino group was recognized as an important parameter in the design of such self-stabilizing units. To evaluate the role of this spacing, a series of maleimides were prepared with the general structure
Figure imgf000121_0001
where x varied from 1 to 4. These maleimides were then conjugated to a fully reduced human !gGI at pH8 and 37 °C and immediately monitored by eiectrospray mass spectrometry to determine the rate of hydrolysis. The distance between the basic group and the maleimide is inversely proportional to the hydrolysis rate— that is, the greater the distance, the slower the hydrolysis. This result illustrates that positioning a basic amino group close to a maleimide results in an increase in the rate of succinimide ring hydrolysis of bioconjugates prepared with the maleimide. However, even with the shortest spacing tested here (x= I), an antibody conjugate would have to be held at pH 8 and 37 °C for approximately 5 hours to achieve complete hydrolysis (about 5 half-lives). Exposure of an antibody or other protein to such conditions for extended periods can potentially result in covalent modifications and misfolding events, and so raaleimides with even faster hydrolysis rates were sought.
[0341] To prepare biocoiijugates with faster hydrolysis rates, a series of maleimides were prepared with the general structure
Figure imgf000122_0001
where x = 1 to 4 and R. = val-cit-PAB-MMAE. These raaleimides were then conjugated to a fully reduced human IgGl at pH8 and 37 °C and immediately monitored by electrospray mass spectrometry to determine the rate of hydrolysis, As shown in Figure 4 (top), the distance between the basic group and the maleimide within this series of structurally related compounds exerts a profound influence on the progress of the hydrolysis reaction. As in the prior example, the shorter the distance between the maleimide and the basic amine, the faster the hydrolysis, Since basic conditions (i.e. high pH) are known to increase the rate of maleimide and succinimide ring hydrolysis, this effect is presumably an example of intramolecular catalysis by a general base mechanism. Within this series the compounds with x=2 and x::::3 did not attain complete hydrolysis during the 3 hour incubation, instead reaching an asymptote at
approximately 80% and 50%, respectively (plots are normalized to the maximally achieved hydrolysis), This phenomenon may arise from a competing reaction such as direct nucleophilic attack of the primary amine on the succinimide ring, or may be due to an isomeric impurity in the maleimide which leads to biphasic hydrolysis kinetics.
Example 6 - Hydrolysis kinetics
[0342] 'i'he previous examples illustrate the influence that a basic group can have over the rate of succinimide ring hydrolysis in a bioconjugate, depending on the distance between the basic group and the maleimide of the parent molecule. However, it is expected that the presence of electron- withdrawing or -donating groups will also influence the rate of ring hydrolysis, since these groups will influence the electron density (and therefore eiectrophilicity) at the carbonyi carbons of the ring. In the conjugates of example 5, a carboxamide group is present in the alpha position relative to the nitrogen of the ring (i.e. a single carbon atom is present between the nitrogen of the ring and the carbonyi carbon of the carboxamide). Because the carboxamide is a weak electron withdrawing group, its presence is likely to influence the observed hydrolysis rates. To better understand the relati ve contributions of the basic amino group and the electron- withdrawing carboxamide group on the observed hydrolysis rates, a series of maleimides were conjugated to a reduced human IgG l antibody at pH 7.4, 22 °C, and the hydrolysis rates determined by mass spectrometry (Figure 5). These maleimides contained j ust the carboxamide in the alpha, position (triangles), just the primary amine in the beta position (inverted triangles), or both the carboxamide and the primary amine (circles). A coniroi maleimide which contained neither group in proximity to the maleimide was also evaluated, although its hydrolysis is so slow that no reaction was observed under these conditions and no data is plotted. Under these conditions the self-stabilizing maleimide which contains both the base and the electron withdrawing group produced a bioconjugate with a hydrolysis tm of just 12 minutes, while the maleimide containing only the amine yielded a t; /2 of 2.5 hours, and the maleimide containing only the carboxamide yielded a tia of 24 hours. This result indicates that the basic group and the electron withdrawing group act in concert to yield a conjugate with the very rapid hydrolysis kinetics which are most convenient for the manufacture of biocon] agates under the desirable mild conditions. Conjugates prepared with the diaminopropionyl maleimide (circles) exhibit ideal hydrolysis characteristics, with a t-, /2 of less than 15 minutes under very gentle conditions and the reaction approaching 100% completion in about 2 hours.
Example 7 - Evaluating spacing between the maleimide and carboxamide group of the self- stabilizing linker assembly
[0343] The rapid and complete succinimide hydrolysis observed in conjugates prepared with self-stabilizing diaminopropionyl maleimido drug-linkers acid (DPR) shown in examples 5 and 6 above indicates the importance of both the basic group and the electron withdrawing group to the design. A second, isomeric maleimido drug-linker was prepared with diaminopropionic acid to further evaluate the role of these two components on the hydrolysis behavior of the resulting conjugates. The structures are termed a-maleimido DPR and β-maleimido DPR and are shown below,
Figure imgf000124_0001
a-maleimido DPR β-maleimido DPR
R - vai-cit-PAB-MMAE
[0344] Both DPR maleimides possess a basic primary amine which is separated from the maleiraido nitrogen by two carbon atoms. Both also possess an electron withdrawing carboxamide group, however the distance from the maleimido nitrogen the carboxaraide varies from 1 to 2 carbon units (a. and β, respectively). Finajly, the separation between the basic amine and the carboxamide also varies from 1 to 2 carbon units (β and a, respectively). Collectively, this means that in β-DPR the carboxamide exerts less electron withdrawing influence on the maleimide ring but more electron withdrawing influence on the primary amine, relative to a- DPR. This is expected to slow the rate of hydrolysis by reducing both the electrophilicity of the maleimide and the basicity of the primary amine. When these maleimido drug-linkers were conjugated to reduced antibody and monitored for suecimmide hydrolysis, a 17-fold lower hydrolysis rate was observed for β-DPR relative to a-DPR (Figure 6). This example illustrates how the relative positioning of a basic group and an electron withdrawing group can be used to 'tune' the hydrolysis rate.
Examples 8 - 15
[0345] To evaluate the stability and pharmacological activity of ADCs prepared with self- stabilizing drug-linkers, a self-stabilizing maleimido-drug-iinker was prepared. This drug-linker contains the maleimido-DPR group coupled to the cytotoxic agent MMAE via a protease- cleavable val-cit PAB self-imrnolative group (referred to herein as maleimido-DPR-val-cit-PAB- MMAE). For comparison, a non self-stabilizing drug-linker was used (referred to herein as maleimido-caproyl-val-cit-PAB-MMAE). The only difference between these agents is the unit between the maleirnide and the valine group of the val-cit linker. The maleimide units of these drug-linkers can be prepared using maleic anhydride and mono -protected diaminopropionic acid and aminocaproic acid, respectively
Figure imgf000125_0001
Example 8 - Evaluating stability of Ligand-Drug Conjugates in Buffer
[0346] in standard buffer systems, maleimide elimination from a bioconjugate prepared using thiol-maleimide chemistry is essentially undetectable because the eliminated maleimide quickly reacts again with the thiol, resulting in an equilibrium which lies far toward the side of the intact conjugate. However, the addition of a thiol scavenger to the buffer creates a system in which maleimide that eliminates from the bioconjugate can instead react with the scavenger, resulting in a persistent, observable loss of the maleirnide from the protein. An experiment using such a system was performed with antibody-drug conjugates prepared with a self-stabilizing
diamine roprionyl (DPR) maleimido drug-linker alongside a non-stabilizing caproyl drug-linker. ADCs were prepared with 8 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyi-val-cit-PAB-MMAE using a fully reduced humanized IgGl . Drug loading was confirmed by reversed-phase HPLC on a polymeric PLRP-S column as described previously (Sun 2005). Complete succinimide hydrolysis of the self-stabilizing linker was also confirmed by electrospray mass spectrometry. These ADCs were placed in 150 mM Tris buffer, pH 8, at 2.5 mg/mL, containing 10 mM N-acetylcysteine as a scavenger, and incubated for 2 weeks at 37 CC. At seven timepoints during the incubation, an aliquot of each ADC was removed and frozen at -80°C Upon completion of the time-course, all samples were analyzed by the above reversed-phase HPLC method to determine the drug : antibody ratio. The results of this study are shown in Figure 7. The ADC prepared with the self-stabilizing DPR maleimido drug-linker exhibited minimal loss of drug over this tirnecourse (from 8.0 to 7.9 drugs per antibody over 14 days), while the ADC prepared with the caproyl maleimido drug-linker lost approximately half of its drug load (from 8.0 to 3.9 drugs per antibody over 14 days) under these conditions.
Example 9 - Ex Vivo Plasma Stability (reversed phase method)
(0347] Assessing drug loading of humanized ADCs in non-human plasma samples by the reversed-phase HPLC method described in Example 8 can be achieved by first isolating the ADCs with IgSelect resin (GE Healthcare), which selectively binds to the human Fc domain. ADCs were prepared with 8 drugs per antibody of either maleimido-DPR-val-eit-PAB-M AE or raaleimido-caproyl-vai-cit-PAB-MMAE using a fully reduced human IgGl. These ADCs (0.25 mg/mL) were incubated in sterile rat plasma for 7 days at 37 °C. At seven timepoints during the incubation, a 50 μΐ, aliquot of each ADC was removed and frozen at -80 °C. Upon completion of the tirnecourse, ADCs were purified from each sample and analyzed by reversed- phase HPLC^ to determine the drug : antibody ratio. The results of this study are plotted in Figure 8. As was observed in buffer, incubation of an ADC prepared with a self-stabilizing maleimide in rat plasma also results in little or no observable loss of drug under conditions which result in the loss of approximately half of the drag from a maleimido-caproyl ADC,
Example 10 - Ex Vivo Plasma Stability (conjugated drug method)
[0348] A second assay format was utilized to assess ADC stability in rat and human plasma ex vivo. ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB- MMAE or maleimido-caproyl-val-cit-PAB-MMAE using a human IgGl partially reduced to a level of 4 thiols per antibody (resulting in an ADC with 4 drugs per antibody). These two ADCs were spiked into rat and human plasma and incubated at 37 °C for 7 days. At seven timepoints during this incubation, aliquots were removed and frozen at -80 °C until completion of the timecourse. ADCs were then isolated from each sample and MMAE released proteolytically from the isolated ADCs as described previously (Sanderson 2005), The released MMAE was then quantified by LC-MS/MS and normalized to the initial value for each ADC (Figure 9). In both rat and human plasma, the ADC prepared with a self- stabilizing maleimide lost little or no drug under these conditions, while approximately half of the drug was lost from a ma!eimido- caproyi ADC.
Example 1 1 - In Vivo Stability
[Θ349] As described in Example 10 above, the drug:antibody ratio can be measured for ADCs in rat plasma by reversed-phase HPLC analysis following purification with IgSeiect resin. This method was applied to samples derived from an in vivo pharmacokinetic experiment in rats. ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl-vai-cit-PAB-MMAE using a humanized IgGl partially reduced to an average of 4 thiols per antibody (resulting in drug : antibody ratio of 4). These ADCs were further purified as described previously (Sanderson 2005) by hydrophobic interaction chromatography to isolate the species containing 4 drugs per antibody. These ADCs were dosed intravenously at 10 nig/kg in Sprague-Dawley rats. At five timepoints, three animals from each dosing group were sacrificed and the collected blood was processed to plasma and frozen at. -80 °C. Upon completion of the study, all samples were processed by the IgSeiect resin method described above, except that the sample volume, varied. The drug : antibody ratio at each timepoint in this study are plotted in Figure 10. As was observed in rat plasma ex vivo, the ADC prepared with a self-stabilizing maleimide exhibits minimal loss of drug in vivo, dropping from an initial value of 4.3 drugs per antibody to a value of 3.6 drugs per antibody ( 12% reduction) after 7 days. During this same timeframe, the drug : antibody ratio of an ADC prepared with a maleimido-caproyl linker dropped from an initial value of 3.9 to a value of 1.5 (61 % reduction). This illustrates that the increased stability of a self-stabilizing drug-linker that is observed ex vivo translates into an in vivo setting.
Example 12 - Pharmacokinetics [0350] Because maleimido-caproyl ADCs are prone to loss of drug through raaleirnide elimination whereas self-stabilizing maleimide ADCs are not, it is reasonable to predict that exposure to antibody-conjugated drug will be greater following equivalent doses of the two ADCs. To confirm this prediction, ADCs were prepared with 4 drugs per antibody of either ma!eimido-DPR-vai-cit-PAB-MMAE or raaleimido-caproyl-val-cit-PAB-MMAE using a human IgG l partially reduced to an average of 4 thiols per antibody (resulting in drug : antibody ratio of 4). These two ADCs were dosed at 2 mg/kg in Sprague-Dawley rats, and blood samples were taken at seven timepoints and processed to plasma. These plasma samples, along with standards of each ADC for the preparation of a calibration curve, were subjected to the mAb Select resin capture and papain release procedure described example 10 above to measure the concentration of antibody-conjugated MMAE. Antibody-conjugated drug concentrations were higher for the ADC prepared with the self-stabilizing drug-linker, with the magnitude of the difference increasing with time (data not shown), initial antibody-conjugated drug concentrations are superimposabie, reflecting the equivalence of the dose and drug : antibody ratio of the ADCs. However, divergence is observed within the first day, reaching a two-fold difference by day 3. These higher concentrations resulted in an approximately 40% greater antibody-conjugated drug AUG tor the self-stabilized ADC relative to the maleimido-caproyl ADC
Example 13 - Toxicology
[0351] To assess the impact of self- stabilizing maleimides on toxicology, ADCs were prepared with 4 drugs per antibody of either maleimido-DPR-val-cit-PAB-MMAE or maleimido-caproyl- val-cit-PAB-MMAE using a humanized IgGl (which has no known binding to any rat antigen) partially reduced to an average of 4 thiols per antibody (resulting in an average drug : antibody ratio of 4). These ADCs wrere dosed intravenously in female CD®IGS rats (Charles River Laboratories) at 10 mg/kg (6 rats per test article plus 6 rats receiving vehicle only). Prior to dosing and at 3 post-dose timepoints, blood samples were taken for hematology and serum chemistry analysis for biomarkers of toxicity. Neutropenia induced by the MMAE ADC appeared less severe for the self-stabilized conjugate than for the maleimido-caproyl ADC (data not shown). Example 14 - Plasma concentration of released drug
(0352] The toxicology experiment described in example 13 above also included blood draws at one hour and 24 hours post-dose, which along with the 4 day and 7 day posi-dose samples were analyzed for unconjugated MMAE in plasma by LC-MS/MS. The results of this analysis indicated that peak concentrations of circulating MMAE are about 2-fold lower for the self- stabilizing maleimido-DPR-val-cit-PAB-MMAE ADC relative to the maleimido-caproyl-val-cit- P AB-MM AE (data not shown) .
Example 15, Xenograft activity
[0353] To evaluate the impact of self-stabilizing drug-linkers on the antitumor activity of ADCs, conjugates were prepared with the anti-CD30 antibody cAC iO using drug-sinkers containing the val-cit-PAB-MMAE cytotoxic pay load linked to the antibody via either a maleimido-caproyl group or a self-stabilizing maleimido-DPR group. These ADCs were evaluated in two separate murine xenograft models of CD30+ human malignancies. In the first mode! (Figure 1 1), Karpas-299 (human ALCL) cells were implanted subcutaneousiy in female SCID mice and tumors were allowed to grow a volume of approximately 250 mm3 before dosing at 1 mg/kg weekly for three doses (six mice per dose group). All six mice dosed with the maleimido-caproyl ADC experienced some tumor growth delay relative to the untreated group, and two animals experienced partial tumor shrinkage; however, all tumors grew out and the entire group was euthanized with large tumors. The self-stabilizing ADC dose group
experienced complete responses (no detectable tumor) in all six animals, with five animals experiencing durable regressions for the course of the study and only one animal sacrificed after its tumor had returned on study day 55. The results of this study indicate a significantly greater in vivo antitumor activity for the ADC prepared with the self-stabilizing drug-linker. In the second model, L428 (human Hodgkin Lymphoma) cells were implanted subcutaneousiy in female NSG mice and tumors were allowed to grow a volume of approximately 100 mm3 before dosing at 1 mg kg ever four days for four doses (six mice per dose group). All animals in both ADC dose groups experienced significant growth dela during treatment, however all tumors began growing out after study day 28 with no significant difference between maleimido-caproyl and self-stabilizing ADCs. Thus, the improvement in antitumor activity observed with the se stabilizing ADC in xenograft studies appears to be model-dependent.

Claims

WHAT IS CLAIMED IS: 1. A Ligand-Functional Agent Conjugate comprising:
a Ligand unit and at least one Functional Agent selected from the group consisting of a Drug unit. Detection Unit, and Stability unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide or a dilactam directly conjugated to the Ligand unit via a thioether linkage; or wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring, a maleimide ring, a hydrolyzed succinimide ring or a hydrolyzed maleimide ring wherein the succinimide ring, maleimide ring. hydrolyzed succinimide ring or hydrolyzed maleimide ring is directly conjugated to the Ligand unit via one or two thioether linkages; and a base and an electron withdrawing group operably linked to stabilize the conjugate in plasma relative to a Ligand-Functional Agent Conjugate lacking the self-stabilizing linker assembly. 2. A Ligand-Functional Agent Conjugate of claim 1, comprising:
a Ligand unit and at least one Functional Agent selected from the group consisting of a Drug unit, a Detection Unit, and a Stability Unit, wherein the Ligand unit and each of the Functional Agent(s) are joined by a self-stabilizing linker assembly comprising a succinimide ring or a hydrolyzed succinimide or a dilactam directly conjugated to the Ligand unit via a thioether linkage; and a base and an electron withdrawing group operably linked to stabilize the conjugate in plasma relative to a Ligand-Functional Agent Conjugate lacking the self-stabilizing linker assembly. 3. A Ligand-Functional Agent Conjugate of claim 1 or claim 2. wherein the Ligand-Functional Agent is a Ligand-Drug Conjugate comprising a Drug unit as the Functional Agent and the Ligand-Drug conjugate further comprises a linker assembly which joins each Drug to a self-stabilizing linker assembly. 4. A Ligand-Functional Agent Conjugate of claim 3, wherein the linker assembly has the formula:
Figure imgf000132_0001
wherein -A- is an optional Stretcher unit, the subscript a' is 0 or 1 ; -W- is an optional Cleavabk unit, the subscript w' is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or 1 ; and the wavy lines indicate the points of attachment to the self-stabilizing linker assembly and the Drug unit.
5. A Ligand-Functional Agent Conjugate of claim 4, wherein the subscript is 1, and W is a dipeptide. tripeptide or tetrapeptide.
6. A Ligand-Functional Agent Conjugate of claim 1, having the formula:
Figure imgf000132_0002
or a salt thereof, wherein
L is a Ligand unit;
D' is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly; and
LSa is the self-stabilizing linker assembly, wherein
M1 is a succinimide ring or hydrolyzed succinimide or together with BU forms a dilactara;
BU is a Basic unit:
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that is C1-g alkylene, Ci-s heteroalkyiene, Ce-io arylene, or CU-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or C4.10 heteroarylene; the subscript a and b are each 0 or 1 , and the sum of a+b is 1 ; and
the subscript ρ ranges from 1 to 20. 7. A Ligand-Functional Agent Conjugate of claim 1 or 2, having the formula:
Figure imgf000133_0001
or a salt thereof, wherein
L is a Ligand unit;
D' is a Drug unit, a Detection unit, or a Stability unit:
Lss is the self-stabilizing linker assembly, wherein
M! is a succinimide ring or hydroiyzed succinimide or together with BU forms a di lactam;
BU is a Basic unit;
HE is a hydrolysis enliancer comprising an electron withdrawing group;
the circle represents a scaffold that is Cj.g alkylene, Ci.g heteroalkylene, C6-io arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C -.o arylene or€4.10 heteroarylene;
the subscript a and b are each 0 or 1, and the sum of a+b is 1 ;
the subscript p ranges from 1 to 20;
-W- is an optional Cleavable unit,
the subscript w! is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1. A is a Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A;
a' is 0 or 1 ; and u is from 1 to 20, wherein when u is from 2 to 20, A is present and when u is 1, A can be present or absent. 8. A Ligand-Functional Agent Conjugate of claim 2, represented by Formula
Figure imgf000134_0001
L is a Ligand unit
LL is a Ligand unit that can be present or absent, wherein L and Li , can be the same or different Ligand units;
D' is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly;
M2 is a maleimide ring, a hydrolyzed maleimide, a succinimide ring, or a hydrolyzed succinimide conjugated to at least one of L or LL via a thioether linkage; and BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can he Q-s alkyiene, Q.g heteroalkylene, C<s-io
arylene. or C4-so heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D*;
the subscripts m, q and r are each 0 or 1, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a Cg-io arylene or C4-10 heteroarylene;
the subscript p ranges from 1 to 20;
-W- is an optional Cieavable unit,
the subscript w' is 0 or 1 ;
-Y~ is an optional Spacer unit,
the subscript y' is 0 or 2,
-A- is an optional Stretcher unit,
-A'- is an optional Stretcher unit component at the terminus of A:
a' is 0 or 1 ; and
u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1. A can be present or absent. 9. A Ligand-Functional Agent Conjugate of any of claims 6, 7 or claim 8, that is a Ligand-Drug Conjugate wherein D' is a Drug unit, q is 1 and m and r are 0. 10. A Ligand-Functional Agent Conjugate of any of claims 6, 7 or claim 8, that is a Ligand-Drug Conjugate wherein D' is a Drug unit, m is 1 and q and r are 0. 11. A Ligand-Functional Agent Conjugate of any of claims 6, 7 or claim 8, that is a Ligand-Drug Conjugate wherein D' is a Drug unit, r is 1 and m and q ar 0 and HE does not comprise a carbonyl group as the electron withdrawing group. 12. A Ligand-Functional Agent Conjugate of any of claims 6 to 11, that is a Ligand-Drug Conjugate wherein D' is a Drug unit and a is 1. 13. A Ligand-Functional Agent Conjugate of any of claims 6, 7 or claim 8, that is a Ligand-Drug Conjugate wherein D' is a Drug unit, a is I, q is 1., and m and r are 0,
14. A Ligand-Functional Agent Conjugate of any of claims 6 to 13, wherein the circle represents a scaffold that is Ct-g alkylene or C i.g heteroalkylene.
15. A Ligand-Functional Agent Conjugate of any of claims 6 to 13, wherein the circle represents a scaffold that is alkylene.
.16. A Ligand-Functional Agent Conjugate of any of claims 6 to 15, wherein the Basic unit comprises a primary or secondary amine.
17. A Ligand-Functional Agent Conjugate of any of claims 6 to 15, wherein the Basic unit is selected from the group consisting of ~(CH2 )XNH2, - Cf-I2 )xNHRa, and -(CH2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Ci.6 alky! and ' C;..6 haloalkyl, or two R!: groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of the succimmide (hydrolyzed or non-hydrolyzed) or dilactam.
18. A Ligand-Functional Agent Conjugate of any of claims 1- 17, wherein the electron withdrawing group comprises a carbonyl, sulfonyl or phosphoryl moiety.
19. A Ligand-Functional Agent Conjugate of any of claims 6, 7 or 8, wherein r is zero, m+q is 1 , and HE is selected from the group consisting
of -CH2C(0>, -C(O)-, -C(0)CH2-, -CH2CH2C(0)- , and -CH2C(0)NH-.
20. A Ligand-Functional Agent Conjugate of any of claims 6 or 7, that is a Ligand-Drug Conjugate having the formula:
Figure imgf000136_0001
Figure imgf000137_0001
Figure imgf000137_0002
or a salt thereof, wherein D is a Drug unit, and Ab is an antibody.
21. A Ligand-Functional Agent Conjugate of claim 2Θ, wherein BU is selected from the group consisting of -(CH2 )ΧΝΗ2, -(CH2 )xNHRa, and -(CH2 )xNRa 2, wherein x is an integer of from 0-4 and each Ra is independently selected from the group consisting of Ct-6 alky] and Cu6 haioalkyl, or two R.a groups are combined with the nitrogen to which they are attached to form an azeiidinyh pyrrolidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the ni trogen atom of the succiniraide (hydrolyzed or non-hydrolyzed) or diiaetam.
22. A Ligand-Functional Agent Conjugate of claim I, that is a Ligand-Drug Conjugate having the formula;
Figure imgf000137_0003
or a pharmaceutically acceptable salt thereof wherein I, is a Ligand unit;
D is a Drug unit;
L° is the optional secondary linker assembly;
Ln is the self-stabilizing linker assembly, wherein
M1 is a succinimide ring or hydro iyzed succinimide or together with B forms a dilactam; V, Q, T, and G are independently selected from -(C(R9)( Rl0))~;
Rs is H or C1.3 alkyl;
Ry and R'° are, in each occurrence, independently selected from H or C j .3 alkyl;
F is C(E')( E2) wherein E1 and E2 are independently selected from hydrogen, an electron withdrawing group, or E1 and E2 together are (:::0);
RS is a reactive site for conjugation to a component of the optional secondary tinker assembly or Drug unit;
g is O to S:
m is O to S;
n is 0 to 5:
d is O or l ;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
and B is a base, provided that when d is zero or El and E~ are hydrogen, RS is an electron withdrawing group, 23. A Ligand-Functional Agent Conjugate of claim 22, wherein ra is 0, and n is 0. 24. A Ligand-Functional Agent Conjugate of any one of claims 20-23, wherein L° has the formula:
Figure imgf000138_0001
wherein -A- is an optional Stretcher unit, the subscript a' is 0 or 1 ; -W- is an optional Cleavable unit, the subscript w' is 0 or 1 ; and -Y- is an optional Spacer unit, and the subscript y' is 0 or 1; and the wavy lines indicate the points of attachment to the self-stabilizing linker assembly and the Drug unit.
25. A Ligand -Functional Agent Conjugate of claim 24, wherein w' is 1 and W is conjugated directly to the Drug unit. 26. A Ligand-Functionai Agent Conjugate of claim 24, wherein w' is 1 and W is conjugated directly to the Spacer unit. 27. A Ligand-Functionai Agent Conjugate of claim 24, wherein w* is 3 and W is conjugated directly to the Drug unit or Spacer unit via a eleavable peptide, disulfide or hydrazine bond, 28. A Ligand-Functionai Agent Conjugate of claim 24, wherein W is absent. 29. A Ligand-Functionai Agent Conjugate of claim 24, wherein W is present and the Spacer unit and Stretcher unit are absent. 30. A Ligand-Functionai Agent Conjugate of any of claims 6 to 29, wherein D is selected from the group consisting of auristatins and PBDs. 31. A Drug-Linker Conjugate having the formula:
Figure imgf000139_0001
or a salt thereof, wherein,
D is a Drug unit;
L° is a an optional secondary linker assembly; and
LSh is the self-stabilizing linker assembly, wherein
BIJ is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group; the circle represents a scaffold that can be Q-g alkylene, d-g heteroalkylene,€5-10 arylene, or C4.10 heteroarylene, and optionally comprises a reactive site suitable for attach merit to the optional secondary linker assembly or Drug unit;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or€4-10 heteroarylene; and the subscript a and b are each 0 or 1, and the sum of a+b is 1.
32. A Drug-Linker Conjugate of claim 31, having the formula:
Figure imgf000140_0001
or a salt thereof.
33. A Functional Agent-Linker Conjugate having the formula:
Figure imgf000141_0001
or a salt thereof, wherein
D' is a Drug unit, a Detection unit, or a Stability unit;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that is Ci.g alkylene, C\.$ heteroalkylene, Cg-io arylene, or C4- 10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D':
the subscripts m, q and r are each 0 or 3, and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a Cg,io arylene or C4-10 heteroarylene;
the subscript a and b are each 0 or 1 , and the sum of a+b is 1 ;
-W- is an optional Cleavable unit,
the subscript w' is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 ,
A is a Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A;
a' is 0 or 1 ; and
u is from 1 to 20, wherein when u is from 2 to 20, A is present and when u is 1, A can be present or absent.
34, A Functional Agent-linker Conjugate having the formula:
Figure imgf000142_0001
Figure imgf000142_0002
or a salt thereof;
wherein
D' is a Drug unit, a Detection unit, or a Stability unit;
L° is an optional secondary linker assembly;
Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be C;.§ alkylene, Ci-g heteroaikylene, C&to arylene, or C4-10 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m -÷· q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a CMO arylene or C4.10 heteroarylene;
-W- is an optional Cleavabie unit,
the subscript w! is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 , -A- is an optional Stretcher unit,
-A - is an optional Stretcher unit component at the terminus of A:
a' is 0 or 1 ; and
u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u, A can be present or absent. 35. A Drug-Linker having the formula:
Figure imgf000143_0001
or a pharmaceutically acceptable salt thereof wherein
D is a Drug unit;
L° is the optional secondary linker assembly;
V, Q, T, and G are independently selected from ~(C(R )( R10))~;
R1 is 11 or C1-3 alkyl;
R9 and R'° are, in each occurrence, independentl selected from H or C'i„3 alkyl;
F is C(E')( E2) wherein E1 and E2 are independently selected from hydrogen, an electron withdrawing group, or E1 and E together are (=0);
RS is a reactive site for conjugation to a component of the optional secondary linker
assembly or Drug unit;
g is 0 to 5;
m is 0 to 5;
n is O to S:
d is 0 or 1;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
and B is a base, provided that when d is zero or E' and E* are hydrogen, RS is an electron withdrawing group. A Linker having the formula:
Figure imgf000144_0001
or a salt thereof, wherein
RG is a reactive group comprising a reactive site at the terminus of L°, suitable for attaching a Drug unit;
L° is an optional secondary linker assembly that is present; and
LoS is the self-stabilizing linker assembly, wherein
BU is a Basic unit:
HE is a hydrolysis enhancer comprising an electron withdrawing group; the circle represents a scaffold that can be Ci-x alkylene, Cj.g heteroaikylene, C6-j0 arylene, or C4.10 heteroarviene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6. io arylene or C io heteroarylene; and the subscript a and b are each 0 or 1, and the sum of a+b is 1.
A Linker of claim 36, having the formula:
Figure imgf000144_0002
Figure imgf000145_0001
|_0_ RG
or a salt thereof.
Figure imgf000145_0002
or a salt thereof, wherein
RG is a reactive group comprising a reactive site at the terminus of
-Α _ Δ
*> 3 ς which is suitabie for attaching a Drug unit, a
Detection unit, or a Stability unit;
BLJ is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group:
the circle represents a scaffold that is Cj .g alkylene, C s heteroalkylene, Cg-io arylene, or C. io heteroarylene, and optionally comprises a reactive site suitable for attachment to
\ ΔΑ the optional secondary linker assembly or to a Drug unit, a Detection unit, or a Stability unit;
the subscripts m, q and r are each 0 or 1 , and the sum of ra + q + r is 0, i or 2 provided that if ni + q + r is 0, the scaffold is a C(,.\o aryiene or (1(. :0 heteroarylene;
the subscript a and b are each 0 or 1 , and the sum of a+b is 1 ;
-\V~ is an optional Cleavable unit,
the subscript w' is 0 or 3 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 ,
-A- is a Stretcher unit,
-A'- is an optional Stretcher unit component at the terminus of A;
a' is 0 or 1 ; and u is from 1 to 20, wherein when u is from 2 to 20, A is present and when u is 1 , A can be present or absent.
39 A Linker represented by a formula selected from the group consisting of:
Figure imgf000146_0001
and
Figure imgf000146_0002
L is an optional secondary linker assembly that is present;
Q and Z are hydrogen or halogen wherein at least one of Q and Z are halogen;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Ci.g alkylene,
Figure imgf000147_0001
heteroalkylene, C&-io arylene, or GMO heteroaryiene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or RG;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or C4.10 heteroaryiene;
W- is an optional. Cleavable unit,
the subscript w' is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 ,
A is an optional Stretcher unit,
A' is an optional Stretcher unit component at the terminus of A;
a' is 0 or 1 ; and
u is from 1 to 20, with the proviso that when u is from 2 to 20, A is present and when u is 1 , A can be present or absent. 40. A Linker having the formula
Figure imgf000147_0002
or a pharmaceutically acceptable salt thereof, wherein
L° is the optional secondary linker assembly that is present;
RG is a. reactive group comprising a reactive site at the terminus of L° which is suitable for attaching a Drug unit; M! is a succinimide ri g or hydrolyzed succinimkle or together with B forms a dilactam; V, Q, T, and G are independently selected from -(C(R9)( R."J))-;
R' ts H or C!-3 alkyl;
R9 and R10 are, in each occurrence, independently selected from H or C1-3 alkyl;
F is C(E!)( E2) wherein E1 and E are independently selected from hydrogen, an electron withdrawing group, or E1 and E2 together are (=0);
RS is a reactive site for conjugation to a component of the optional secondary linker assembly;
g is O to S;
m is O to S:
n is i) to 5;
d is O or l ;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
and B is a base, provided that when d is zero or E1 and E2 are hydrogen, RS is an electron withdrawing group.
41. A Ligand-Linker Conjugate having the formula:
Figure imgf000148_0001
or a salt thereof, wherein
L is a Ligand unit;
the subscript ranges from 1 to 32;
L° is an optional secondary linker assembly that is present;
RG is a reactive group comprising a reactive site at the terminus of L° which is suitable for attaching a Drug unit;
Lss is the self-stabilizing linker assembly, wherein
M1 is a succinimide; BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Cj.g alkylene, Q.s heteroalkylene, C0-10 arylene, or€4.50 heteroatylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or Drug unit;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m + q + r is 0, the scaffold is a C6-io arylene or C4-10 heteroarylene; and the subscript a and b are each 0 or 1, and the sum of a+b is 1. 42, A Ligand-Linker Conjugate having the formula:
Figure imgf000149_0001
or a salt thereof, wherein
L is a Ligand unit;
RG is a reactive group comprising a reactive site at the terminus of
Figure imgf000149_0002
h is suitable for attaching a Drug unit, a Detection unit, or a Stability unit:
Lah is the self-stabilizing linker assembly, wherein
M1 is a succinimide ring or hydrolyzed succinimiae or together with BU forms a dilactam;
BU is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group; the circle represents a scaffold that is C;_g alkylene, Ci.g heteroalkylene, C O arylene, or €4.10 heteroarylene, and optionally comprises a reactive site suitable for attachment to the optional secondary linker assembly or D';
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if m ÷ q + r is 0, the scaffold is a Ce- io aryJene or C4.10 heteroarylene;
the subscript a and b are each 0 or 3 , and the sum of a+b is 1 ;
the subscript p ranges from 3 to 20;
-W- is an optional Cieavable unit,
the subscript w' is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y' is 0 or 1 ,
-A- is a Stretcher unit,
-A'- is an optional Stretcher unit component at the terminus of A;
a' is 0 or 1 ; and
u is from 1 to 20. wherein when u is from 2 to 20, A is present and when u is 1 , A can be present or absent, 43. L gand-Linker Conjugate represented by a formula selected from the group consisting of:
Figure imgf000150_0001
Figure imgf000151_0001
or a pharmaceutically acceptable salt thereof; wherein
L is a Li and unit;
L(. is a Ligand unit that can be present or absent, wherein L and LL can be the same or
different Ligand units;
RG is a reactive group (comprising a reactive site) at the terminus of L° or
- - A Aa. Ww. Yy - > which is suitable tor attaching a Drug unit, Detection unit or Stability unit;
L° is an optional secondary linker assembly that is present;
M2 is a maleimide ring, a hydrolyzed maleimide, a succinimide ring, or a hydrolyzed
succinimide conjugated to at least one of L or LL via a thioether linkage; and
BLJ is a Basic unit;
HE is a hydrolysis enhancer comprising an electron withdrawing group;
the circle represents a scaffold that can be Cj.g alk lene, C).g heteroalkylene, C6-1o arylene, or C4- 10 heteroarylene, and optionally comprises a reactive site suitable for attachment to L°, A, W, Y or FA;
the subscripts m, q and r are each 0 or 1 , and the sum of m + q + r is 0, 1 or 2 provided that if ra + q + r is 0, the scaffold is a C -io arylene or C4.10 heteroarylene;
the subscript p ranges from 1 to 20;
-W- is an optional Cleavabie unit,
the subscri t w' is 0 or 1 ;
-Y- is an optional Spacer unit,
the subscript y* is 0 or I ,
-A- is an optional Stretcher unit,
-A'- is an optional Stretchier unit component at the terminus of A; a' is 0 or 1 ; and
u is from 1 to 20, with the proviso that when u is from 2 to 20. A is present and when u is 1, A can be present or absent, 44. A Ligand-Linker Conjugate of claim 41,wherein L is an antibody (Ab) having the formula:
Figure imgf000152_0001
or a pharmaceutically acceptable salt thereof wherein L is a Ligand uni t;
L° is the optional secondary linker assembly that is present;
RG is a reactive site at the terminus of L° which is suitable for attaching a Drug unit;
L1 1 is the self-stabilizing linker assembly, wherein
M1 is a non-hydrolyzed succinimide or hvdrolyzed succinimide or together with B forms a dilactam:
V, Q, T, and G are independently selected from. -<C(Ry)( R10))-;
R* is H or C aikyl;
R9 and Rk' are, in each occurrence, independently selected from H or C1-3 alkyi;
F is C(E'')( E2) wherein E1 and Ei are independently selected from hydrogen, an electron withdrawing group, or E1 and E2 together are (=0);
RS is a reactive site for conjugation to a component of the optional secondary linker assembly;
g is 0 to 5;
m is () to 5;
n is 0 to 5;
d is 0 or 1 ;
x is 0 to 4, provided that when m is 0, x is 1 to 4;
and B is a base, provided that when d is zero or E' and E" are hydrogen, RS acts as an electron withdrawing group. 46. A Ligand-Functional Agent Conjugate of any of one of claims 1-3Θ, or a Ligancl-Linker Conjugate of any one of claims 41-45, wherein the Ligand unit is a monoclonal antibody. 47. A Ligand-Functional Agent Conjugate of any of one of claims 6-3 ), or a Ligand-Linker Conjugate of any one of claims 41-45, wherein p is about 4 to about 8. 48. A Ligand-Functional Agent Conjugate of any of one of claims 6-30, or a . Drug-Linker Conjugate of claims 31 or 32, or a Functional Agent-Linker Conjugate of claims 33 or 34, or a Drug-Linker of claim 35, or a Linker of any one of claims 36-39, or Ligand-Linker Conjugate of any one of claims 41-45, wherein the Basic unit is selected from the group consisting of -(CI )XN¾, -(CH2 )xNHRa, and (CI 1., )xNRa 2, wherein x is an integer of from 0-4 and each 8 is independently selected from the group consisting of C1-6 alkyl and haloalky!, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrrolidinyl or piperidinyl group, provided that there are no less than 2 intervening atoms between the base of the Basic unit and the nitrogen atom of M1.
49. A Ligand-Functional Agent Conjugate of any of one of claims 6-30, or a Drug-Linker Conjugate of claims 31 or 32, or a Functional Agent-Linker Conjugate of claims 33 or 34, or a Drug-Linker of claim 35, or a Linker of any one of claims 36-39, or Ligand-Linker Conjugate of any one of claims 41-45, wherein the Basic unit, comprises:
Figure imgf000154_0001
Figure imgf000154_0002
JJHR' R¾R - NH R7HN . /,NR8
N'""' "'NHR8
Figure imgf000154_0003
wherein R5, R6, R'' and R8 are, at each occurrence, independently selected from the group consisting of hydrogen and Ci_6 alkyl, and e is 0-4 and the wavy line indicates point of attachment to a group tethering the Basic unit to the Conjugate, Drug-Linker, or Linker.
50 A Ligand-Functionai Agent Conjugate of any of one of claims 6-30, or a Drug-Linker Conjugate of claims 31 or 32, or a Functional Agent-Linker Conjugate of claims 33 or 34, or a Drug-Linker of claim 35. or a Linker of any one of claims 36-39, or Ligand-Linker Conjugate of any one of claims 41-45, wherein there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydro lyzed or non-hydro lyzed) or diJactam and there are no more than 5 intervening atoms between the electron withdrawing group and the nitrogen atom of the succinimide ring
(hvdrolvzed or non-hydrolyzed) or ilactam. 51. A Ligand-Functionai A gent Conjugate of any of one of claims 6-30, or a Drug-Linker Conjugate of claims 31 or 32, or a Functional Agent-Linker Conjugate of claims 33 or 34, or a Drug-Linker of claim 35, or a Linker of any one of claims 36-39, or Ligand-Linker Conjugate of any one of claims 41-45, wherein there are no less than 2 and no more than 6 intervening atoms between the base of the Basic unit and the nitrogen atom of the succinimide (hydroiyzed or non-hydrolyzed) or dilactam and there are no more than 3 intervening atoms between the electron withdrawing group and the nitrogen atom of the succinimide ring
(hydroiyzed or non-hydrolyzed) or dilactam. -Drug Conjugate of claim 20, having the formula:
Figure imgf000155_0001
or a pharmaceutically acceptable salt thereof wherein Bu is selected from the group consisting of -(CH2 )XN¾, -~(CH2 )xNHRa, and ~(CH2 )xNRa ?, wherein x is an integer of from 1 -4 and each Ra is independently selected from the group consisting of C1-6 alkyl and Cj_6 haloaikyl, or two Ra groups are combined with the nitrogen to which they are attached to form an azetidinyl, pyrro!idinyl or piperidinyl group.
53. A Ligand-Drug Conjugate of claim 52, wherein x is 1 , 2, 3, or 4 and Ra is C,_6 alkyl. 54. A Ligand-Drug Conjugate of claim 53, wherein Lu is <
wherein
-A- is an optional Stretcher unit, the subscript a' is 0 or 1 ;
~W~ is ail optional Cleavable unit, the subscript w' is 1 ; and
-Y- is an optional Spacer unit, and the subscript y' is 0 or 1. 55. A Ligand-Drug Conjugate of claim 53, wherein p is about 4 or about 8. 56. A Ligand-Drug Conjugate of claim 55, wherein D is an aurisvatin. 57. A Ligand-Drug Conjugate of claim 56, wherein Ab is a monoclonal antibody. 58. A method of treating cancer, an autoimmune disease or an infectious disease that expresses a target antigen comprising administering a Ligand-Functional Agent Conjugate of any one of claims 1 to 30, wherein the Ligand is a monoclonal antibody that specifically binds to the target antigen. 59. The Ligand-Functional Agent Conjugate of any one of claims 1 to 30, wherein the self-stabilizing linker assembly comprises a succinimide ring or a hydrolyzed succinimide directly conjugated to the Ligand unit. 60. The Ligand-Functional Agent Conjugate of claim 59, wherein the self- stabilizing linker assembly comprises a succinimide ring and wherein the electron withdrawing group is positioned to increase the electrophiiicity of the succinimide ring rendering it more reactive with water and the base is positioned to assist the hydrolysis of the succinimide ring. 61. The Ligand-Functional Agent Conjugate of claim 59, wherein the self- stabilizing linker assembly comprises a hydrolyzed succinimide ring and wherein the electron withdrawing group and base are positioned such that they increased the rate of succinimide ring hydrolysis as compared to succinimide ring hydrolysis in a ligand drug conjugate lacking the self-stabilizing linker assembly, 62. The Ligand-Functional Agent Conjugate of claim 59, wherein the self- stabilizing linkers is conjugated to thiol residues on the Ligand at sites that are susceptible to elimination reaction and subsequent transfer of drug- linker if non-self- stabilizing aikyl maleimides are used as the drug- linker.
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Cited By (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9144615B2 (en) 2011-06-10 2015-09-29 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US9254339B2 (en) 2011-06-10 2016-02-09 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
WO2016040684A1 (en) 2014-09-11 2016-03-17 Seattle Genetics, Inc Targeted delivery of tertiary amine-containing drug substances
WO2016054315A1 (en) 2014-10-01 2016-04-07 Medimmune, Llc Method of conjugating a polypeptide
US20160310612A1 (en) * 2013-10-15 2016-10-27 Seattle Genetics, Inc. Pegylated drug-linkers for improved ligand-drug conjugate pharmacokinetics
WO2016192528A1 (en) 2015-05-29 2016-12-08 Newbio Therapeutics, Inc. Trimaleimide Linkers and Uses Thereof
JP2017531027A (en) * 2014-07-16 2017-10-19 メドシャイン ディスカバリー インク Linker and its application to ADC
WO2017214301A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
WO2017214282A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
WO2017214339A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
WO2017214335A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
WO2017214233A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
WO2017214458A2 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
WO2017214456A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
WO2017214462A2 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
US9849191B2 (en) 2013-10-11 2017-12-26 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
WO2018031690A1 (en) 2016-08-09 2018-02-15 Seattle Genetics, Inc. Drug conjugates with self-stabilizing linkers having improved physiochemical properties
JP2018507168A (en) * 2014-12-03 2018-03-15 ジェネンテック, インコーポレイテッド Quaternary amine compounds and antibody-drug conjugates thereof
WO2018075692A2 (en) 2016-10-19 2018-04-26 Invenra Inc. Antibody constructs
EP3210623A4 (en) * 2014-10-24 2018-07-04 NOF Corporation Antibody-drug complex having cyclic benzylidene acetal linker
EP3270965A4 (en) * 2015-03-18 2018-09-05 Seattle Genetics, Inc. Cd48 antibodies and conjugates thereof
WO2019033773A1 (en) 2017-08-14 2019-02-21 Newbio Therapeutics, Inc. Tetramaleimide linkers and use thereof
US10316080B2 (en) 2013-10-11 2019-06-11 Asana Biosciences, Llc Protein-polymer-drug conjugates
WO2019236954A1 (en) 2018-06-07 2019-12-12 Seattle Genetics, Inc. Camptothecin conjugates
WO2019234136A1 (en) 2018-06-05 2019-12-12 King's College London Btnl3/8 targeting constructs for delivery of payloads to the gastrointestinal system
US10689458B2 (en) 2015-11-30 2020-06-23 Pfizer Inc. Site specific HER2 antibody drug conjugates
CN111533778A (en) * 2015-07-28 2020-08-14 上海皓元生物医药科技有限公司 Industrial production method of antibody-conjugated drug linker
JP2020128378A (en) * 2014-12-09 2020-08-27 アッヴィ・インコーポレイテッド Bcl-xl inhibitory compounds and antibody drug conjugates including the same
JP2020143062A (en) * 2014-12-09 2020-09-10 アッヴィ・インコーポレイテッド Bcl-xL INHIBITORY COMPOUNDS HAVING LOW CELL PERMEABILITY AND ANTIBODY DRUG CONJUGATES INCLUDING THE SAME
WO2021067861A1 (en) 2019-10-04 2021-04-08 Seagen Inc. Camptothecin peptide conjugates
US11091498B2 (en) 2016-04-04 2021-08-17 Rutgers, The State University Of New Jersey Topoisomerase poisons
US11116847B2 (en) 2013-12-19 2021-09-14 Seagen Inc. Methylene carbamate linkers for use with targeted-drug conjugates
US11147852B2 (en) 2011-12-23 2021-10-19 Pfizer Inc. Engineered antibody constant regions for site-specific conjugation and methods and uses therefor
WO2021226454A1 (en) * 2020-05-08 2021-11-11 Mpeg La, L.L.C. Linker compounds
US11174318B2 (en) 2016-12-22 2021-11-16 Università Degli Studi Magna Graecia Catanzaro Monoclonal antibody targeting a unique sialoglycosylated cancer-associated epitope of CD43
US11229708B2 (en) 2015-12-04 2022-01-25 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11364303B2 (en) 2017-09-29 2022-06-21 Pfizer Inc. Cysteine engineered antibody drug conjugates
US11389543B2 (en) 2017-09-08 2022-07-19 Seagen Inc. Process for the preparation of tubulysins and intermediates thereof
US11541128B2 (en) 2016-12-14 2023-01-03 Seagen Inc. Multi-drug antibody drug conjugates
WO2023280227A2 (en) 2021-07-06 2023-01-12 Profoundbio Us Co. Linkers, drug linkers and conjugates thereof and methods of using the same
WO2023033129A1 (en) 2021-09-03 2023-03-09 東レ株式会社 Pharmaceutical composition for treating and/or preventing cancer
EP3991754A4 (en) * 2019-06-28 2023-05-17 Shanghai Fudan-Zhangjiang Bio-Pharmaceutical Co., Ltd. Antibody-drug conjugate, intermediate thereof, preparation method therefor and application thereof
RU2800137C1 (en) * 2019-06-28 2023-07-19 Шанхай Фудань-Чжанцзян Био-Фармасьютикал Ко., Лтд. Antibody-drug conjugate, intermediate for its production, method of its production and its use
US11730822B2 (en) 2017-03-24 2023-08-22 Seagen Inc. Process for the preparation of glucuronide drug-linkers and intermediates thereof
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates
WO2023178289A2 (en) 2022-03-17 2023-09-21 Seagen Inc. Camptothecin conjugates
US11793880B2 (en) 2015-12-04 2023-10-24 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11806405B1 (en) 2021-07-19 2023-11-07 Zeno Management, Inc. Immunoconjugates and methods
US11844839B2 (en) 2016-03-25 2023-12-19 Seagen Inc. Process for the preparation of pegylated drug-linkers and intermediates thereof
EP4321522A1 (en) 2022-08-12 2024-02-14 Seagen Inc. Cytotoxic compounds and conjugates thereof
WO2024092219A1 (en) * 2022-10-28 2024-05-02 Eli Lilly And Company Self-hydrolyzing maleimides for bioconjugation
WO2024108053A1 (en) 2022-11-17 2024-05-23 Sanofi Ceacam5 antibody-drug conjugates and methods of use thereof
WO2024129756A1 (en) 2022-12-13 2024-06-20 Seagen Inc. Site-specific engineered cysteine antibody drug conjugates
US12036286B2 (en) 2021-03-18 2024-07-16 Seagen Inc. Selective drug release from internalized conjugates of biologically active compounds
WO2024165045A1 (en) * 2023-02-09 2024-08-15 Beigene, Ltd. Self-stabilizing linker conjugates

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA037203B1 (en) * 2012-05-15 2021-02-18 Сиэтл Джинетикс, Инк. Antibody-drug conjugates with self-stabilizing linkers
CN109069665A (en) * 2016-05-10 2018-12-21 百时美施贵宝公司 The antibody-drug conjugates of the tubulysin analog of stability with enhancing
SG11202106122QA (en) * 2018-12-21 2021-07-29 Seagen Inc Adcs with thiol multiplex linkers
CN114306633A (en) * 2020-09-29 2022-04-12 迈威(上海)生物科技股份有限公司 Preparation method of disubstituted bridging antibody coupling drug

Citations (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0012023A1 (en) 1978-12-05 1980-06-11 Claude Peter Windsor-Smith Change speed gear
EP0171496A2 (en) 1984-08-15 1986-02-19 Research Development Corporation of Japan Process for the production of a chimera monoclonal antibody
EP0173494A2 (en) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by DNA splicing and expression
WO1986001533A1 (en) 1984-09-03 1986-03-13 Celltech Limited Production of chimeric antibodies
EP0184187A2 (en) 1984-12-04 1986-06-11 Teijin Limited Mouse-human chimaeric immunoglobulin heavy chain, and chimaeric DNA encoding it
WO1987002671A1 (en) 1985-11-01 1987-05-07 International Genetic Engineering, Inc. Modular assembly of antibody genes, antibodies prepared thereby and use
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4880935A (en) 1986-07-11 1989-11-14 Icrf (Patents) Limited Heterobifunctional linking agents derived from N-succinimido-dithio-alpha methyl-methylene-benzoates
EP0446071A2 (en) 1990-03-09 1991-09-11 Hybritech Incorporated Tris-maleimido compounds as intermediates in trifunctional antibody synthesis
US5122368A (en) 1988-02-11 1992-06-16 Bristol-Myers Squibb Company Anthracycline conjugates having a novel linker and methods for their production
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
US5622929A (en) 1992-01-23 1997-04-22 Bristol-Myers Squibb Company Thioether conjugates
WO1997034631A1 (en) 1996-03-18 1997-09-25 Board Of Regents, The University Of Texas System Immunoglobin-like domains with increased half lives
US5824805A (en) 1995-12-22 1998-10-20 King; Dalton Branched hydrazone linkers
US5973166A (en) 1998-03-02 1999-10-26 The Dexter Corporation Method for the preparation of maleimides
US6130237A (en) 1996-09-12 2000-10-10 Cancer Research Campaign Technology Limited Condensed N-aclyindoles as antitumor agents
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
WO2002088172A2 (en) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
US20030083263A1 (en) 2001-04-30 2003-05-01 Svetlana Doronina Pentapeptide compounds and uses related thereto
WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US20050238649A1 (en) 2003-11-06 2005-10-27 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2005112919A2 (en) 2004-05-19 2005-12-01 Medarex, Inc. Self-immolative linkers and drug conjugates
US20060024317A1 (en) 2004-05-19 2006-02-02 Medarex, Inc Chemical linkers and conjugates thereof
WO2007038658A2 (en) 2005-09-26 2007-04-05 Medarex, Inc. Antibody-drug conjugates and methods of use
WO2007085930A1 (en) 2006-01-25 2007-08-02 Sanofi-Aventis Cytotoxic agents comprising new tomaymycin derivatives and their therapeutic use
US20090018086A1 (en) 2005-07-07 2009-01-15 Seattle Genetics, Inc. Monomethylvaline Compounds Having Phenylalanine Side-Chain Replacements at the C-Terminus
US20090111756A1 (en) 2005-07-07 2009-04-30 Seattle Genectics, Inc. Monomethylvaline Compounds Having Phenylalanine Carboxy Modifications at the C-Terminus
US20090274713A1 (en) 2008-04-30 2009-11-05 Immunogen Inc. Cross-linkers and their uses
WO2010091150A1 (en) 2009-02-05 2010-08-12 Immunogen, Inc. Novel benzodiazepine derivatives
US20110020343A1 (en) 2008-03-18 2011-01-27 Seattle Genetics, Inc. Auristatin drug linker conjugates
WO2011023883A1 (en) 2009-08-25 2011-03-03 Sanofi-Aventis Conjugates of pyrrolo[1,4]benzodiazepine dimers as anticancer agents
US7968687B2 (en) 2007-10-19 2011-06-28 Seattle Genetics, Inc. CD19 binding agents and uses thereof
US20110256157A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
US8142784B2 (en) 2004-06-01 2012-03-27 Genentech, Inc. Antibody-drug conjugates and methods
US8163888B2 (en) 2003-10-10 2012-04-24 Immunogen, Inc. Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates
WO2012112708A1 (en) 2011-02-15 2012-08-23 Immunogen, Inc. Cytotoxic benzodiazepine derivatives and methods of preparation

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH07508550A (en) * 1992-07-01 1995-09-21 アクゾ ノーベル ナムローゼ フェンノートシャップ Method of modifying polyphenylene ether resin composition
US8877901B2 (en) * 2002-12-13 2014-11-04 Immunomedics, Inc. Camptothecin-binding moiety conjugates
US7432331B2 (en) * 2002-12-31 2008-10-07 Nektar Therapeutics Al, Corporation Hydrolytically stable maleimide-terminated polymers
US20110288011A1 (en) * 2008-12-05 2011-11-24 Jean-Paul Castaigne Peptide therapeutic conjugates and uses thereof
PL3903829T3 (en) * 2009-02-13 2023-08-14 Immunomedics, Inc. Immunoconjugates with an intracellularly-cleavable linkage
EP2629801B3 (en) * 2010-10-22 2019-11-27 Seattle Genetics, Inc. Synergistic effects between auristatin-based antibody drug conjugates and inhibitors of the pi3k-akt mtor pathway
EA037203B1 (en) 2012-05-15 2021-02-18 Сиэтл Джинетикс, Инк. Antibody-drug conjugates with self-stabilizing linkers
EP2968591A1 (en) * 2013-03-15 2016-01-20 Novartis AG Cell proliferation inhibitors and conjugates thereof

Patent Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0012023A1 (en) 1978-12-05 1980-06-11 Claude Peter Windsor-Smith Change speed gear
US4816397A (en) 1983-03-25 1989-03-28 Celltech, Limited Multichain polypeptides or proteins and processes for their production
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
EP0171496A2 (en) 1984-08-15 1986-02-19 Research Development Corporation of Japan Process for the production of a chimera monoclonal antibody
EP0173494A2 (en) 1984-08-27 1986-03-05 The Board Of Trustees Of The Leland Stanford Junior University Chimeric receptors by DNA splicing and expression
WO1986001533A1 (en) 1984-09-03 1986-03-13 Celltech Limited Production of chimeric antibodies
EP0184187A2 (en) 1984-12-04 1986-06-11 Teijin Limited Mouse-human chimaeric immunoglobulin heavy chain, and chimaeric DNA encoding it
WO1987002671A1 (en) 1985-11-01 1987-05-07 International Genetic Engineering, Inc. Modular assembly of antibody genes, antibodies prepared thereby and use
US5225539A (en) 1986-03-27 1993-07-06 Medical Research Council Recombinant altered antibodies and methods of making altered antibodies
US4880935A (en) 1986-07-11 1989-11-14 Icrf (Patents) Limited Heterobifunctional linking agents derived from N-succinimido-dithio-alpha methyl-methylene-benzoates
US5122368A (en) 1988-02-11 1992-06-16 Bristol-Myers Squibb Company Anthracycline conjugates having a novel linker and methods for their production
US5585089A (en) 1988-12-28 1996-12-17 Protein Design Labs, Inc. Humanized immunoglobulins
EP0446071A2 (en) 1990-03-09 1991-09-11 Hybritech Incorporated Tris-maleimido compounds as intermediates in trifunctional antibody synthesis
US5622929A (en) 1992-01-23 1997-04-22 Bristol-Myers Squibb Company Thioether conjugates
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US5824805A (en) 1995-12-22 1998-10-20 King; Dalton Branched hydrazone linkers
WO1997034631A1 (en) 1996-03-18 1997-09-25 Board Of Regents, The University Of Texas System Immunoglobin-like domains with increased half lives
US6130237A (en) 1996-09-12 2000-10-10 Cancer Research Campaign Technology Limited Condensed N-aclyindoles as antitumor agents
US5973166A (en) 1998-03-02 1999-10-26 The Dexter Corporation Method for the preparation of maleimides
US20030083263A1 (en) 2001-04-30 2003-05-01 Svetlana Doronina Pentapeptide compounds and uses related thereto
WO2002088172A2 (en) 2001-04-30 2002-11-07 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
US20050009751A1 (en) 2001-04-30 2005-01-13 Seattle Genetics, Inc. Pentapeptide compounds and uses related thereto
WO2004010957A2 (en) 2002-07-31 2004-02-05 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US7659241B2 (en) 2002-07-31 2010-02-09 Seattle Genetics, Inc. Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease
US8163888B2 (en) 2003-10-10 2012-04-24 Immunogen, Inc. Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates and methods of making said conjugates
US20050238649A1 (en) 2003-11-06 2005-10-27 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
US7498298B2 (en) 2003-11-06 2009-03-03 Seattle Genetics, Inc. Monomethylvaline compounds capable of conjugation to ligands
WO2005112919A2 (en) 2004-05-19 2005-12-01 Medarex, Inc. Self-immolative linkers and drug conjugates
US20060024317A1 (en) 2004-05-19 2006-02-02 Medarex, Inc Chemical linkers and conjugates thereof
US8142784B2 (en) 2004-06-01 2012-03-27 Genentech, Inc. Antibody-drug conjugates and methods
US20090111756A1 (en) 2005-07-07 2009-04-30 Seattle Genectics, Inc. Monomethylvaline Compounds Having Phenylalanine Carboxy Modifications at the C-Terminus
US20090018086A1 (en) 2005-07-07 2009-01-15 Seattle Genetics, Inc. Monomethylvaline Compounds Having Phenylalanine Side-Chain Replacements at the C-Terminus
US8343928B2 (en) 2005-07-07 2013-01-01 Seattle Genetics, Inc. Monomethylvaline compounds having phenylalanine side-chain replacements at the C-terminus
WO2007038658A2 (en) 2005-09-26 2007-04-05 Medarex, Inc. Antibody-drug conjugates and methods of use
WO2007085930A1 (en) 2006-01-25 2007-08-02 Sanofi-Aventis Cytotoxic agents comprising new tomaymycin derivatives and their therapeutic use
US7968687B2 (en) 2007-10-19 2011-06-28 Seattle Genetics, Inc. CD19 binding agents and uses thereof
US20110020343A1 (en) 2008-03-18 2011-01-27 Seattle Genetics, Inc. Auristatin drug linker conjugates
US20090274713A1 (en) 2008-04-30 2009-11-05 Immunogen Inc. Cross-linkers and their uses
WO2010091150A1 (en) 2009-02-05 2010-08-12 Immunogen, Inc. Novel benzodiazepine derivatives
WO2011023883A1 (en) 2009-08-25 2011-03-03 Sanofi-Aventis Conjugates of pyrrolo[1,4]benzodiazepine dimers as anticancer agents
US20110256157A1 (en) 2010-04-15 2011-10-20 Spirogen Limited Pyrrolobenzodiazepines and conjugates thereof
WO2012112708A1 (en) 2011-02-15 2012-08-23 Immunogen, Inc. Cytotoxic benzodiazepine derivatives and methods of preparation

Non-Patent Citations (35)

* Cited by examiner, † Cited by third party
Title
ALLEY ET AL., BIOCONJUGATE CHEM, vol. 19, 2008, pages 759 - 765
AMSBERRY ET AL., J. ORG. CHEM., vol. 55, 1990, pages 5867
BEIDLER ET AL., J. IMMUNOL., vol. 141, 1988, pages 4053 - 4060
CHARI ET AL., CANCER RES., vol. 52, 1992, pages 127 - 131
DUBOWCHIKWALKER, PHARM. THERAPEUTICS, vol. 83, 1999, pages 67 - 123
H. N. BORAH ET AL., J. CHEM. RESEARCH (S, 1998, pages 272 - 272
HAY ET AL., BIOORG. MED. CHEM. LETT., vol. 9, 1999, pages 2237
JONES ET AL., NATURE, vol. 321, 1986, pages 552 - 525
KABAT E ET AL., J. IMMUNOLOGY, vol. 125, no. 3, 1980, pages 961 - 969
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTE OF HEALTH
KINGSBURY ET AL., J. MED. CHEM., vol. 27, 1984, pages 1447
KOZBOR ET AL., IMMUNOLOGY TODAY, vol. 4, 1983, pages 72 - 79
LIU ET AL., J. IMMUNOL., vol. 139, 1987, pages 3521 - 3526
M.A. WALKER, TETRAHEDRON LETTERS, vol. 35, no. 5, 1994, pages 665 - 668
MORRISON, SCIENCE, vol. 229, 1985, pages 1202 - 1207
NEVILLE ET AL., BIOL. CHEM., vol. 264, 1989, pages 14653 - 14661
NISHIMURA ET AL., CANCER. RES., vol. 47, 1987, pages 999 - 1005
OI ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 214
OLSSON ET AL., METH. ENZYMOL., vol. 92, 1982, pages 3 - 16
PAGE ET AL., INTL. J. OF ONCOLOGY, vol. 3, 1993, pages 473 - 476
RODRIGUES ET AL., CHEMISTRY BIOLOGY, vol. 2, 1995, pages 223
RYAN ET AL., CHEM. COMMUN., vol. 47, 2011, pages 5452 - 5454
SHAW ET AL., J. NATL. CANCER INST., vol. 80, 1988, pages 1553 - 1559
SHEN ET AL., NATURE BIOTECH, vol. 30, no. 2, 2012, pages 184 - 9
SKEHAN ET AL., J. NAT'L CANCER INST., vol. 82, 1990, pages 1107 - 12
SMITH ET AL., J. AM. CHEM. SOC., vol. 132, no. 6, 2010, pages 1960 - 1965
STEPHEN C. ALLEY ET AL., BIOCONJUGATE CHEMISTRY, vol. 19, no. 3, 1 March 2008 (2008-03-01), pages 759 - 765
STORM ET AL., J. AMER. CHEM. SOC., vol. 94, 1972, pages 5815
SUN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 3439 - 3443
TENG ET AL., PROC. NATL. ACAD. SCI. USA., vol. 80, 1983, pages 7308 - 7312
THORPE ET AL., CANCER RES., vol. 47, 1987, pages 5924 - 5931
V. ONDRUS, ARKIVOC, vol. 60-67, 2001
VERHOEYAN ET AL., SCIENCE, vol. 239, 1988, pages 1534 - 1043
WAWRZYNCZAK ET AL.: "Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer", 1987, OXFORD U. PRESS
WOOD ET AL., NATURE, vol. 314, 1985, pages 446 - 449

Cited By (85)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9770519B2 (en) 2011-06-10 2017-09-26 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US9254339B2 (en) 2011-06-10 2016-02-09 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US10603386B2 (en) 2011-06-10 2020-03-31 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US10537645B2 (en) 2011-06-10 2020-01-21 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US9943609B2 (en) 2011-06-10 2018-04-17 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US9144615B2 (en) 2011-06-10 2015-09-29 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US11147852B2 (en) 2011-12-23 2021-10-19 Pfizer Inc. Engineered antibody constant regions for site-specific conjugation and methods and uses therefor
US10316080B2 (en) 2013-10-11 2019-06-11 Asana Biosciences, Llc Protein-polymer-drug conjugates
US11434278B2 (en) 2013-10-11 2022-09-06 Asana Biosciences, Llc Protein-polymer-drug conjugates
US9849191B2 (en) 2013-10-11 2017-12-26 Mersana Therapeutics, Inc. Protein-polymer-drug conjugates
US11103593B2 (en) 2013-10-15 2021-08-31 Seagen Inc. Pegylated drug-linkers for improved ligand-drug conjugate pharmacokinetics
EP3057585B1 (en) 2013-10-15 2020-07-22 Seattle Genetics, Inc. Pegylated drug-linkers for improved ligand-drug conjugate pharmacokinetics
US20160310612A1 (en) * 2013-10-15 2016-10-27 Seattle Genetics, Inc. Pegylated drug-linkers for improved ligand-drug conjugate pharmacokinetics
US11116847B2 (en) 2013-12-19 2021-09-14 Seagen Inc. Methylene carbamate linkers for use with targeted-drug conjugates
JP2017531027A (en) * 2014-07-16 2017-10-19 メドシャイン ディスカバリー インク Linker and its application to ADC
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JP7042291B2 (en) 2014-07-16 2022-03-25 メドシャイン ディスカバリー インク Its application to linkers and ADCs
TWI701049B (en) * 2014-09-11 2020-08-11 美商西雅圖遺傳學公司 Targeted delivery of tertiary amine-containing drug substances
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CN114732916A (en) * 2014-09-11 2022-07-12 西雅图基因公司 Targeted delivery of tertiary amine-containing drug substances
EP3191502A4 (en) * 2014-09-11 2018-04-25 Seattle Genetics, Inc. Targeted delivery of tertiary amine-containing drug substances
TWI794617B (en) * 2014-09-11 2023-03-01 美商西雅圖遺傳學公司 Targeted delivery of tertiary amine-containing drug substances
WO2016040684A1 (en) 2014-09-11 2016-03-17 Seattle Genetics, Inc Targeted delivery of tertiary amine-containing drug substances
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EP3900742A1 (en) * 2014-09-11 2021-10-27 Seagen Inc. Targeted delivery of tertiary amine-containing drug substances
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WO2016054315A1 (en) 2014-10-01 2016-04-07 Medimmune, Llc Method of conjugating a polypeptide
US10377837B2 (en) 2014-10-24 2019-08-13 Nof Corporation Antibody-drug conjugate having cyclic benzylidene acetal linker
US10808050B2 (en) 2014-10-24 2020-10-20 Nof Corporation Antibody-drug conjugate having cyclic benzylidene acetal linker
EP3210623A4 (en) * 2014-10-24 2018-07-04 NOF Corporation Antibody-drug complex having cyclic benzylidene acetal linker
JP2018507168A (en) * 2014-12-03 2018-03-15 ジェネンテック, インコーポレイテッド Quaternary amine compounds and antibody-drug conjugates thereof
JP2020143062A (en) * 2014-12-09 2020-09-10 アッヴィ・インコーポレイテッド Bcl-xL INHIBITORY COMPOUNDS HAVING LOW CELL PERMEABILITY AND ANTIBODY DRUG CONJUGATES INCLUDING THE SAME
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US10722592B2 (en) 2015-03-18 2020-07-28 Seattle Genetics, Inc. CD48 antibodies and conjugates thereof
EP3270965A4 (en) * 2015-03-18 2018-09-05 Seattle Genetics, Inc. Cd48 antibodies and conjugates thereof
WO2016192528A1 (en) 2015-05-29 2016-12-08 Newbio Therapeutics, Inc. Trimaleimide Linkers and Uses Thereof
CN111533778B (en) * 2015-07-28 2023-04-25 上海皓元生物医药科技有限公司 Industrial production method of antibody-coupled drug connector
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US10689458B2 (en) 2015-11-30 2020-06-23 Pfizer Inc. Site specific HER2 antibody drug conjugates
US11229708B2 (en) 2015-12-04 2022-01-25 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11793880B2 (en) 2015-12-04 2023-10-24 Seagen Inc. Conjugates of quaternized tubulysin compounds
US11844839B2 (en) 2016-03-25 2023-12-19 Seagen Inc. Process for the preparation of pegylated drug-linkers and intermediates thereof
US11091498B2 (en) 2016-04-04 2021-08-17 Rutgers, The State University Of New Jersey Topoisomerase poisons
WO2017214335A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
WO2017214301A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
WO2017214282A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
EP3888689A1 (en) 2016-06-08 2021-10-06 AbbVie Inc. Anti-egfr antibody drug conjugates
WO2017214462A2 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
WO2017214456A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
EP4104865A1 (en) 2016-06-08 2022-12-21 AbbVie Inc. Anti-egfr antibody drug conjugates
WO2017214339A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
EP4364754A2 (en) 2016-06-08 2024-05-08 AbbVie Inc. Anti-b7-h3 antibodies and antibody drug conjugates
WO2017214458A2 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-cd98 antibodies and antibody drug conjugates
US10640563B2 (en) 2016-06-08 2020-05-05 Abbvie Inc. Anti-B7-H3 antibodies and antibody drug conjugates
WO2017214233A1 (en) 2016-06-08 2017-12-14 Abbvie Inc. Anti-egfr antibody drug conjugates
AU2017310436B2 (en) * 2016-08-09 2022-08-25 Seagen Inc. Drug conjugates with self-stabilizing linkers having improved physiochemical properties
WO2018031690A1 (en) 2016-08-09 2018-02-15 Seattle Genetics, Inc. Drug conjugates with self-stabilizing linkers having improved physiochemical properties
EP3496747A4 (en) * 2016-08-09 2020-04-15 Seattle Genetics, Inc. Drug conjugates with self-stabilizing linkers having improved physiochemical properties
US11944689B2 (en) 2016-08-09 2024-04-02 Seagen Inc. Drug conjugates with self-stabilizing linkers having improved physiochemical properties
WO2018075692A2 (en) 2016-10-19 2018-04-26 Invenra Inc. Antibody constructs
US11541128B2 (en) 2016-12-14 2023-01-03 Seagen Inc. Multi-drug antibody drug conjugates
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US11730822B2 (en) 2017-03-24 2023-08-22 Seagen Inc. Process for the preparation of glucuronide drug-linkers and intermediates thereof
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US11389543B2 (en) 2017-09-08 2022-07-19 Seagen Inc. Process for the preparation of tubulysins and intermediates thereof
US11364303B2 (en) 2017-09-29 2022-06-21 Pfizer Inc. Cysteine engineered antibody drug conjugates
WO2019234136A1 (en) 2018-06-05 2019-12-12 King's College London Btnl3/8 targeting constructs for delivery of payloads to the gastrointestinal system
WO2019236954A1 (en) 2018-06-07 2019-12-12 Seattle Genetics, Inc. Camptothecin conjugates
RU2800137C1 (en) * 2019-06-28 2023-07-19 Шанхай Фудань-Чжанцзян Био-Фармасьютикал Ко., Лтд. Antibody-drug conjugate, intermediate for its production, method of its production and its use
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WO2021067861A1 (en) 2019-10-04 2021-04-08 Seagen Inc. Camptothecin peptide conjugates
WO2021226454A1 (en) * 2020-05-08 2021-11-11 Mpeg La, L.L.C. Linker compounds
US11759527B2 (en) 2021-01-20 2023-09-19 Abbvie Inc. Anti-EGFR antibody-drug conjugates
US12036286B2 (en) 2021-03-18 2024-07-16 Seagen Inc. Selective drug release from internalized conjugates of biologically active compounds
WO2023280227A2 (en) 2021-07-06 2023-01-12 Profoundbio Us Co. Linkers, drug linkers and conjugates thereof and methods of using the same
US11806405B1 (en) 2021-07-19 2023-11-07 Zeno Management, Inc. Immunoconjugates and methods
WO2023033129A1 (en) 2021-09-03 2023-03-09 東レ株式会社 Pharmaceutical composition for treating and/or preventing cancer
WO2023178289A2 (en) 2022-03-17 2023-09-21 Seagen Inc. Camptothecin conjugates
EP4321522A1 (en) 2022-08-12 2024-02-14 Seagen Inc. Cytotoxic compounds and conjugates thereof
WO2024092219A1 (en) * 2022-10-28 2024-05-02 Eli Lilly And Company Self-hydrolyzing maleimides for bioconjugation
WO2024108053A1 (en) 2022-11-17 2024-05-23 Sanofi Ceacam5 antibody-drug conjugates and methods of use thereof
WO2024129756A1 (en) 2022-12-13 2024-06-20 Seagen Inc. Site-specific engineered cysteine antibody drug conjugates
WO2024165045A1 (en) * 2023-02-09 2024-08-15 Beigene, Ltd. Self-stabilizing linker conjugates

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