US20200407440A1 - Amatoxin antibody-drug conjugates and uses thereof - Google Patents

Amatoxin antibody-drug conjugates and uses thereof Download PDF

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US20200407440A1
US20200407440A1 US16/858,509 US202016858509A US2020407440A1 US 20200407440 A1 US20200407440 A1 US 20200407440A1 US 202016858509 A US202016858509 A US 202016858509A US 2020407440 A1 US2020407440 A1 US 2020407440A1
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antibody
seq
amino acid
variable region
acid sequence
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Charlotte Fenton McDonagh
Rajiv Panwar
Ganapathy N. SARMA
Torsten HECHLER
Michael Kulke
Andreas Pahl
Christoph Mueller
Werner Simon
Christian Lutz
Francesca Gallo
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Dianthus Therapeutics Inc
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Magenta Therapeutics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6831—Fungal toxins, e.g. alpha sarcine, mitogillin, zinniol or restrictocin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to amatoxins, Antibody-Drug Conjugates (ADCs) comprising an amatoxin, compositions comprising such ADCs, and methods for using the same.
  • ADCs Antibody-Drug Conjugates
  • Monoclonal antibodies can be conjugated to a therapeutic agent to form an antibody drug conjugate (ADC).
  • ADCs can exhibit increased efficacy, as compared to an unconjugated antibody.
  • the linkage of the antibody to the drug can be direct, or indirect via a linker.
  • An important aspect of successful therapeutic ADCs is that the ADC be not only effective but well-tolerated. Often the cytotoxin impacts both efficacy and tolerability.
  • ADCs have been proposed as a therapeutic regimen for preparing patients for transplant and stem cell therapy.
  • stem cells or immune cells can be selectively depleted while leaving the patient's remaining immune system largely intact.
  • Palchaudhuri et al. (2016) Nat. Biotechnol. 34, 738-745 describes the use of a single dose of an anti-CD45 ADC where an anti-CD45 antibody was conjugated to saporin, and its ability to enable engraftment of donor cells and for treatment in a sickle-cell anemia model.
  • the CD45-SAP ADC was reported to have avoided neutropenia and anemia, and provided for rapid recovery of T and B cells with minimal overall toxicity. There remains a need for toxins that can be used for non-genotoxic targeted ADC-conditioning where the toxin is potent to the target cells while minimizing patient side effects.
  • the present invention provides an amatoxin that can be used in an antibody drug conjugate (ADC), e.g., for delivery of the amatoxin to a target cell.
  • ADC antibody drug conjugate
  • ADC antibody-drug conjugate
  • an ADC disclosed herein has the structure of formula (Ia):
  • an ADC disclosed herein has the structure of formula (b):
  • L comprises one or more of a bond, —(C ⁇ O)—, a —C(O)NH— group, an —OC(O)NH— group, C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, a —(CH 2 CH 2 O) p — group where p is an integer from 1-6, or a solubility enhancing group; wherein each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3
  • the solubility enhancing group has the formula —O a —C(O)NH—SO 2 —NR 1 —, wherein:
  • L comprises a —(CH 2 ) n — unit, where n is an integer from 2-6, e.g., n is 6.
  • S is the sulfur atom of a cysteine residue present in the antibody, or the antigen-binding fragment thereof.
  • ADC antibody-drug conjugate
  • ADC comprising an antibody, or an antigen-binding fragment thereof, conjugated to an amatoxin via a linker, the ADC having the structure of formula (I):
  • the ADC has a structure of formula (Ia):
  • the ADC has the structure of formula (b):
  • L comprises one or more of a hydrazine, a disulfide, a thioether, an amino acid, a peptide consisting of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, heteroaryl, a —(C ⁇ O)— group, a —C(O)NH— group, an —OC(O)NH— group, a —(CH 2 CH 2 O) p — group where p is an integer from 1-6, or a solubility enhancing group; wherein each C 1 -C
  • the solubility enhancing group has the formula —O a —C(O)NH—SO 2 —NR 1 —, wherein:
  • L comprises a peptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg.
  • an ADC described herein further comprises a PAB group.
  • L is represented by the formula:
  • an ADC comprising an antibody conjugated to an amatoxin, the ADC having a structure according to formula (II):
  • the ADC has the structure of formula (IIa):
  • the ADC has the structure of formula (Ib):
  • an antibody, or antigen-binding fragment of an antibody, used in the ADCs described herein has binding regions disclosed in the Sequence Table,
  • the antibody, or the antigen binding fragment thereof comprises either an Fc region comprising at least one mutation selected from the group consisting of D265C, H435A, L234A, or L235A (according to EU index); or an Fc region comprising D265C, H435A, L234A, or L235A (according to EU index) mutations.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD45.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD137.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD5.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD2.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 or a variable region from the heavy chain variable region amino acid sequence of Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, Ab69, Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, or Ab249; and a light chain comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 or a variable region from the light chain variable region amino acid sequence of Ab55, Ab54, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, Ab69, Ab85, Ab86, Ab87, Ab88, Ab89, Ab77, Ab79, Ab81, Ab85, or Ab249;
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising an HC-CDR1, an HC-CDR2, and an HC-CDR3 or a variable region from the heavy chain variable region amino acid sequence of SEQ ID NO: 147, 164, 166, 168, 170, 172, 174, 176, 178, 180, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 238, or 243; and a light chain comprising an LC-CDR1, an LC-CDR2, and an LC-CDR3 or a variable region from the light chain variable region amino acid sequence of SEQ ID NO: 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising a heavy chain (HC)-CDR1, HC-CDR2, and HC-CDR3 comprising an amino acid sequence as set forth in SEQ ID No: 11, 12, and 13, respectively, and a light chain comprising a light chain (LC)-CDR1, LC-CDR2, and LC-CDR3 comprising an amino acid sequence as set forth in SEQ ID Nos: 14, 15, and 16, respectively.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising a heavy chain (HC)-CDR1, HC-CDR2, and HC-CDR3 comprising an amino acid sequence as set forth in SEQ ID Nos: 245, 246, and 247, respectively, and a light chain comprising a light chain (LC)-CDR1, LC-CDR2, and LC-CDR3 comprising an amino acid sequence as set forth in SEQ ID Nos: 248, 249, and 250, respectively.
  • HC heavy chain
  • HC-CDR2 heavy chain
  • HC-CDR3 comprising an amino acid sequence as set forth in SEQ ID Nos: 245, 246, and 247, respectively
  • LC light chain
  • LC-CDR3 comprising an amino acid sequence as set forth in SEQ ID Nos: 248, 249, and 250, respectively.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 9 and a light chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 10.
  • the antibody, or the antigen binding fragment thereof specifically binds to human CD117 and comprises a heavy chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 243, and a light chain comprising a variable region comprising an amino acid sequence as set forth in SEQ ID NO: 244.
  • Also disclosed herein is a method of depleting a population of cells in a human subject, said method comprising administering an ADC disclosed herein to the subject, wherein the ADC comprises an antibody, or an antigen-binding fragment thereof, that specifically binds to an extracellular antigen expressed by cells in the population of cells.
  • Also provided is a method of conditioning a human subject for cell transplantation comprising administering an ADC disclosed herein, to the human subject such that the endogenous stem or endogenous immune cells in the human subject are depleted, wherein the ADC specifically binds to an extracellular antigen expressed by the endogenous stem or endogenous immune cells.
  • the method further comprises administering to the human subject allogenic stem cells or allogeneic immune cells.
  • composition comprising an ADC disclosed herein, and a pharmaceutically acceptable carrier.
  • FIG. 1 depicts the structures of formulae IV ( FIG. 1A ), VI ( FIG. 1B ), and IIa ( FIG. 1C ).
  • “Ab” in FIGS. 1A to 1C represents an antibody.
  • FIGS. 1A, 1B, and 1C represent Conjugates A, B, and C, respectively, referred to in the examples. Conjugates A, B, and C are also referred to as ADC A, ADC B, and ADC C.
  • FIG. 2 graphically depicts results from in vitro cytoxicity assays using Kasumi-1 cells in the presence of ADC with pre-incubation of ADC in media (A) or 50% human serum (B) to evaluate serum stability of conjugates.
  • FIG. 3 graphically depicts results from in vitro cytoxicity assays using Kasumi-1 cells in the presence of ADC with iteration on the time of incubation with titrated ADC samples to evaluate differences in the kinetics of cytotoxicity between cleavable and non-cleavable conjugates.
  • FIG. 4 graphically depicts results from two in vitro cell killing assays using Kasumi-1 cells ( 4 A) and CD34+ cells ( 4 B).
  • the ADC tested was anti-CD117 ADC C.
  • FIG. 5 graphically depicts the ability of anti-CD117 ADC C to potently deplete human CD34+ in the bone marrow of humanized NSG mice.
  • FIG. 6 are Kaplan Meier plots reflecting the survival of Kasumi-1 implanted humanized NSG mice treated with the indicated doses of conjugates or controls.
  • FIG. 7 graphically depicts the efficacy of Conjugate C as evaluated in male cynomolgus monkeys. 2.0 mg/kg dose (LALA) 30.2867 batch issues may have resulted in a decrease in HSC sensitivity
  • FIG. 8 graphically depicts tolerability of ADCs containing either Conjugate A or Conjugate C in male cynomolgus monkeys.
  • FIG. 9 graphically depicts pharmacokinetic analysis of conjugate A and conjugate C administered to male cynomolgus monkeys.
  • FIG. 10 graphically depicts that anti-CD2 and CD5 ADC C are able to deplete T cells.
  • FIG. 11 graphically depicts that while anti-CD2 ADCs A and C are saturated by Day 5, some cells still express CD5.
  • FIGS. 12A and 12B graphically depict results showing anti-CD45 ADCs A or C ( FIG. 14A ) or anti-CD45 ADCs A or B ( FIG. 14B ) in in vitro cell killing assays.
  • FIG. 13 graphically depicts results showing anti-CD45 ADCs A or C in in vitro cell killing assays.
  • FIG. 14 graphically depicts results of anti-CD45 ADCs A or C in in vivo cell depletion experiments.
  • FIG. 15 graphically depicts results from administration of anti-CD45 ADCs A or C to mice at a number of dose amounts. Levels of peripheral lymphocytes, HSCs, and lymphocytes are shown. All ADCs were administered at 1 mg/kg.
  • FIG. 16 graphically depicts results showing anti-CD137 ADCs A and C in a T cell killing assay.
  • FIG. 17 graphically depicts cell line serum stability of anti-CD137 ADCs A and C over 48 hours.
  • acyl refers to —C( ⁇ O)R, wherein R is hydrogen (“aldehyde”), C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 7 carbocyclyl, C 6 -C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, as defined herein.
  • R is hydrogen (“aldehyde”), C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 3 -C 7 carbocyclyl, C 6 -C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, as defined herein.
  • Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.
  • C 1 -C 12 alkyl refers to a straight chain or branched, saturated hydrocarbon having from 1 to 12 carbon atoms.
  • Representative C 1 -C 12 alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C 1 -C 12 alkyls include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl.
  • a C 1 -C 12 alkyl group can be unsubstituted or substituted.
  • alkenyl refers to C 2 -C 12 hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp 2 double bond. Examples include, but are not limited to: ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, and the like.
  • An alkenyl group can be unsubstituted or substituted.
  • Alkynyl refers to a C 2 -C 12 hydrocarbon containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp triple bond. Examples include, but are not limited to acetylenic and propargyl. An alkynyl group can be unsubstituted or substituted.
  • Aryl refers to a C 6 -C 20 carbocyclic aromatic group.
  • aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl.
  • An aryl group can be unsubstituted or substituted.
  • Arylalkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with an aryl radical.
  • Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like.
  • the arylalkyl group comprises 6 to 20 carbon atoms, e.g.
  • the alkyl moiety including alkanyl, alkenyl or alkynyl groups
  • the arylalkyl group is 1 to 6 carbon atoms and the aryl moiety is 5 to 14 carbon atoms.
  • An alkaryl group can be unsubstituted or substituted.
  • Cycloalkyl refers to a saturated carbocyclic radical, which may be mono- or bicyclic. Cycloalkyl groups include a ring having 3 to 7 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. A cycloalkyl group can be unsubstituted or substituted.
  • Cycloalkenyl refers to an unsaturated carbocyclic radical, which may be mono- or bicyclic. Cycloalkenyl groups include a ring having 3 to 6 carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. A cycloalkenyl group can be unsubstituted or substituted.
  • Heteroaralkyl refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl radical.
  • Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like.
  • the heteroarylalkyl group comprises 6 to 20 carbon atoms, e.g.
  • the alkyl moiety, including alkanyl, alkenyl or alkynyl groups, of the heteroarylalkyl group is 1 to 6 carbon atoms and the heteroaryl moiety is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S.
  • the heteroaryl moiety of the heteroarylalkyl group may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6] system.
  • Heteroaryl and “heterocycloalkyl” as used herein refer to an aromatic or non-aromatic ring system, respectively, in which one or more ring atoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur.
  • the heteroaryl or heterocycloalkyl radical comprises 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S.
  • a heteroaryl or heterocycloalkyl may be a monocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or [6,6] system.
  • Heteroaryl and heterocycloalkyl can be unsubstituted or substituted.
  • Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds, A series of Monographs” (John Wiley & Sons, New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.
  • heteroaryl groups include by way of example and not limitation pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carb
  • heterocycloalkyls include by way of example and not limitation dihydroypyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.
  • carbon bonded heteroaryls and heterocycloalkyls are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline.
  • carbon bonded heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.
  • nitrogen bonded heteroaryls and heterocycloalkyls are bonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of a morpholine, and position 9 of a carbazole, or beta-carboline.
  • nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.
  • Substituted as used herein and as applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, and the like, means that one or more hydrogen atoms are each independently replaced with a substituent.
  • the foregoing chemical moieties such as “alkyl”, “alkylene”, “heteroalkyl”, “heteroalkylene”, “alkenyl”, “alkenylene”, “heteroalkenyl”, “heteroalkenylene”, “alkynyl”, “alkynylene”, “heteroalkynyl”, “heteroalkynylene”, “cycloalkyl”, “cycloalkylene”, “heterocycloalkyl”, heterocycloalkylene”, “aryl,” “arylene”, “heteroaryl”, and “heteroarylene” groups can optionally be substituted.
  • Typical substituents include, but are not limited to, —X, —R, —OH, —OR, —SH, —SR, NH 2 , —NHR, —N(R) 2 , —N + (R) 3 , —CX 3 , —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO 2 , —N 3 , —NC( ⁇ O)H, —NC( ⁇ O)R, —C( ⁇ O)H, —C( ⁇ O)R, —C( ⁇ O)NH 2 , —C( ⁇ O)N(R) 2 , —SO 3 —, —SO 3 H, —S( ⁇ O) 2 R, —OS( ⁇ O) 2 OR, —S( ⁇ O) 2 NH 2 , —S( ⁇ O) 2 N(R) 2 , —S( ⁇ O)R, —OP( ⁇ O)(OH)
  • radical naming conventions can include either a mono-radical or a di-radical, depending on the context.
  • a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a di-radical.
  • a substituent identified as alkyl that requires two points of attachment includes di-radicals such as —CH 2 —, —CH 2 CH 2 —, —CH 2 CH(CH 3 )CH 2 —, and the like.
  • Other radical naming conventions clearly indicate that the radical is a di-radical such as “alkylene,” “alkenylene,” “arylene,” “heterocycloalkylene,” and the like.
  • a substituent is depicted as a di-radical (i.e., has two points of attachment to the rest of the molecule), it is to be understood that the substituent can be attached in any directional configuration unless otherwise indicated.
  • “Isomerism” means compounds that have identical molecular formulae but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereoisomers,” and stereoisomers that are non-superimposable mirror images of each other are termed “enantiomers,” or sometimes “optical isomers.”
  • a carbon atom bonded to four non-identical substituents is termed a “chiral center.”
  • “Chiral isomer” means a compound with at least one chiral center. Compounds with more than one chiral center may exist either as an individual diastereomer or as a mixture of diastereomers, termed “diastereomeric mixture.”
  • a stereoisomer may be characterized by the absolute configuration (R or S) of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are ranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.
  • the compounds disclosed in this description and in the claims may comprise one or more asymmetric centers, and different diastereomers and/or enantiomers of each of the compounds may exist.
  • the description of any compound in this description and in the claims is meant to include all enantiomers, diastereomers, and mixtures thereof, unless stated otherwise.
  • the description of any compound in this description and in the claims is meant to include both the individual enantiomers, as well as any mixture, racemic or otherwise, of the enantiomers, unless stated otherwise.
  • the structure of a compound is depicted as a specific enantiomer, it is to be understood that the invention of the present application is not limited to that specific enantiomer.
  • the structural formula of the compound represents a certain isomer for convenience in some cases, but the present disclosure includes all isomers, such as geometrical isomers, optical isomers based on an asymmetrical carbon, stereoisomers, tautomers, and the like, it being understood that not all isomers may have the same level of activity.
  • the compounds may occur in different tautomeric forms.
  • the compounds according to the disclosure are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the invention of the present application is not limited to that specific tautomer.
  • a salt for example, can be formed between an anion and a positively charged group (e.g., amino) on a compound of the disclosure.
  • Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate).
  • pharmaceutically acceptable anion refers to an anion suitable for forming a pharmaceutically acceptable salt.
  • a salt can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a compound of the disclosure.
  • Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and an ammonium cation such as tetramethylammonium ion.
  • Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • the compounds of the disclosure also include those salts containing quaternary nitrogen atoms.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
  • the compounds of the present disclosure can exist in either hydrated or unhydrated (the anhydrous) form or as solvates with other solvent molecules.
  • Non-limiting examples of hydrates include monohydrates, dihydrates, etc.
  • Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.
  • “Solvate” means solvent addition forms that contain either stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thus forming a solvate. If the solvent is water the solvate formed is a hydrate; and if the solvent is alcohol, the solvate formed is an alcoholate.
  • Hydrates are formed by the combination of one or more molecules of water with one molecule of the substance in which the water retains its molecular state as H 2 O.
  • a hydrate refers to, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • a crystal polymorphism may be present for the compounds or salts thereof represented by the formulae disclosed herein. It is noted that any crystal form, crystal form mixture, or anhydride or hydrate thereof, is included in the scope of the present disclosure.
  • the term “about” refers to a value that is within 10% above or below the value being described.
  • the term “about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.
  • amatoxin refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or a variant or derivative thereof, such as a variant or derivative thereof capable of inhibiting RNA polymerase II activity. Suitable amatoxins and derivatives thereof are further described herein below. As described herein, amatoxins may be conjugated to an antibody, or antigen-binding fragment thereof, for instance, by way of a linker moiety (L) (thus forming a conjugate (i.e., ADC)). Exemplary methods of amatoxin conjugation and linkers useful for such processes are described below.
  • L linker moiety
  • antibody refers to an immunoglobulin molecule that specifically binds to, or is immunologically reactive with, a particular antigen, and includes monoclonal, genetically engineered, and otherwise modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi- tri- and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antigen binding fragments of antibodies, including, for example, Fab′, F(ab′) 2 , Fab, Fv, rIgG, and scFv fragments.
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • Fab and F(ab′) 2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • the antibodies of the present invention are generally isolated or recombinant. “Isolated,” when used herein refers to a polypeptide, e.g., an antibody, that has been separated and/or recovered from a cell or cell culture from which it was expressed. Thus, an “isolated antibody,” refers to an antibody which is substantially free of other antibodies having different antigenic specificities. For instance, an isolated antibody that specifically binds to CD117 is substantially free of antibodies that specifically bind antigens other than CD117.
  • antigen-binding fragment refers a fragment of an antibody that retains the ability to specifically bind to a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragment can be, for example, a Fab, F(ab′) 2 , scFv, diabody, a triabody, an affibody, a nanobody, an aptamer, or a domain antibody.
  • binding fragments encompassed of the term “antigen-binding fragment” of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H 1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H 1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody, (v) a dAb including V H and V L domains; (vi) a dAb fragment that consists of a V H domain (see, e.g., Ward et al., Nature 341:544-546, 1989); (vii) a dAb which consists of a V H or a V L domain; (viii) an isolated complementarity determining
  • the two domains of the Fv fragment, V L and V H are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).
  • Antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in certain cases, by chemical peptide synthesis procedures known in the art.
  • anti-CD117 antibody or “an antibody that binds to CD117” refers to an antibody that is capable of binding CD117, e.g., human CD117, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD117.
  • the amino acid sequences of the two main isoforms of human CD117 are provided in SEQ ID NO: 145 (isoform 1) and SEQ ID NO: 146 (isoform 2).
  • An “anti-CD117 ADC” refers to an ADC wherein the antibody is an anti-CD117 antibody.
  • anti-CD45 antibody or “an antibody that binds to CD45” refers to an antibody that is capable of binding CD45, e.g., human CD117, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD45.
  • An “anti-CD45 ADC” refers to an ADC wherein the antibody is an anti-CD45 antibody.
  • anti-CD137 antibody or “an antibody that binds to CD137” refers to an antibody that is capable of binding CD137, e.g., human CD137, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD137.
  • An “anti-CD137 ADC” refers to an ADC wherein the antibody is an anti-CD137 antibody.
  • anti-CD2 antibody or “an antibody that binds to CD2” refers to an antibody that is capable of binding CD2, e.g., human CD2, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD2.
  • An “anti-CD2 ADC” refers to an ADC wherein the antibody is an anti-CD2 antibody.
  • anti-CD5 antibody or “an antibody that binds to CD5” refers to an antibody that is capable of binding CD5, e.g., human CD5, with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting CD5.
  • An “anti-CD5 ADC” refers to an ADC wherein the antibody is an anti-CD5 antibody.
  • the term “bispecific antibody” refers to, for example, a monoclonal, often a human or humanized antibody that is capable of binding at least two different antigens or at; least two different epitopes that may be on the same antigen or different antigens.
  • one of the binding specificities can be directed towards a hematopoietic stem cell surface antigen, CD117 (e.g., GNNK+ CD117), and the other can specifically bind a different hematopoietic stem cell surface antigen or another cell surface protein, such as a receptor or receptor subunit involved in a signal transduction pathway that potentiates cell growth, among others.
  • CDR complementarity determining region
  • FRs framework regions
  • the amino acid positions that delineate a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions.
  • variable domains of native heavy and light chains each contain four framework regions that primarily adopt a ⁇ -sheet configuration, connected by three CDRs, which form loops that connect, and in some cases form part of, the ⁇ -sheet structure.
  • the CDRs in each chain are held together in close proximity by the framework regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, with the CDRs from the other antibody chains, contribute to the formation of the target binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md., 1987).
  • numbering of immunoglobulin amino acid residues is performed according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated (although any antibody numbering scheme, including, but not limited to IMGT and Chothia, can be utilized).
  • condition refers to processes by which a patient is prepared for receipt of a transplant, e.g., a transplant containing hematopoietic stem cells. Such procedures promote the engraftment of a hematopoietic stem cell transplant (for instance, as inferred from a sustained increase in the quantity of viable hematopoietic stem cells within a blood sample isolated from a patient following a conditioning procedure and subsequent hematopoietic stem cell transplantation.
  • a patient may be conditioned for hematopoietic stem cell transplant therapy by administration to the patient of an ADC capable of binding an antigen expressed by hematopoietic stem cells, such as CD117 (e.g., GNNK+ CD117).
  • an ADC capable of binding an HSC antigen to a patient in need of hematopoietic stem cell transplant therapy can promote the engraftment of a hematopoietic stem cell graft, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating a vacancy filled by an exogenous hematopoietic stem cell transplant.
  • conjugate refers to a compound formed by the chemical bonding of a reactive functional group of one molecule, such as an antibody or antigen-binding fragment thereof, with an appropriately reactive functional group of another molecule, such as a cytotoxin described herein.
  • Conjugates may include a linker between the two molecules bound to one another.
  • linkers that can be used for the formation of a conjugate include peptide-containing linkers, such as those that contain naturally occurring or non-naturally occurring amino acids, such as D-amino acids. Linkers can be prepared using a variety of strategies described herein and known in the art.
  • a linker may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).
  • conjugate when referring to a compound is also referred to interchangeably herein as a “drug conjugate”, “antibody drug conjugate” or “ADC”.
  • the term “coupling reaction” refers to a chemical reaction in which two or more substituents suitable for reaction with one another react so as to form a chemical moiety that joins (e.g., covalently) the molecular fragments bound to each substituent.
  • Coupling reactions include those in which a reactive substituent bound to a fragment that is a cytotoxin, such as a cytotoxin known in the art or described herein, reacts with a suitably reactive substituent bound to a fragment that is an antibody, or antigen-binding fragment thereof, such as an antibody, antigen-binding fragment thereof, or specific anti-CD117 antibody that binds CD117 (such as GNNK+ CD117) known in the art or described herein.
  • suitably reactive substituents include a nucleophile/electrophile pair (e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thio/ ⁇ , ⁇ -unsaturated carbonyl pair, among others), a diene/dienophile pair (e.g., an azide/alkyne pair, among others), and the like.
  • a nucleophile/electrophile pair e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thio/ ⁇ , ⁇ -unsaturated carbonyl pair, among others
  • diene/dienophile pair e.g., an azide/alkyne pair, among others
  • Coupling reactions include, without limitation, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive modalities known in the art or described herein.
  • cycloaddition e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others
  • nucleophilic aromatic substitution e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others
  • nucleophilic aromatic substitution e.g., [4+2] Diels-Alder cycloa
  • CRU competitive repopulating unit
  • the term “donor” refers to a human or animal from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient.
  • the one or more cells may be, for example, a population of hematopoietic stem cells.
  • the term “diabody” refers to a bivalent antibody containing two polypeptide chains, in which each polypeptide chain includes V H and V L domains joined by a linker that is too short (e.g., a linker composed of five amino acids) to allow for intramolecular association of V H and V L domains on the same peptide chain. This configuration forces each domain to pair with a complementary domain on another polypeptide chain so as to form a homodimeric structure.
  • the term “triabody” refers to trivalent antibodies containing three peptide chains, each of which contains one V H domain and one V L domain joined by a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain.
  • a linker that is exceedingly short (e.g., a linker composed of 1-2 amino acids) to permit intramolecular association of V H and V L domains within the same peptide chain.
  • peptides configured in this way typically trimerize so as to position the V H and V L domains of neighboring peptide chains spatially proximal to one another (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).
  • drug-to-antibody ratio refers to the number of drugs, e.g., amatoxin, attached to the antibody of a conjugate.
  • the DAR of an ADC can range from 1 to 8, although higher loads are also possible depending on the number of linkage sites on an antibody.
  • the conjugate has a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.
  • the term “endogenous” describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is found naturally in a particular organism, such as a human patient.
  • a hematopoietic stem cell or a cell of hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte,
  • the term “engraftment potential” is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate a tissue, whether such cells are naturally circulating or are provided by transplantation.
  • the term encompasses all events surrounding or leading up to engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest.
  • the engraftment efficiency or rate of engraftment can be evaluated or quantified using any clinically acceptable parameter as known to those of skill in the art and can include, for example, assessment of competitive repopulating units (CRU); incorporation or expression of a marker in tissue(s) into which stem cells have homed, colonized, or become engrafted; or by evaluation of the progress of a subject through disease progression, survival of hematopoietic stem and progenitor cells, or survival of a recipient.
  • Engraftment can also be determined by measuring white blood cell counts in peripheral blood during a post-transplant period. Engraftment can also be assessed by measuring recovery of marrow cells by donor cells in a bone marrow aspirate sample.
  • exogenous describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte) that is not found naturally in a particular organism, such as a human patient.
  • Exogenous substances include those that are provided from an external source to an organism or to cultured matter extracted therefrom.
  • Fc refers to the portion of an IgG antibody that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule.
  • the Fc region comprises the C-terminal half of two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and binding sites for complement and Fc receptors, including the FcRn receptor.
  • An Fc region contains the second constant domain CH2 (e.g., residues at EU positions 231-340 of IgG1) and the third constant domain CH3 (e.g., residues at EU positions 341-447 of human IgG1).
  • the Fc region includes the “lower hinge region” (e.g., residues at EU positions 233-239 of IgG1).
  • Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of positions in Fc domains, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and thus slight differences between the sequences presented in the instant application and sequences known in the art can exist.
  • a “wild type IgG Fc domain” or “WT IgG Fc domain” refers to any naturally occurring IgG Fc region (i.e., any allele).
  • sequences of the heavy chains of human IgG1, IgG2, IgG3 and IgG4 can be found in a number of sequence databases, for example, at the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG1_HUMAN), respectively.
  • An example of a “WT” Fc region is provided in SEQ ID NO: 122 (which provides a heavy chain constant region containing an Fc region).
  • modified Fc region or “variant Fc region” as used herein refers to an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc region.
  • an ADC described herein comprises an intact antibody.
  • an intact antibody comprises two heavy chains each comprising a variable region, a constant region and an Fc region, and two light chains each comprising a variable region and a constant region.
  • an intact IgG comprises two light chains each comprising a light chain variable region (VL) and alight chain constant region (CL), and comprises two heavy chains each comprising a heavy chain variable region (VH) and three heavy chain constant regions (CH1, CH2, and CH3).
  • CH2 and CH3 represent the Fc region of the heavy chain.
  • frame region includes amino acid residues that are adjacent to the CDRs of an antibody or antigen-binding fragment thereof.
  • FW region residues may be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies, among others.
  • HSCs hematopoietic stem cells
  • granulocytes e.g., promyelocytes, neutrophils, eosinophils, basophils
  • erythrocytes e.g., reticulocytes, erythrocytes
  • thrombocytes e.g., megakaryoblasts, platelet producing megakaryocytes, platelets
  • monocytes e.g., monocytes, macrophages
  • dendritic cells e.g., NK cells, B-cells and T-cells.
  • Such cells may include CD34 + cells.
  • CD34 + cells are immature cells that express the CD34 cell surface marker.
  • CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above, whereas in mice, HSCs are CD34 ⁇ .
  • HSCs also refer to long term repopulating HSCs (LT-HSC) and short term repopulating HSCs (ST-HSC). LT-HSCs and ST-HSCs are differentiated, based on functional potential and on cell surface marker expression.
  • human HSCs are CD34+, CD38 ⁇ , CD45RA ⁇ , CD90+, CD49F+, and lin ⁇ (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A).
  • bone marrow LT-HSCs are CD34 ⁇ , SCA-1+, C-kit+, CD135 ⁇ , Slamfl/CD150+, CD48 ⁇ , and lin ⁇ (negative for mature lineage markers including Ter119, CD11b, Gr, CD3, CD4, CD8, B220, IL7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135 ⁇ , Slamfl/CD150+, and lin ⁇ (negative for mature lineage markers including Ter119, CD11b, Gr, CD3, CD4, CD8, B220, IL7ra).
  • ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions.
  • LT-HSC have greater self-renewal potential (i.e., they survive throughout adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time, and do not possess serial transplantation potential). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and thus, can more quickly give rise to differentiated progeny.
  • hematopoietic stem cell functional potential refers to the functional properties of hematopoietic stem cells which include 1) multi-potency (which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal (which refers to the ability of hematopoietic stem cells to give rise to daughter cells that have equivalent potential as the mother cell, and further that this ability can
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • a human antibody may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or during gene rearrangement or by somatic mutation in vivo).
  • the term “human antibody”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • a human antibody can be produced in a human cell (for example, by recombinant expression) or by a non-human animal or a prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (such as heavy chain and/or light chain) genes.
  • a human antibody when a human antibody is a single chain antibody, it can include a linker peptide that is not found in native human antibodies.
  • an Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, for example, PCT Publication Nos. WO 1998/24893; WO 1992/01047; WO 1996/34096; WO 1996/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).
  • a “humanized” antibody refers to an antibody that contains minimal sequences derived from non-human immunoglobulin.
  • “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. All or substantially all of the FW regions may also be those of a human immunoglobulin sequence.
  • the humanized antibody can also contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence.
  • Fc immunoglobulin constant region
  • patients that are “in need of” a hematopoietic stem cell transplant include patients that exhibit a defect or deficiency in one or more blood cell types, as well as patients having a stem cell disorder, autoimmune disease, cancer, or other pathology described herein.
  • Hematopoietic stem cells generally exhibit 1) multi-potency, and can thus differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells), 2) self-renewal, and can thus give rise to daughter cells that have equivalent potential as the mother cell, and 3) the ability to be reintroduced into a transplant recipient whereupon they home to the hematopoietic stem cell niche and re-establish productive and sustained hematop
  • Hematopoietic stem cells can thus be administered to a patient defective or deficient in one or more cell types of the hematopoietic lineage in order to re-constitute the defective or deficient population of cells in vivo.
  • the patient may be suffering from cancer, and the deficiency may be caused by administration of a chemotherapeutic agent or other medicament that depletes, either selectively or non-specifically, the cancerous cell population.
  • the patient may be suffering from a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome.
  • a hemoglobinopathy e.g., a non-malignant hemoglobinopathy
  • the subject may be one that is suffering from adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
  • ADA SCID adenosine deaminase severe combined immunodeficiency
  • the subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder.
  • the subject may have or be affected by a malignancy, such as neuroblastoma or a hematologic cancer.
  • the subject may have a leukemia, lymphoma, or myeloma.
  • the subject has acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • the subject has myelodysplastic syndrome.
  • the subject has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, Type 1 diabetes, or another autoimmune pathology described herein.
  • the subject is in need of chimeric antigen receptor T-cell (CART) therapy.
  • the subject has or is otherwise affected by a metabolic storage disorder.
  • the subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, metachromatic leukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it
  • a patient “in need of” a hematopoietic stem cell transplant may one that is or is not suffering from one of the foregoing pathologies, but nonetheless exhibits a reduced level (e.g., as compared to that of an otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes.
  • endogenous cell types within the hematopoietic lineage such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eos
  • FACS fluorescence activated cell sorting
  • a neutral antibody refers to an antibody, or an antigen binding fragment thereof, that is not capable of significantly neutralizing, blocking, inhibiting, abrogating, reducing or interfering with the activities of a particular or specified target (e.g., CD117), including the binding of receptors to ligands or the interactions of enzymes with substrates.
  • a neutral anti-CD117 antibody, or fragment thereof is an anti-CD117 antibody that does not substantially inhibit SCF-dependent cell proliferation and does not cross block SCF binding to CD117.
  • An example of a neutral antibody is Ab67 (or an antibody having the binding regions of Ab67).
  • an “antagonist” anti-CD117 antibody inhibits SCF-dependent proliferation and is able to cross block SCF binding to CD117.
  • An example of an antagonist antibody is Ab55 (or an antibody having the binding regions of Ab55).
  • the term “recipient” refers to a patient that receives a transplant, such as a transplant containing a population of hematopoietic stem cells.
  • the transplanted cells administered to a recipient may be, e.g., autologous, syngeneic, or allogeneic cells.
  • sample refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells) taken from a subject.
  • a specimen e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, and cells
  • scFv refers to a single chain Fv antibody in which the variable domains of the heavy chain and the light chain from an antibody have been joined to form one chain.
  • scFv fragments contain a single polypeptide chain that includes the variable region of an antibody light chain (V L ) (e.g., CDR-L1, CDR-L2, and/or CDR-L3) and the variable region of an antibody heavy chain (V H ) (e.g., CDR-H1, CDR-H2, and/or CDR-H3) separated by a linker.
  • V L variable region of an antibody light chain
  • V H variable region of an antibody heavy chain
  • the linker that joins the V L and V H regions of a scFv fragment can be a peptide linker composed of proteinogenic amino acids.
  • linkers can be used to so as to increase the resistance of the scFv fragment to proteolytic degradation (for example, linkers containing D-amino acids), in order to enhance the solubility of the scFv fragment (for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues), to improve the biophysical stability of the molecule (for example, a linker containing cysteine residues that form intramolecular or intermolecular disulfide bonds), or to attenuate the immunogenicity of the scFv fragment (for example, linkers containing glycosylation sites).
  • linkers containing D-amino acids for example, hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • hydrophilic linkers such as polyethylene glycol-containing linkers or polypeptides containing repeating glycine and serine residues
  • variable regions of the scFv molecules described herein can be modified such that they vary in amino acid sequence from the antibody molecule from which they were derived.
  • nucleotide or amino acid substitutions leading to conservative substitutions or changes at amino acid residues can be made (e.g., in CDR and/or framework residues) so as to preserve or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.
  • telomere binding refers to the ability of an antibody to recognize and bind to a specific protein structure (epitope) rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody. By way of example, an antibody “binds specifically” to a target if the antibody, when labeled, can be competed away from its target by the corresponding non-labeled antibody.
  • an antibody specifically binds to a target, e.g., CD117, CD45, CD2, CD5, CD137, CD134, or CD252, if the antibody has a K D for the target of at least about 10 ⁇ 4 M, 10 ⁇ 5 M, 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M, 10 ⁇ 12 M, or less (less meaning a number that is less than 10 ⁇ 12 , e.g. 10 ⁇ 13 ).
  • K D (M) is determined according to standard bio-layer interferometery (BLI).
  • K off (1/s) is determined according to standard bio-layer interferometery (BLI).
  • the antibody may be capable of specifically binding to two or more antigens which are related in sequence.
  • an antibody can specifically bind to both human and a non-human (e.g., mouse or non-human primate) orthologs of CD117, CD45, CD2, CD5, CD137, CD134, or CD252.
  • the terms “subject” and “patient” refer to an organism, such as a human, that receives treatment for a particular disease or condition as described herein.
  • a patient such as a human patient, may receive treatment prior to hematopoietic stem cell transplant therapy in order to promote the engraftment of exogenous hematopoietic stem cells.
  • the phrase “substantially cleared from the blood” refers to a point in time following administration of a therapeutic agent, e.g., an ADC comprising an amatoxin, to a patient when the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent is not detectable by conventional means (for instance, such that the therapeutic agent is not detectable above the noise threshold of the device or assay used to detect the therapeutic agent).
  • a therapeutic agent e.g., an ADC comprising an amatoxin
  • stem cell disorder broadly refers to any disease, disorder, or condition that may be treated or cured by conditioning a subject's target tissues, and/or by ablating an endogenous stem cell population in a target tissue (e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue) and/or by engrafting or transplanting stem cells in a subject's target tissues.
  • a target tissue e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue
  • stem cells in a subject's target tissues e.g., ablating an endogenous hematopoietic stem or progenitor cell population from a subject's bone marrow tissue
  • Type I diabetes has been shown to be cured by hematopoietic stem cell transplant and may benefit from conditioning in accordance with the compositions and methods described herein.
  • Additional disorders that can be treated using the compositions and methods described herein include, without limitation, sickle cell anemia, thalassemias, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV/AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome.
  • Additional diseases that may be treated using the patient conditioning and/or hematopoietic stem cell transplant methods described herein include inherited blood disorders (e.g., sickle cell anemia) and autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease.
  • Additional diseases that may be treated using the conditioning and/or transplantation methods described herein include a malignancy, such as a neuroblastoma or a hematologic cancer, such as leukemia, lymphoma, and myeloma.
  • the cancer may be acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.
  • Additional diseases treatable using the conditioning and/or transplantation methods described herein include myelodysplastic syndrome.
  • the subject has or is otherwise affected by a metabolic storage disorder.
  • the subject may suffer or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's Disease, Hurlers Disease, sphingolipidoses, metachromatic leukodystrophy, or any other diseases or disorders which may benefit from the treatments and therapies disclosed herein and including, without limitation, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyper immunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis and those diseases, or disorders described in “Bone Marrow Transplantation for Non-Malignant Disease,” ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety
  • transfection refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, such as electroporation, lipofection, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • treat refers to reducing the severity and/or frequency of disease symptoms, eliminating disease symptoms and/or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and/or their underlying cause, and improving or remediating damage caused, directly or indirectly, by disease.
  • beneficial or desired clinical results include, but are not limited to, promoting the engraftment of exogenous hematopoietic cells in a patient following antibody conditioning therapy as described herein and subsequent hematopoietic stem cell transplant therapy.
  • Additional beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem cell transplant following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to the patient.
  • Beneficial results of therapy described herein may also include an increase in the cell count or relative concentration of one or more cells of hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte, following conditioning therapy and subsequent hematopoietic stem cell transplant therapy.
  • hematopoietic lineage such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte,
  • Additional beneficial results may include the reduction in quantity of a disease-causing cell population, such as a population of cancer cells (e.g., CD117+ leukemic cells) or autoimmune cells (e.g., CD117+ autoimmune lymphocytes, such as a CD117+ T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen).
  • a disease-causing cell population such as a population of cancer cells (e.g., CD117+ leukemic cells) or autoimmune cells (e.g., CD117+ autoimmune lymphocytes, such as a CD117+ T-cell that expresses a T-cell receptor that cross-reacts with a self-antigen).
  • a disease-causing cell population such as a population of cancer cells (e.g., CD117+ leukemic cells) or autoimmune cells (e.g., CD117+ autoimmune lymphocytes, such as a CD117+ T-cell that expresses a T-cell receptor that cross-re
  • variants and “derivative” are used interchangeably and refer to naturally-occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein.
  • a variant or derivative of a compound, peptide, protein, or other substance described herein may retain or improve upon the biological activity of the original material.
  • vector includes a nucleic acid vector, such as a plasmid, a DNA vector, a plasmid, a RNA vector, virus, or other suitable replicon.
  • Expression vectors described herein may contain a polynucleotide sequence as well as, for example, additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • kits for expression of antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements may include, for example, 5′ and 3′ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.
  • ADCs Antibody-Drug Conjugates
  • Antibodies, and antigen-binding fragments thereof, as described herein can be conjugated (linked) to a cytotoxic molecule (i.e., a cytotoxin), thus forming an antibody-drug conjugate (ADC).
  • a cytotoxic molecule i.e., a cytotoxin
  • ADC antibody-drug conjugate
  • the ADCs as disclosed herein include an antibody (including an antigen-binding fragment thereof) conjugated to an amatoxin, e.g., amatoxin set forth in Formula (V), wherein the cytotoxic moiety, when not conjugated to an antibody, has a cytotoxic or cytostatic effect.
  • the cytotoxic moiety exhibits reduced or no cytotoxicity when bound in a conjugate, but resumes cytotoxicity after cleavage from the linker.
  • the cytotoxic moiety maintains cytotoxicity without cleavage from the linker.
  • the cytotoxic molecule is conjugated to a cell internalizing antibody, or antigen-binding fragment thereof as disclosed herein, such that following the cellular uptake of the antibody, or fragment thereof, the cytotoxin may access its intracellular target and, e.g., mediate hematopoietic cell death.
  • ADCs of the present disclosure therefore may be of the general formula
  • an antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to linker (L), through a chemical moiety (Z), to a cytotoxic moiety (Cy).
  • the antibody or antigen-binding fragment thereof may be conjugated to a number of drug moieties as indicated by integer n, which represents the average number of cytotoxins per antibody, which may range, e.g., from about 1 to about 20. Any number of cytotoxins can be conjugated to the antibody, e.g., 1, 2, 3, 4, 5, 6, 7, or 8.
  • n is from 1 to 4.
  • n is 1.
  • n is 2.
  • the average number of drug moieties per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • the quantitative distribution of ADC in terms of n may also be determined. In some instances, separation, purification, and characterization of homogeneous ADC where n is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis.
  • n may be limited by the number of attachment sites on the antibody.
  • an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached.
  • antibodies do not contain many free and reactive cysteine thiol groups which may be linked to a drug moiety; primarily, cysteine thiol residues in antibodies exist as disulfide bridges.
  • an antibody may be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups.
  • a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP)
  • an antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Only the most reactive lysine groups may react with an amine-reactive linker reagent. In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups such as lysine or cysteine.
  • the loading (drug/antibody ratio) of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reductive conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachments.
  • the cytotoxin of the antibody-drug conjugates described herein is an amatoxin or derivative thereof.
  • Amatoxins are potent and selective inhibitors of RNA polymerase II and thereby also inhibit the transcription and protein biosynthesis of the affected cells.
  • the term “amatoxin” refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or a variant or derivative thereof, such as a variant or derivative thereof capable of inhibiting RNA polymerase II activity.
  • Amatoxins are rigid bicyclic octapeptides having the basic sequence Ile-Trp-Gly-Ile-Gly-Cys-Asn(or Asp)-Pro, crosslinked by an attachment between the Cys sulfur and position 2 of the Trp indole ring, forming a tryptathionine.
  • certain amino acid substituents are varied by post-translational modification (i.e. Pro to Hyp; Ile to DHIle; and Trp to 5-OH Trp).
  • Amatoxins may be isolated from a variety of mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiota brunneo - incarnata ) or may be prepared semi-synthetically or synthetically. Different mushroom species contain varying amounts of different amatoxin family members. A member of this family, ⁇ -amanitin, is known to be an extremely potent inhibitor of eukaryotic RNA polymerase II and to a lesser degree, RNA polymerase Ill, thereby inhibiting transcription and protein biosynthesis. Wieland, Int. J. Pept. Protein Res. 1983, 22(3):257-276.
  • Antibodies, or antigen-binding fragments thereof, that recognize and bind to an antigen expressed on the cell surface of a human stem cell or a T cell can be conjugated to an amatoxin, such as an ⁇ -amanitin or a derivative thereof, as described in, for example, U.S. Pat. Nos. 9,233,173 and 9,399,681 and US Patent Application Publication Nos. 2016/0089450, 2016/0002298, 2015/0218220, 2014/0294865, the disclosure of each of which is incorporated herein by reference as it pertains, for example, to amatoxins, such as ⁇ -amanitin, as well as covalent linkers that can be used for covalent conjugation. Exemplary methods of amatoxin conjugation and linkers useful for such processes are described herein. Exemplary linker-containing amatoxins useful for conjugation to an antibody, or antigen-binding fragment, in accordance with the compositions and methods are also described herein.
  • amatoxin derivative or “amanitin derivative” refers to an amatoxin that has been chemically modified at one or more positions relative to a naturally occurring amatoxin, such as ⁇ -amanitin, ⁇ -amanitin, ⁇ -amanitin, ⁇ -amanitin, amanin, amaninamide, amanullin, amanullinic acid, or proamanullin.
  • the derivative may be obtained by chemical modification of a naturally occurring compound (“semi-synthetic”), or may be obtained from an entirely synthetic source. Synthetic routes to various amatoxin derivatives are disclosed in, for example, U.S. Pat. No.
  • amatoxin or derivative thereof is represented by formula (V):
  • Q is S.
  • amatoxin or derivative thereof is represented by formula (Va):
  • Q is S.
  • amatoxin or derivative thereof is represented by formula (Vb):
  • Q is S.
  • Linker means a divalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody or fragment thereof (Ab) to an amatoxin as described herein, e.g., an amatoxin of formulae (IV), (IVa), (IVb), (V), (Va), or (Vb), to form an antibody-drug conjugate (ADC).
  • bivalent linker reagents which are useful to attach two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups are known, and methods have been described their resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, p. 234-242).
  • present linkers have two reactive termini, one for conjugation to an antibody and the other for conjugation to an amatoxin.
  • the antibody conjugation reactive terminus of the linker (reactive moiety, defined herein as Z′) is typically a chemical moiety that is capable of conjugation to the antibody through, e.g., a cysteine thiol or lysine amine group on the antibody, and so is typically a thiol-reactive group such as a Michael acceptor (as in maleimide), a leaving group, such as a chloro, bromo, iodo, or an R-sulfanyl group, or an amine-reactive group such as a carboxyl group. Conjugation of the linker to the antibody is described more fully herein below.
  • the amatoxin conjugation reactive terminus of the linker is typically a chemical moiety that is capable of conjugation to the amatoxin through formation of a bond with a reactive substituent within the amatoxin molecule.
  • Non-limiting examples include, for example, formation of an amide bond with a basic amine or carboxyl group on the amatoxin, via a carboxyl or basic amine group on the linker, respectively, or formation of an ether or the like, via alkylation of an OH group on the amatoxin via e.g., a leaving group on the linker.
  • linker when the term “linker” is used in describing the linker in conjugated form, one or both of the reactive termini will be absent (such as reactive moiety Z′, having been converted to chemical moiety Z, as described herein below) or incomplete (such as being only the carbonyl of the carboxylic acid) because of the formation of the bonds between the linker and/or the amatoxin, and between the linker and/or the antibody or antigen-binding fragment thereof.
  • conjugation reactions are described further herein below.
  • linkers suitable for the present disclosure may be substantially stable in circulation, but allow for release of the amatoxin within or in close proximity to the target cells.
  • certain linkers suitable for the present disclosure may be categorized as “cleavable” or “non-cleavable”.
  • cleavable linkers contain one or more functional groups that is cleaved in response to a physiological environment.
  • a cleavable linker may contain an enzymatic substrate (e.g., valine-alanine) that degrades in the presence of an intracellular enzyme (e.g., cathepsin B), an acid-cleavable group (e.g., a hydrozone) that degrades in the acidic environment of a cellular compartment, or a reducible group (e.g., a disulfide) that degrades in an intracellular reducing environment.
  • an intracellular enzyme e.g., cathepsin B
  • an acid-cleavable group e.g., a hydrozone
  • a reducible group e.g., a disulfide
  • Non-cleavable linkers suitable for use herein further may include one or more groups selected from a bond, —(C ⁇ O)—, C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted, and/or may include one or more heteroatoms (e.g., S, N, or O) in place of one or more carbon atoms.
  • heteroatoms e.g., S, N, or O
  • Non-limiting examples of such groups include (CH 2 ) p , (C ⁇ O)(CH 2 ) p , and polyethyleneglycol (PEG; (CH 2 CH 2 O) p ), units, wherein p is an integer from 1-6, independently selected for each occasion.
  • the linker L comprises one or more of a bond, —(C ⁇ O)—, a —C(O)NH— group, an —OC(O)NH— group, C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, a —(CH 2 CH 2 O) p — group where p is an integer from 1-6, or a solubility enhancing group;
  • each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may optionally be substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, tri
  • each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may optionally be interrupted by one or more heteroatoms selected from O, S and N.
  • each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may optionally be interrupted by one or more heteroatoms selected from O, S and N and may be optionally substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl,
  • the linker L comprises a solubility enhancing group of the formula —O a —C(O)NH—SO 2 —N(R 1 )—, wherein:
  • a is 0 or 1
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, each of which may be optionally substituted or optionally interrupted by one or more heteroatoms selected from O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl.
  • solubility enhancing groups are described in, for example, U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017/0298145, the disclosures of each of which are incorporated herein by reference in their entirety.
  • the solubility enhancing group of the formula —O a —C(O)NH—SO 2 —N(R′)— further comprises a C 1 -C 6 alkylene or a —(CH 2 CH 2 O) p — group, where p is an integer from 1-6.
  • solubility enhancing groups include those depicted in Table 2, above.
  • the non-cleavable linker comprises a —(CH 2 ) n — unit, where n is an integer from, 2-12, e.g., 2-6. In some embodiments, the non-cleavable linker comprises a —(CH 2 ) n — where n is 1, 2, 3, 4, 5, or 6. In some embodiments, the non-cleavable linker is —(CH 2 ) n — where n is 6, represented by the formula:
  • the linker conjugating the antibody or antigen binding fragment thereof and the amatoxin is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment.
  • Cleavable linkers are designed to exploit the differences in local environments, e.g., extracellular and intracellular environments, including, for example, pH, reduction potential or enzyme concentration, to trigger the release of the amatoxin in the target cell.
  • cleavable linkers are relatively stable in circulation, but are particularly susceptible to cleavage in the intracellular environment through one or more mechanisms (e.g., including, but not limited to, activity of proteases, peptidases, and glucuronidases).
  • Cleavable linkers used herein are substantially stable in circulating plasma and/or outside the target cell and may be cleaved at some efficacious rate inside the target cell or in close proximity to the target cell.
  • Suitable cleavable linkers include those that may be cleaved, for instance, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it pertains to linkers suitable for covalent conjugation).
  • Suitable cleavable linkers may include, for example, chemical moieties such as a hydrazine, a disulfide, a thioether or a dipeptide.
  • Linkers hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like.
  • linkers suitable for covalent conjugation include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, or the like.
  • Linkers cleavable under reducing conditions include, for example, a disulfide.
  • a variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT (See, e.g., Thorpe et al., 1987, Cancer Res.
  • Linkers susceptible to enzymatic hydrolysis can be, e.g., a peptide-containing linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.
  • intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stabilities of the conjugates are typically high.
  • the peptidyl linker is at least two amino acids long or at least three amino acids long.
  • Exemplary amino acid linkers include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
  • suitable peptides include those containing amino acids such as Valine, Alanine, Citrulline (Cit), Phenylalanine, Lysine, Leucine, and Glycine.
  • Amino acid residues which comprise an amino acid linker component include those occurring naturally, as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline.
  • Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe).
  • Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly).
  • the linker includes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit.
  • Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety as it pertains to linkers suitable for covalent conjugation.
  • the linker comprises a dipeptide selected from Val-Ala and Val-Cit.
  • Linkers suitable for conjugating the antibodies, antigen-binding fragments, described herein to a cytotoxic molecule include those capable of releasing an amatoxin by a 1,6-elimination process.
  • Chemical moieties capable of this elimination process include the p-aminobenzyl (PAB) group, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents as described in Jain et al., Pharm. Res. 32:3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety as it pertains to linkers suitable for covalent conjugation.
  • PAB p-aminobenzyl
  • the linker includes a “self-immolative” group such as the afore-mentioned PAB or PABC (para-aminobenzyloxycarbonyl), which are disclosed in, for example, Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644; U.S. Pat. No. 6,214,345; US20030130189; US20030096743; U.S. Pat. No. 6,759,509; US20040052793; U.S. Pat. Nos.
  • PAB para-aminobenzyloxycarbonyl
  • self-immolative linkers include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed in, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114, and U.S. Pat. No. 7,754,681; Hay et al.
  • a dipeptide is used in combination with a self-immolative linker.
  • the linker L comprises one or more of a hydrazine, a disulfide, a thioether, an amino acid, a peptide consisting of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, heteroaryl, a —(C ⁇ O)— group, a —C(O)NH— group, an —OC(O)NH— group, a —(CH 2 CH 2 O) p — group where p is an integer from 1-6, or a solubility enhancing group;
  • PAB p-a
  • each C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be optionally substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy,
  • each C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may optionally be interrupted by one or more heteroatoms selected from O, S and N.
  • each C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may optionally be interrupted by one or more heteroatoms selected from O, S and N and may be optionally substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl
  • one or more of the groups listed may be present in the form of a bivalent (diradical) species, e.g., C 1 -C 6 alkylene and the like.
  • the linker L comprises a solubility enhancing group of the formula —O a —C(O)NH—SO 2 —N(R 1 )—, wherein:
  • a is 0 or 1
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, each of which may be optionally substituted or optionally interrupted by one or more heteroatoms selected from O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl.
  • solubility enhancing groups are described in, for example, U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017/0298145, the disclosures of each of which are incorporated herein by reference in their entirety.
  • the solubility enhancing group of the formula —O a —C(O)NH—SO 2 —N(R 1 )— further comprises a C 1 -C 6 alkylene or a —(CH 2 CH 2 O) p — group, where p is an integer from 1-6.
  • Such solubility enhancing groups include those depicted in Table 2, above.
  • the linker includes a p-aminobenzyl group (PAB).
  • PAB p-aminobenzyl group
  • the p-aminobenzyl group is disposed between the cytotoxic drug and a protease cleavage site in the linker.
  • the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit.
  • the p-aminobenzyl group is part of a p-aminobenzylamido unit.
  • the linker comprises a dipeptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg.
  • the linker comprises one or more of PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.
  • the linker comprises a combination of one or more of a peptide, oligosaccharide, —(CH 2 ) p —, —(CH 2 CH 2 O) p —, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.
  • the linker comprises a —(C ⁇ O)(CH 2 ) p — unit, wherein p is an integer from 1-6.
  • the linker comprises PAB-Ala-Val-propionyl, represented by the formula:
  • the linker comprises PAB-Cit-Val-propionyl, represented by the formula:
  • PAB-dipeptide-propionyl linkers are disclosed in, e.g., International Patent Application Publication No. WO2017/149077, which is incorporated by reference herein in its entirety. Further, the cytotoxins disclosed in WO2017/149077 are incorporated by reference herein.
  • the linker is reacted with an amatoxin or derivative thereof according to any of formulae (V), (Va), or (Vb) under appropriate conditions to form a linker-amatoxin conjugate.
  • reactive groups are used on the amatoxin or linker to form a covalent attachment.
  • the amatoxin-linker conjugate is subsequently reacted with the antibody, derivatized antibody, or antigen-binding fragment thereof, under appropriate conditions to form the ADC.
  • the linker may first be reacted with the antibody, derivatized antibody or antigen-binding fragment thereof, to form a linker-antibody conjugate, and then reacted with the amatoxin to form the ADC.
  • Suitable attachment points on the antibody molecule include, but are not limited to, the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of aromatic amino acids.
  • non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a linker to an amino (or carboxy) group on an antibody moiety.
  • bifunctional agents such as dialdehydes or imidoesters may also be used to link the amino group on a linker to an amino group on an antibody moiety.
  • Schiff base reaction also available for attachment of amatoxins to antibody moieties. This method involves the periodate oxidation of a glycol or hydroxy group on either the antibody or linker, thus forming an aldehyde which is then reacted with the linker or antibody, respectively. Covalent bond formation occurs via formation of a Schiff base between the aldehyde and an amino group.
  • Isothiocyanates may also be used as coupling agents for covalently attaching amatoxins or antibody moieties to linkers. Other techniques are known to the skilled artisan and within the scope of the present disclosure.
  • Linkers useful in for conjugation to the antibodies or antigen-binding fragments as described herein include, without limitation, linkers containing a chemical moiety Z, formed by a coupling reaction between the antibody and a reactive chemical moiety (referred to herein as a reactive substituent, Z′) on the linker as depicted in Table 3, below. Wavy lines designate points of attachment to the antibody or antigen-binding fragment, and the cytotoxic molecule, respectively.
  • antibody-drug conjugates useful in conjunction with the methods described herein may be formed by the reaction of an antibody, or antigen-binding fragment thereof, with a linker or amatoxin-linker conjugate, as described herein, the linker or amatoxin-linker conjugate including a reactive substituent Z′, suitable for reaction with a reactive substituent on the antibody, or antigen-binding fragment thereof, to form the chemical moiety Z.
  • examples of suitably reactive substituents Z′ on the linker and reactive substituents on the antibody or antigen-binding fragment thereof include a nucleophile/electrophile pair (e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thiol/ ⁇ , ⁇ -unsaturated carbonyl pair, and the like), a diene/dienophile pair (e.g., an azide/alkyne pair, or a diene/ ⁇ , ⁇ -unsaturated carbonyl pair, among others), and the like.
  • a nucleophile/electrophile pair e.g., a thiol/haloalkyl pair, an amine/carbonyl pair, or a thiol/ ⁇ , ⁇ -unsaturated carbonyl pair, and the like
  • a diene/dienophile pair e.g., an azide/alkyne pair, or a diene/
  • Coupling reactions between the reactive substitutents to form the chemical moiety Z include, without limitation, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive modalities known in the art or described herein.
  • the reactive substituent Z′ is an electrophilic functional group suitable for reaction with a nucleophilic functional group on the antibody, or antigen-binding fragment thereof.
  • Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, nucleophilic groups such as (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • nucleophilic groups such as (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated.
  • Reactive substituents that may be present within an antibody, or antigen-binding fragment thereof, as disclosed herein include, without limitation, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine residues, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.
  • haloaryl e.g., fluoroaryl
  • haloheteroaryl e.g., fluoroheteroaryl
  • haloalkyl e.g., fluoroheteroaryl
  • the reactive substituents present within an antibody, or antigen-binding fragment thereof as disclosed herein include, are amine or thiol moieties.
  • Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges.
  • Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol).
  • DTT dithiothreitol
  • Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.
  • Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues).
  • cysteine residues e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues.
  • U.S. Pat. No. 7,521,541 teaches engineering antibodies by introduction of reactive cysteine amino acids.
  • the reactive substituent Z′ attached to the linker is a nucleophilic group which is reactive with an electrophilic group present on an antibody.
  • Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups.
  • a nucleophilic group (e.g., a) heteroatom of can react with an electrophilic group on an antibody and form a covalent bond to the antibody.
  • Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • chemical moiety Z is the product of a reaction between reactive nucleophilic substituents present within the antibodies, or antigen-binding fragments thereof, such as amine and thiol moieties, and a reactive electrophilic substituent Z′ attached to the linker.
  • Z′ may be a Michael acceptor (e.g., maleimide), activated ester, electron-deficient carbonyl compound, or an aldehyde, among others.
  • linkers suitable for the synthesis of linker-antibody conjugates and ADCs include, without limitation, reactive substituents Z′ attached to the linker, such as a maleimide or haloalkyl group. These may be attached to the linker by, for example, reagents such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others described, in for instance, Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
  • reagents such as succinimidyl 4-(N-maleimidomethyl)
  • the reactive substituent Z′ attached to linker L is a maleimide, azide, or alkyne.
  • a maleimide-containing linker is the non-cleavable maleimidocaproyl-based linker, which is particularly useful for the conjugation of microtubule-disrupting agents such as auristatins.
  • Such linkers are described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it pertains to linkers for chemical conjugation.
  • the reactive substituent Z′ is —(C ⁇ O)— or —NH(C ⁇ O)—, such that the linker may be joined to the antibody, or antigen-binding fragment thereof, by an amide or urea moiety, respectively, resulting from reaction of the —(C ⁇ O)— or —NH(C ⁇ O)— group with an amino group of the antibody or antigen-binding fragment thereof.
  • the reactive substituent Z′ is an N-maleimidyl group, halogenated N-alkylamido group, sulfonyloxy N-alkylamido group, carbonate group, sulfonyl halide group, thiol group or derivative thereof, alkynyl group comprising an internal carbon-carbon triple bond, (hetero)cycloalkynyl group, bicyclo[6.1.0]non-4-yn-9-yl group, alkenyl group comprising an internal carbon-carbon double bond, cycloalkenyl group, tetrazinyl group, azido group, phosphine group, nitrile oxide group, nitrone group, nitrile imine group, diazo group, ketone group, (O-alkyl)hydroxylamino group, hydrazine group, halogenated N-maleimidyl group, 1,1-bis (sulfonylmethyl)methylcarbonyl group or elimination
  • the chemical moiety Z is selected from Table 3 or Table 4. In some embodiments, the chemical moiety Z is:
  • S is a sulfur atom which represents the reactive substituent present within an antibody, or antigen-binding fragment thereof, that specifically binds to an antigen expressed on the cell surface of a human stem cell or a T cell (e.g., from the —SH group of a cysteine residue).
  • the linker-reactive substituent group taken together as L-Z′, prior to conjugation with the antibody or antigen binding fragment thereof, has the structure:
  • linker-Z′ N-beta-maleimidopropyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB).
  • BMP-Val-Ala-PAB N-beta-maleimidopropyl-Val-Ala-para-aminobenzyl
  • the wavy line at the linker terminus indicates the point of attachment to the amatoxin.
  • the linker L and the chemical moiety Z, after conjugation to the antibody, taken together as L-Z-Ab has the structure:
  • S is a sulfur atom which represents the reactive substituent present within an antibody, or antigen-binding fragment thereof, that specifically binds to an antigen expressed on the cell surface of a human stem cell or a T cell (e.g., from the —SH group of a cysteine residue.
  • the wavy line at the linker terminus indicates the point of attachment to the amatoxin.
  • the linker-reactive substituent group taken together as L-Z′, prior to conjugation with the antibody or antigen binding fragment thereof, has the structure:
  • This linker-reactive substituent group may alternatively be referred to as 1-n-hexyl-maleimide, which is a non-cleavable linker.
  • the wavy line at the linker terminus indicates the point of attachment to the amatoxin.
  • the linker L and the chemical moiety Z after conjugation to the antibody, taken together as L-Z-Ab, has the structure:
  • S is a sulfur atom which represents the reactive substituent present within an antibody, or antigen-binding fragment thereof, that specifically binds to an antigen expressed on the cell surface of a human stem cell or a T cell (e.g., from the —SH group of a cysteine residue).
  • the wavy line at the linker terminus indicates the point of attachment to the amatoxin.
  • the linker-reactive substituent group structure L-Z′ prior to conjugation with the antibody or antigen binding fragment thereof, includes a maleimide as the group Z′.
  • the cytotoxin of the ADC as disclosed herein is an amatoxin or derivative thereof as represented by any of formulae (V), (Va), or (Vb).
  • amatoxin or derivative thereof as represented by any of formulae (V), (Va), or (Vb).
  • the amatoxin has the structure of formula (V), and the linker is attached by an ether bond to the OH group of the hydroxyl tryptophan residue.
  • the ADC may be represented by formula (I):
  • L is a linker
  • Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody, or antigen-binding fragment thereof;
  • Ab is an antibody
  • the amatoxin has the structure of formula (Va), and the linker is attached by an ether bond to the OH group of the hydroxyl tryptophan residue.
  • the ADC may be represented by formula (Ia):
  • the amatoxin has the structure of formula (Vb), and the linker is attached by an ether bond to the OH group of the hydroxyl tryptophan residue.
  • the ADC may be represented by formula (Ib):
  • the linker L of the ADC of formula (I), (Ia), or (Ib) is a non-cleavable linker.
  • the non-cleavable linker L comprises one or more of a bond, —(C ⁇ O)—, a —C(O)NH— group, an —OC(O)NH— group, C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, a —(CH 2 CH 2 O) p — group where p is an integer from 1-6, or a solubility enhancing group;
  • each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may optionally be substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, tri
  • each C 1 -C 6 alkylene, C 1 -C 6 heteroalkylene, C 2 -C 6 alkenylene, C 2 -C 6 heteroalkenylene, C 2 -C 6 alkynylene, C 2 -C 6 heteroalkynylene, C 3 -C 6 cycloalkylene, heterocycloalkylene, arylene, or heteroarylene may optionally be interrupted by one or more heteroatoms selected from O, S and N.
  • the non-cleavable linker L comprises a solubility enhancing group of the formula —O a —C(O)NH—SO 2 —NR 1 —, wherein:
  • a is 0 or 1
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, each of which may be optionally substituted, optionally interrupted, or both, by one or more heteroatoms selected from O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl.
  • the non-cleavable linker L comprises a —(CH 2 ) n — unit, where n is an integer from 2-6. In some embodiments, the non-cleavable linker L is —(CH 2 ) n —, where n is 6.
  • Ab, Z, and the non-cleavable linker L, taken together as Ab-Z-L is represented by the formula:
  • S is a sulfur atom which represents the reactive substituent present within the antibody, or antigen-binding fragment thereof, that specifically binds to an antigen expressed on a cell, e.g., surface of a human stem cell or a T cell (e.g., from the —SH group of a cysteine residue).
  • a cell e.g., surface of a human stem cell or a T cell (e.g., from the —SH group of a cysteine residue).
  • the wavy line at the linker terminus indicates the point of attachment to the amatoxin.
  • the ADC according to formula (I) is represented by formula (II):
  • the ADC according to formula (II) is represented by formula (IIa):
  • the ADC according to formula (II) is represented by formula (Ib):
  • ADCs comprising an amatoxin and a non-cleavable linker conjugating the amatoxin to the antibody moiety of the ADC have improved tolerability as compared to an ADC comprising an amatoxin and a cleavable linker.
  • the improved tolerability may be increased therapeutic index.
  • the improved tolerability may be a smaller elevation, or absence of elevation, of one or more blood liver enzyme levels (e.g., AST, ALT, ADH, or total bilirubin) at a particular dose of the ADC comprising a non-cleavable linker as compared to an ADC comprising a cleavable linker.
  • blood liver enzyme levels e.g., AST, ALT, ADH, or total bilirubin
  • the linker L of the ADC of formula (I), (Ia), or (Ib) is a cleavable linker.
  • the cleavable linker L comprises one or more of a hydrazine, a disulfide, a thioether, an amino acid, a peptide consisting of up to 10 amino acids, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, heteroaryl, a —(C ⁇ O)— group, a —C(O)NH— group, an —OC(O)NH
  • each C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may be optionally substituted with from 1 to 5 substituents independently selected for each occasion from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy,
  • each C 1 -C 6 alkyl, C 1 -C 6 heteroalkyl, C 2 -C 6 alkenyl, C 2 -C 6 heteroalkenyl, C 2 -C 6 alkynyl, C 2 -C 6 heteroalkynyl, C 3 -C 6 cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group may optionally be interrupted by one or more heteroatoms selected from O, S and N.
  • the cleavable linker L comprises a solubility enhancing group of the formula —O a —C(O)NH—SO 2 —NR 1 —, wherein:
  • a is 0 or 1
  • R 1 is selected from the group consisting of hydrogen, C 1 -C 24 alkyl groups, C 3 -C 24 cycloalkyl groups, C 2 -C 24 (hetero)aryl groups, C 3 -C 24 alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, each of which may be optionally substituted or optionally interrupted by one or more heteroatoms selected from O, S and NR 3 , wherein R 3 is independently selected from the group consisting of hydrogen and C 1 -C 4 alkyl.
  • the cleavable linker L comprises a peptide selected from the group consisting of Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg.
  • the cleavable linker L further comprises a PAB group.
  • the cleavable linker L is represented by the formula:
  • an antibody or antigen binding fragment thereof is conjugated to one or more cytotoxic drug moieties (Cy; e.g., an amatoxin), for example, from about 1 to about 20 cytotoxic moieties per antibody, through a linker L and a chemical moiety Z, each as disclosed herein.
  • cytotoxic drug moieties e.g., an amatoxin
  • n is 1.
  • n is about 1 to about 5, about 1 to about 4, about 1 to about 3, or about 3 to about 5.
  • n is about 1, about 2, about 3, or about 4.
  • the ADCs of the present disclosure may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a reactive substituent of an antibody or antigen binding fragment thereof with a bivalent linker reagent to form Ab-Z-L as described herein above, followed by reaction with a cytotoxic moiety Cy; or (2) reaction of a reactive substituent of a cytotoxic moiety with a bivalent linker reagent to form Cy-L-Z′, followed by reaction with a reactive substituent of an antibody or antigen binding fragment thereof as described herein above, to form an ADC of formula Ab-(Z-L-Cy) n . Additional methods for preparing ADC are described herein.
  • the antibody or antigen binding fragment thereof can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups.
  • the ADC is then formed by conjugation through the sulfhydryl group's sulfur atom as described herein above.
  • the antibody can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (—CHO) group (see, for e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55).
  • the ADC is then formed by conjugation through the corresponding aldehyde as described herein above.
  • Other protocols for the modification of proteins for the attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.
  • linker-drug moieties to cell-targeted proteins such as antibodies, immunoglobulins or fragments thereof are found, for example, in U.S. Pat. Nos. 5,208,020; 6,441,163; WO2005037992; WO2005081711; and WO2006/034488, all of which are hereby expressly incorporated by reference in their entirety.
  • a fusion protein comprising the antibody and cytotoxic agent may be made, e.g., by recombinant techniques or peptide synthesis.
  • the length of DNA may comprise respective regions encoding the two portions of the conjugate either adjacent one another or separated by a region encoding a linker peptide which does not destroy the desired properties of the conjugate.
  • the ADC of formula (IIa) may be prepared by conjugation of a thiol group on the antibody to the amatoxin-linker conjugate Cy-L-Z′, represented by the structure:
  • This amatoxin-linker conjugate may be prepared according to Schemes 1 to 3, starting from commercially available 6-hydroxytryptophan (1).
  • Compound 1 may be protected with tert-butyloxycarbonyl anhydride ((BOC) 2 O).
  • Hexahydropyrroloindole 3 may be produced as a mixture of cis and trans isomers upon irradiation of protected hydroxytryptophan 2 in the presence of oxygen and a sensitizer (Rose Bengal).
  • Preparation of compound 3 (as well as procedure for preparation of compounds 2 to Va) are provided in International Patent Application Publication No. WO2019/030173, the disclosure of which is incorporated by reference herein in its entirety.
  • FMOC protected 4-hydroxyproline (4) may be alkylated with allyl bromide and subsequently attached to tetrahydropyranyl (THP) polystyrene resin under mild acidic conditions (pyridinium para-toluenesulfonate; PPTS) to give the resin bound allyl ester 5, which may be deprotected with palladium tetrakistriphenylphosphine palladium and dimethyl barbituric acid to give intermediate 6.
  • THP tetrahydropyranyl
  • PPTS pyridinium para-toluenesulfonate
  • Protected amino acid 7 may be prepared according to the method reported in International Patent Application Publication No. WO2014/009025, the disclosure of which is incorporated herein in its entirety. Protected amine 7 may then be coupled to resin bound hydroxyproline 6 to afford peptide 8.
  • Peptide 8 may then be subjected to multiple coupling and deprotection reactions to provide monocyclic intermediate 9 (Scheme 2).
  • Amino acids FmocAsn(Trt)OH, FmocCys(OTrt)OH, FmocGlyOH, FmocIleOH, FmocGlyOH, and hexahydropyrroloindole 3 may be utilized in sequential solid phase coupling reactions to yield intermediate 9.
  • Each amino acid may be coupled using PyBOP/HOBT in dichloromethane and dimethylformamide (DMF) in the presence of diisopropylethylamine (DIEA).
  • DIEA diisopropylethylamine
  • Deprotection may be performed with 20% piperidine in DMF.
  • intermediate 9 may be cleaved from the solid phase support resin with trifluoroacetic acid in the presence of triisopropylsilane to give peptide 10.
  • DPPA diphenylphosphorylazide
  • DIEA DPPA diphenylphosphorylazide
  • the maleimidohexyl amatoxin conjugate 14 may be prepared from compound 11 according to Scheme 3.
  • Diels-Alder adduct 12 may be prepared from maleimide and 2,5-dimethylfuran and then alkylated with 1,6-dibromohexane to give protected linker 13.
  • Compound 11 may be alkylated with compound 13 in dimethyl sulfoxide (DMSO) in the presence of sodium hydroxide, followed by heating to 100° C. in DMSO to afford the amatoxin-linker conjugate 14.
  • DMSO dimethyl sulfoxide
  • Procedures for preparing compounds 12 and 13, as well as O-alkylation of a related amatoxin ( ⁇ -amanitin) have been previously reported in U.S. Patent Application Publication No. 2018/0043033, the disclosure of which is incorporated by reference herein in its entirety.
  • the ADC compositions and methods disclosed herein comprise an agent to facilitate the selective delivery of such ADCs to a population of cells in the target tissues (e.g., hematopoietic stem cells of the bone marrow stem cell niche).
  • the cell target specificity of the ADC is determined by an antigen binding protein such as an antibody, or antigen binding portion thereof.
  • the invention includes ADCs comprising antibodies, and antigen-binding fragments thereof, that specifically bind to human CD45, CD49d (VLA-4), CD49f(VLA-6), CD51, CD84, CD90, CD117, CD133, CD134, CD184 (CXCR4), HLA-DR, CD11a, CD18, CD34, CD41/61, CD43, CD58, CD71, CD97, CD162, CD166, CD205 and CD361, CD13, CD33, CD34, CD44, CD4, CD59, CD84/CD150, CD90/Thy1, CD93, CD105/Endoglin, CD123/IL-3R, CD126/IL-6R, CD133, CD135/Flt3 receptor, CD166/ALCAM, Prominin 2, Erythropoietin R, Endothelial Cell-Selective Adhesion Molecule, CD244, Tie1, Tie2, MPL, G-CSFR, CSF3R, IL-1R, g
  • antigens that can be bound by the ADCs disclosed herein include, but are not limited to, CD7, CDw12, CD13, CD15, CD19, CD21, CD22, CD29, CD30, CD33, CD34, CD36, CD38, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD45RA, CD45RB, CD45RC, CD45RO, CD48, CD49b, CD49d, CD49e, CD49f, CD50, CD53, CD55, CD64a, CD68, CD71, CD72, CD73, CD81, CD82, CD85A, CD85K, CD90, CD99, CD104, CD105, CD109, CD110, CD111, CD112, CD114, CD115, CD123, CD124, CD126, CD127, CD130, CD131, CD133, CD135, CD138, CD151, CD157, CD162, CD164, CD168, CD172a, CD173, CD174, CD175, CD175s,
  • antigens that can be bound by the ADCs disclosed herein include, but are not limited to, CD11a, CD18, CD37, CD47, CD52, CD58, CD62L, CD69, CD74, CD97, CD103, CD132, CD156a, CD179a, CD79b, CD184, CD232, CD244, CD252, CD302, CD305, CD317 and CD361.
  • an antibody, or antigen binding fragment thereof, in an ADC described herein has a certain dissociation rate which is particularly advantageous when used as a part of a conjugate.
  • an anti-CD117 antibody has, in certain embodiments, an off rate constant (Koff) for human CD117 and/or rhesus CD117 of 1 ⁇ 10 ⁇ 2 to 1 ⁇ 10 ⁇ 3 , 1 ⁇ 10 ⁇ 3 to 1 ⁇ 10 ⁇ 4 , 1 ⁇ 10 ⁇ 5 to 1 ⁇ 10 ⁇ 6 , 1 ⁇ 10 ⁇ 6 to 1 ⁇ 10 ⁇ 7 or 1 ⁇ 10 ⁇ 7 to 1 ⁇ 10 ⁇ 8 , as measured by bio-layer interferometry (BLI).
  • Koff off rate constant
  • the antibody or antigen-binding fragment thereof binds a cell surface antigen (e.g., human CD117 and/or rhesus CD117) with a K D of about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 8 nM or less, about 6 nM or less, about 4 nM or less, about 2 nM or less, about 1 nM or less as determined by a Bio-Layer Interferometry (BLI) assay.
  • a cell surface antigen e.g., human CD117 and/or rhesus CD117
  • the present invention includes ADCs comprising antibodies, and antigen-binding fragments thereof, that specifically bind to CD117, such as GNNK+ CD117.
  • ADCs may be used as therapeutic agents to, for example, (i) treat cancers and autoimmune diseases characterized by CD117+ cells and (ii) promote the engraftment of transplanted hematopoietic stem cells in a patient in need of transplant therapy.
  • These therapeutic activities can be caused, for instance, by the binding of isolated anti-CD117 antibodies, antigen-binding fragments thereof, that bind to CD117 (e.g., GNNK+ CD117) expressed on the surface of a cell, such as a cancer cell, autoimmune cell, or hematopoietic stem cell and subsequently inducing cell death.
  • CD117 e.g., GNNK+ CD117
  • the depletion of endogenous hematopoietic stem cells can provide a niche toward which transplanted hematopoietic stem cells can home, and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells may successfully engraft in a patient, such as human patient suffering from a stem cell disorder described herein.
  • Antibodies and antigen-binding fragments capable of binding human CD117 can be used in conjunction with the compositions and methods described herein in order to condition a patient for hematopoietic stem cell transplant therapy.
  • Polymorphisms affecting the coding region or extracellular domain of CD117 in a significant percentage of the population are not currently well-known in non-oncology indications.
  • GNNK+ and GNNK ⁇ Two of the CD117 isoforms are located on the intracellular domain of the protein, and two are present in the external juxtamembrane region.
  • the two extracellular isoforms, GNNK+ and GNNK ⁇ differ in the presence (GNNK+) or absence (GNNK ⁇ ) of a 4 amino acid sequence. These isoforms are reported to have the same affinity for the ligand (SCF), but ligand binding to the GNNK ⁇ isoform was reported to increase internalization and degradation.
  • SCF ligand
  • the GNNK+ isoform can be used as an immunogen in order to generate antibodies capable of binding CD117, as antibodies generated against this isoform will be inclusive of the GNNK+ and GNNK ⁇ proteins.
  • anti-human CD117 (hCD117) antibodies disclosed herein are able to bind to both isoform 1 and isoform 2 of human CD117.
  • Antibody 54 (Ab54), Antibody 55 (Ab55), Antibody 56 (Ab56), Antibody 57 (Ab57), Antibody 58 (Ab58), Antibody 61 (Ab61), Antibody 66 (Ab66), Antibody 67 (Ab67), Antibody 68 (Ab68), and Antibody 69 (Ab69) were human antibodies that were identified in this screen. These antibodies cross react with human CD117 and rhesus CD117. Further, these antibodies disclosed herein are able to bind to both isoforms of human CD117, i.e., isoform 1 (SEQ ID NO: 145) and isoform 2 (SEQ ID NO: 146).
  • amino acid sequences for the various binding regions of anti-CD117 antibodies including Ab54, Ab55, Ab56, Ab57, Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69 are described in the Sequence Table below. Included in the invention are ADCs comprising human anti-CD117 antibodies comprising the CDRs as set forth in the Sequence Table below, as well as human anti-CD117 antibodies comprising the variable regions set forth in the Sequence Table below.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 55.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 55 i.e., Ab55
  • the VH CDR domain amino acid sequences of Antibody 55 are set forth in SEQ ID NO: 21 (VH CDR1); SEQ ID NO: 22 (VH CDR2), and SEQ ID NO: 23 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 55 is described in SEQ ID NO: 20 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 21, 22, and 23, and a light chain variable region CDR set as set forth in SEQ ID Nos: 24, 25, and 26.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 20, and a heavy chain variable region as set forth in SEQ ID NO: 19.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 54.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 54 i.e., Ab54
  • the VH CDR domain amino acid sequences of Antibody 54 are set forth in SEQ ID NO: 31 (VH CDR1); SEQ ID NO: 32 (VH CDR2), and SEQ ID NO: 33 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 54 is described in SEQ ID NO: 30 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 31, 32, and 33, and a light chain variable region CDR set as set forth in SEQ ID Nos: 34, 35, and 36.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 30, and a heavy chain variable region as set forth in SEQ ID NO: 29.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 56.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 56 i.e., Ab56
  • the VH CDR domain amino acid sequences of Antibody 56 are set forth in SEQ ID NO: 41 (VH CDR1); SEQ ID NO: 42 (VH CDR2), and SEQ ID NO: 43 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 56 is described in SEQ ID NO: 40 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 41, 42, and 43, and a light chain variable region CDR set as set forth in SEQ ID Nos: 44, 45, and 46.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 40, and a heavy chain variable region as set forth in SEQ ID NO: 39.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 57.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 57 i.e., Ab57
  • the VH CDR domain amino acid sequences of Antibody 57 are set forth in SEQ ID NO: 51 (VH CDR1); SEQ ID NO: 52 (VH CDR2), and SEQ ID NO: 53 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 57 is described in SEQ ID NO: 50 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 51, 52, and 53, and a light chain variable region CDR set as set forth in SEQ ID Nos: 54, 55, and 56.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 50, and a heavy chain variable region as set forth in SEQ ID NO: 49.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 58.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 58 i.e., Ab58
  • SEQ ID NO: 59 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 58 are set forth in SEQ ID NO: 61 (VH CDR1); SEQ ID NO: 62 (VH CDR2), and SEQ ID NO: 63 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 58 is described in SEQ ID NO: 60 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 58 are set forth in SEQ ID NO: 64 (VL CDR1); SEQ ID NO: 65 (VL CDR2), and SEQ ID NO: 66 (VL CDR3).
  • the heavy chain constant region of Antibody 58 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 58 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 61, 62, and 63, and a light chain variable region CDR set as set forth in SEQ ID Nos: 64, 65, and 66.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 60, and a heavy chain variable region as set forth in SEQ ID NO: 59.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 61.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 61 i.e., Ab61
  • SEQ ID NO: 69 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 61 are set forth in SEQ ID NO: 71 (VH CDR1); SEQ ID NO: 72 (VH CDR2), and SEQ ID NO: 73 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 61 is described in SEQ ID NO: 70 (see Sequence Table).
  • VL CDR domain amino acid sequences of Antibody 61 are set forth in SEQ ID NO: 74 (VL CDR1); SEQ ID NO: 75 (VL CDR2), and SEQ ID NO: 76 (VL CDR3).
  • the heavy chain constant region of Antibody 61 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 61 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 71, 72, and 73, and a light chain variable region CDR set as set forth in SEQ ID Nos: 74, 75, and 76.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 70, and a heavy chain variable region as set forth in SEQ ID NO: 69.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 66.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 66 i.e., Ab66
  • SEQ ID NO: 79 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 66 are set forth in SEQ ID NO: 81 (VH CDR1); SEQ ID NO: 82 (VH CDR2), and SEQ ID NO: 83 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 66 is described in SEQ ID NO: 80 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 66 are set forth in SEQ ID NO: 84 (VL CDR1); SEQ ID NO: 85 (VL CDR2), and SEQ ID NO: 86 (VL CDR3).
  • the heavy chain constant region of Antibody 66 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 66 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 81, 82, and 83, and a light chain variable region CDR set as set forth in SEQ ID Nos: 84, 85, and 86.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 80, and a heavy chain variable region as set forth in SEQ ID NO: 79.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 67.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 67 is set forth in SEQ ID NO: 9 (see Sequence Table).
  • the VH CDR domain amino acid sequences of Antibody 67 are set forth in SEQ ID NO 11 (VH CDR1); SEQ ID NO: 12 (VH CDR2), and SEQ ID NO: 13 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 67 is described in SEQ ID NO: 10 (see Table 2).
  • VL CDR domain amino acid sequences of Antibody 67 are set forth in SEQ ID NO 14 (VL CDR1); SEQ ID NO: 15 (VL CDR2), and SEQ ID NO: 16 (VL CDR3).
  • the full length heavy chain (HC) of Antibody 67 is set forth in SEQ ID NO: 110, and the full length heavy chain constant region of Antibody 67 is set forth in SEQ ID NO: 122.
  • the light chain (LC) of Antibody 67 is set forth in SEQ ID NO: 109.
  • the light chain constant region of Antibody 67 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 11, 12, and 13, and alight chain variable region CDR set as set forth in SEQ ID Nos: 14, 15, and 16.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain comprising the amino acid residues set forth in SEQ ID NO: 9, and a heavy chain variable region as set forth in SEQ ID NO: 10.
  • an anti-CD117 antibody comprises a heavy chain comprising SEQ ID NO: 110 and a light chain comprising SEQ ID NO: 109.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 68.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 68 i.e., Ab68
  • SEQ ID NO: 89 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 68 are set forth in SEQ ID NO: 91 (VH CDR1); SEQ ID NO: 92 (VH CDR2), and SEQ ID NO: 93 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 68 is described in SEQ ID NO: 90 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 68 are set forth in SEQ ID NO: 94 (VL CDR1); SEQ ID NO: 95 (VL CDR2), and SEQ ID NO: 96 (VL CDR3).
  • the heavy chain constant region of Antibody 68 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 68 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 91, 92, and 93, and a light chain variable region CDR set as set forth in SEQ ID Nos: 94, 95, and 96.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 90, and a heavy chain variable region as set forth in SEQ ID NO: 89.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 69.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 69 i.e., Ab69
  • the VH CDR domain amino acid sequences of Antibody 69 are set forth in SEQ ID NO: 101 (VH CDR1); SEQ ID NO: 102 (VH CDR2), and SEQ ID NO: 103 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 69 is described in SEQ ID NO: 100 (see Sequence Table).
  • VL CDR domain amino acid sequences of Antibody 69 are set forth in SEQ ID NO: 104 (VL CDR1); SEQ ID NO: 105 (VL CDR2), and SEQ ID NO: 106 (VL CDR3).
  • the heavy chain constant region of Antibody 69 is set forth in SEQ ID NO: 122.
  • the light chain constant region of Antibody 69 is set forth in SEQ ID NO: 121.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 101, 102, and 103, and a light chain variable region CDR set as set forth in SEQ ID Nos: 104, 105, and 106.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 100, and a heavy chain variable region as set forth in SEQ ID NO: 99.
  • amino acid sequences for the various binding regions of the anti-CD117 antibodies Ab77, Ab79, Ab81, Ab85, Ab86, Ab87, Ab88, and Ab89 are described in the Sequence Table provided below.
  • Anti-CD117 antibodies having these sequences can also be used in the ADCs described herein.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 77.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 77 i.e., Ab77
  • the VH CDR domain amino acid sequences of Antibody 77 are set forth in SEQ ID NO: 263 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 77 is described in SEQ ID NO: 231 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 263, 2, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 264, 265, and 266.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 231, and a heavy chain variable region as set forth in SEQ ID NO: 147.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 79.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 79 i.e., Ab79
  • the VH CDR domain amino acid sequences of Antibody 79 are set forth in SEQ ID NO: 263 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 79 is described in SEQ ID NO: 233 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 263, 2, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 267, 265, and 266.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 233, and a heavy chain variable region as set forth in SEQ ID NO: 147.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 81.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 81 i.e., Ab81
  • the VH CDR domain amino acid sequences of Antibody 81 are set forth in SEQ ID NO: 263 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 81 is described in SEQ ID NO: 235 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 263, 2, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 264, 268, and 266.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 235, and a heavy chain variable region as set forth in SEQ ID NO: 147.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 85.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 85 i.e., Ab86
  • SEQ ID NO: 243 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 85 are set forth in SEQ ID NO: 245 (VH CDR1); SEQ ID NO: 246 (VH CDR2), and SEQ ID NO: 247 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 85 is described in SEQ ID NO: 242 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 245, 246, and 247, and a light chain variable region CDR set as set forth in SEQ ID Nos: 248, 249, and 250.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 244, and a heavy chain variable region as set forth in SEQ ID NO: 243.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 86.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 86 i.e., Ab86
  • SEQ ID NO: 251 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 86 are set forth in SEQ ID NO: 245 (VH CDR1); SEQ ID NO: 253 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 86 is described in SEQ ID NO: 252 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 86 are set forth in SEQ ID NO: 254 (VL CDR1); SEQ ID NO: 249 (VL CDR2), and SEQ ID NO: 255 (VL CDR3).
  • the heavy chain constant region of Antibody 86 is set forth in SEQ ID NO: 269.
  • the light chain constant region of Antibody 86 is set forth in SEQ ID NO: 283.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 245, 253, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 254, 249, and 255.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 252, and a heavy chain variable region as set forth in SEQ ID NO: 251.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 87.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 87 i.e., Ab87
  • SEQ ID NO: 243 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 87 are set forth in SEQ ID NO: 245 (VH CDR1); SEQ ID NO: 246 (VH CDR2), and SEQ ID NO: 247 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 87 is described in SEQ ID NO: 256 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 87 are set forth in SEQ ID NO: 257 (VL CDR1); SEQ ID NO: 5 (VL CDR2), and SEQ ID NO: 255 (VL CDR3).
  • the heavy chain constant region of Antibody 87 is set forth in SEQ ID NO: 269.
  • the light chain constant region of Antibody 87 is set forth in SEQ ID NO: 283.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 245, 246, and 247, and alight chain variable region CDR set as set forth in SEQ ID Nos: 257, 5, and 255.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a heavy chain variable region as set forth in SEQ ID NO: 243.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 88.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 88 i.e., Ab88
  • SEQ ID NO: 258 see Sequence Table.
  • the VH CDR domain amino acid sequences of Antibody 88 are set forth in SEQ ID NO: 245 (VH CDR1); SEQ ID NO: 259 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 88 is described in SEQ ID NO: 256 (see Sequence Table).
  • the VL CDR domain amino acid sequences of Antibody 88 are set forth in SEQ ID NO: 257 (VL CDR1); SEQ ID NO: 5 (VL CDR2), and SEQ ID NO: 255 (VL CDR3).
  • the heavy chain constant region of Antibody 88 is set forth in SEQ ID NO: 269.
  • the light chain constant region of Antibody 88 is set forth in SEQ ID NO: 283.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 245, 259, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 257, 5, and 255.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 256, and a heavy chain variable region as set forth in SEQ ID NO: 258.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 89.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 89 i.e., Ab89
  • the VH CDR domain amino acid sequences of Antibody 89 are set forth in SEQ ID NO: 245 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 3 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 89 is described in SEQ ID NO: 252 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 245, 2, and 3, and a light chain variable region CDR set as set forth in SEQ ID Nos: 254, 249, and 255.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 252, and a heavy chain variable region as set forth in SEQ ID NO: 260.
  • the invention provides an ADC comprising an anti-CD117 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs, variable regions, corresponding to those of Antibody 249.
  • the heavy chain variable region (VH) amino acid sequence of Antibody 249 i.e., Ab249) is set forth in SEQ ID NO: 238 (see Sequence Table).
  • the VH CDR domain amino acid sequences of Antibody 249 are set forth in SEQ ID NO: 286 (VH CDR1); SEQ ID NO: 2 (VH CDR2), and SEQ ID NO: 287 (VH CDR3).
  • the light chain variable region (VL) amino acid sequence of Antibody 249 is described in SEQ ID NO: 242 (see Sequence Table).
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable heavy chain CDR set (CDR1, CDR2, and CDR3) as set forth in SEQ ID Nos: 286, 2, and 287, and a light chain variable region CDR set as set forth in SEQ ID Nos: 288, 249, and 289.
  • an anti-CD117 antibody, or antigen-binding portion thereof comprises a variable light chain comprising the amino acid residues set forth in SEQ ID NO: 242, and a heavy chain variable region as set forth in SEQ ID NO: 238.
  • anti-CD117 antibody drug conjugates comprising binding regions (heavy and light chain CDRs or variable regions) as set forth in SEQ ID Nos: 147 to 168.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 148.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 149.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 150.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 151.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 152. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 153.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 154. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 155.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 156. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 157.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 158. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 159.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 160.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 161.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 162.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 163.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 164, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 165. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 166, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 167.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 168, and alight chain variable region as set forth in the amino acid sequence of SEQ ID NO: 169.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 170, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 171.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 172, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 173.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 174, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 175.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 176, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 177. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 178, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 179.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 180, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 181.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 172, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 182.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 183, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 184. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 185, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 186.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 187, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 188. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 189, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 190.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 191, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 192.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 193, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 194.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 195, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 196. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 197, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 198.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 199, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 200.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 201, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 190.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 202, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 203.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 204, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 205.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 206, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 207.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 208, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 209.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 210, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 211.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 212, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 213.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 214, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 215.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 216, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 217.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 218, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 219.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 220, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 221.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 222, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 223.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 224, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 225.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 226, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 227.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 228.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 229.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 230.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 231.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 232.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 233. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 234.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 235. In one embodiment, the anti-CD117 antibody, or antigen binding portion thereof, comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 236.
  • the anti-CD117 antibody, or antigen binding portion thereof, of an ADC described herein comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 237.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 243, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 244.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 251, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 252.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 243, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 256.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 258, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 256.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 260, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 252.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 238, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 239.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 239.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 147, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 240.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 238, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 241.
  • the anti-CD117 antibody, or antigen binding portion thereof comprises a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO: 238, and a light chain variable region as set forth in the amino acid sequence of SEQ ID NO: 242.
  • Certain of the anti-CD117 antibodies described herein are neutral antibodies, in that the antibodies do not substantially inhibit CD117 activity on a CD117 expressing cell.
  • Neutral antibodies can be identified using, for example, an in in vitro stem cell factor (SCF)-dependent cell proliferation assay.
  • SCF stem cell factor
  • a neutral CD117 antibody will not kill CD34+ cells that are dependent on SCF to divide, as a neutral antibody will not block SCF from binding to CD117 such as to inhibit CD117 activity.
  • Neutral antibodies can be used for diagnostic purposes, given their ability to specifically bind to human CD117, but are also effective for killing CD117 expressing cells when conjugated to a cytotoxin, such as those described herein.
  • antibodies used in conjugates have agonistic or antagonistic activity that is unique to the antibody. Described herein, however, is a unique approach to conjugates, especially in the context wherein the conjugate is being used as a conditioning agent prior to a stem cell transplantation.
  • cytotoxin While antagonistic antibodies alone or in combination with a cytotoxin as a conjugate can be effective given the killing ability of the antibody alone in addition to the cytotoxin, conditioning with a conjugate comprising a neutral anti-CD117 antibody presents an alternative strategy where the activity of the antibody is secondary to the effect of the cytotoxin, but the internalizing and affinity characteristics, e.g., dissociation rate, of the antibody are important for effective delivery of the cytotoxin.
  • neutral anti-CD117 antibodies examples include Ab58, Ab61, Ab66, Ab67, Ab68, and Ab69.
  • a comparison of the amino acid sequences of the CDRs of neutral, anti-CD117 antibody CDRs reveals consensus sequences among two groups of neutral antibodies identified.
  • Ab58 and Ab61 share the same light chain CDRs and HC CDR3, with slight variations in the HC CDR1 and HC CDR2.
  • Consensus sequences for the HC CDR1 and CDR2 are described in SEQ ID Nos: 133 and 134.
  • Ab66, Ab67, Ab68, and Ab69 are also neutral antibodies.
  • Antagonist antibodies are also provided herein, including Ab54, Ab55, Ab56, and Ab57. While Ab54, Ab55, Ab56, and Ab57 share the same light chain CDRs and the same HC CDR3, these antibodies have variability within their HC CDR1 and HC CDR2 regions. Consensus sequences for these antibodies in the HC CDR1 and HC CDR2 regions are provided in SEQ ID Nos: 127 and 128, respectively.
  • Anti-CD117 antibodies that may be used in the compositions and methods disclosed herein are also described in US 2019-0153114 and US 2019-0144558, the antibody sequences of which are hereby incorporated by reference.
  • the anti-CD117 antibody, or antigen binding fragment thereof comprises variable regions having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the anti-CD117 antibody, or antigen binding fragment thereof comprises CDRs comprising the SEQ ID Nos disclosed herein with framework regions of the variable regions described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the anti-CD117 antibodies described herein can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multiple chain or single chain antibodies, and/or binding fragments that specifically bind human CD117, including but not limited to Fab, Fab′, (Fab′)2, Fv), scFv (single chain Fv), surrobodies (including surrogate light chain construct), single domain antibodies, camelized antibodies and the like. They also can be of, or derived from, any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g. IgG1, IgG2, IgG3 or IgG4), or IgM. In some embodiments, the anti-CD117 antibody is an IgG (e.g. IgG1, IgG2, IgG3 or IgG4).
  • Antibodies for use in conjunction with the methods described herein include variants of those antibodies described above, such as antibody fragments that contain or lack an Fc domain, as well as humanized variants of non-human antibodies described herein and antibody-like protein scaffolds (e.g., 10 Fn3 domains) containing one or more, or all, of the CDRs or equivalent regions thereof of an antibody, or antibody fragment, described herein.
  • antibody-like protein scaffolds e.g. 10 Fn3 domains
  • Exemplary antigen-binding fragments of the foregoing antibodies include a dual-variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, a Fv fragment, a Fab fragment, a F(ab′) 2 molecule, and a tandem di-scFv, among others.
  • scFv single-chain Fv molecule
  • anti-CD117 antibodies comprising one or more radiolabeled amino acids are provided.
  • a radiolabeled anti-CD117 antibody may be used for both diagnostic and therapeutic purposes (conjugation to radiolabeled molecules is another possible feature).
  • labels for polypeptides include, but are not limited to 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re.
  • Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see for instance Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. Nos.
  • a radioisotope may be conjugated by a chloramine T method.
  • the present invention includes ADCs comprising antibodies, and antigen-binding fragments thereof, that specifically bind to a CD45 polypeptide, e.g., a human CD45 polypeptide, and uses thereof.
  • a CD45 polypeptide e.g., a human CD45 polypeptide
  • the antibody, or antigen-binding fragment thereof, that specifically binds to a CD45 polypeptide comprises a heavy chain variable region and a light chain variable region.
  • CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase essential for T and B cell antigen receptor-mediated signaling.
  • CD45 includes a large extracellular domain, and a phosphatase containing cytosolic domain.
  • CD45 may act as both a positive and negative regulator depending on the nature of the stimulus and the cell type involved.
  • the isoforms are RA, RO, RB, RAB, RBC and RABC (Hermiston et al. 2003 “CD45: a critical regulator of signaling thresholds in immune cells.” Annu Rev Immunol. 2:107-137.).
  • CD45RA is expressed on na ⁇ ve T cells
  • CD45RO is expressed on activated and memory T cells, some B cell subsets, activated monocytes/macrophages, and granulocytes.
  • CD45RB is expressed on peripheral B cells, na ⁇ ve T cells, thymocytes, weakly on macrophages, and dendritic cells.
  • the anti-CD45 antibody is selected from apamistamab (also known 90Y-BC8, lomab-B, BC8; as described in, e.g., US20170326259, WO2017155937, and Orozco et al. Blood. 127.3 (2016): 352-359.) or BC8-B10 (as described, e.g., in Li et al. PloS one 13.10 (2016): e0205135.), each of which is incorporated by reference.
  • Other anti-CD45 antibodies have been described, for example, in WO2003048327, WO2016016442, US20170226209, US20160152733, U.S. Pat. No. 9,701,756; US20110076270, or U.S. Pat. No. 7,825,222, each of which is incorporated by reference.
  • the present invention includes ADCs comprising antibodies, and antigen-binding fragments thereof, that specifically bind to a CD137 polypeptide, e.g., a human CD137 polypeptide, and uses thereof.
  • a CD137 polypeptide e.g., a human CD137 polypeptide
  • the antibody, or antigen-binding fragment thereof, that specifically binds to a CD137 polypeptide comprises a heavy chain variable region and a light chain variable region.
  • T cells have been shown to express CD137, as this antigen is a transmembrane TNF receptor superfamily of costimulatory molecules and is expressed on a variety of hematopoietic cells and promotes T cell activation and regulates proliferation and survival of T cells (see, e.g., Cannons et al., J. Immunol. 167:1313-1324, 2001, the disclosure of which is incorporated herein by reference as it pertains to the expression of CD137 by T cells).
  • CD137 is alternatively named tumor necrosis factor receptor superfamily member 9 (TNFRSF9), 4-1BB, or induced by lymphocyte activation (ILA).
  • TNFRSF9 tumor necrosis factor receptor superfamily member 9
  • 4-1BB 4-1BB
  • IVA lymphocyte activation
  • the anti-CD137 antibody is selected from ADG106 (as described in, e.g., US20190055314, WO2019037711, WO2019036855); AGEN2373 (as described in, e.g., WO2018191502, US20180344870); ATOR-1017 (as described in, e.g., WO2018091740, US20180118841) PE0166 (as described in, e.g., Song et al. AACR 2019, Abstract 2397/21), urelumab (also known as BMS-663513; as described in, e.g., WO2004010947, WO2005035584, US20090068192, U.S. Pat.
  • ADG106 as described in, e.g., US20190055314, WO2019037711, WO2019036855
  • AGEN2373 as described in, e.g., WO2018191502, US20180344870
  • ATOR-1017 as
  • utomilumab also known as PF-05082566, MOR-7480.; as described in, e.g., WO2012032433, US20120237498, US20140178368, WO2012145183, WO2015119923, WO2015179236, US20160152722, US20190031765, WO2017130076, Chin et al. Nature communications. 9.1 (2018): 4679.; Segal et al. Clinical Cancer Research. 24.8 (2016): 1816-1823; Fisher et al. Cancer Immunology, Immunotherapy. 61.10 (2012): 1721-1733), each of which is incorporated by reference.
  • anti-CD137 antibodies have been described, for example, in WO2018134787, WO2019020774, WO2017077085, US20180327504, US20190099488, US2019006045, US20190015508, WO2019014328, US20190071510, WO2018127787, US20180258177, U.S. Ser. No. 10/174,122, WO2016110584, WO2018017761, WO2018098370, US20130149301, WO2019027754, WO2018156740, US20160244528, WO2016134358, U.S. Ser. No.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to an anti-CD137 antibody herein, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an anti-CD137 antibody herein.
  • an antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain of an anti-CD137 antibody herein, or a variant thereof, which variant (i) differs from the anti-CD137 antibody in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from the anti-CD137 antibody in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from the anti-CD137 antibody in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the anti-CD137 antibody, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region can have an enhanced biological activity relative to the heavy chain variable region
  • an anti-CD137 antibody that may be used in the methods and compositions (including ADCs) described herein is the murine anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX) or an anti-CD137 antibody comprising antigen binding regions corresponding to the BBK2 antibody.
  • the BBK2 antibody (which may also be referred to as a BBK-2 antibody or an anti-4-1BB antibody), is a mouse monoclonal antibody (IgG1, kappa) that binds to the ectodomain of human 4-1BB recombinant protein (4-1BB is also known as CD137).
  • the methods and compositions of the disclosure include an anti-CD137 antibody comprising the binding regions (e.g., the CDRs) of the BBK2 antibody.
  • the methods and compositions of the disclosure comprise an antibody that competitively inhibits the binding of the BBK2 antibody to its epitope on CD137.
  • the anti-CD137 antibody is humanized BBK2 or chimeric BBK2.
  • the methods and compositions described herein include a chimeric anti-CD137 (ch-BBK2) antibody comprising the variable heavy and light chain regions of BBK2.
  • the chimeric BBK2 antibody is an IgG1 antibody comprising human constant regions.
  • the heavy chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 290, and the light chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 291.
  • the CDR regions (CDR1, CDR2, and CDR3) of each of the heavy and light chain sequences are described in bold below.
  • the CDR regions of BBK2 can be defined according to Kabat numbering. CDRs as defined by Kabat numbering are described below for each of the heavy and light chain sequences (described in bold below).
  • the variable regions of BBK2 are italicized.
  • the VH CDR amino acid sequences of anti-CD137 antibody BBK2 are as follows: SYWIN (VH CDR1; SEQ ID NO: 292); NIYPSDSYTNYNQKFKD (VH CDR2; SEQ ID NO: 293) and NGVEGYPHYYAMEY (VH CDR3; SEQ ID NO: 294), and the VL CDR amino acid sequences of anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: RASQDLSNHLY (VL CDR1; SEQ ID NO: 295); YTSRLHS (VL CDR2; SEQ ID NO: 296) and QQGYTLPYT (VL CDR3; SEQ ID NO: 297).
  • the heavy chain variable region of BBK2 is set forth in SEQ ID NO: 298 as QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFK DKATLTVDKSSNTVYMQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS.
  • the light chain variable region of BBK2 is set forth in SEQ ID NO: 299 as DIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSG SGTDYSLTIRNLEQEDVATYFCQQGYTLPYTFGGGTKLEIK.
  • Anti-CD137 antibodies can comprise the heavy and light chain variable region amino acid sequences as set forth in SEQ ID Nos: 298 and 299, respectively.
  • the anti-CD137 antibody e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 294; and comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 295, a CDR2 comprising the amino acid sequence of SEQ ID NO: 296, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 297.
  • ch-BBK2 chimeric antibody
  • humanized BBK2 antibody comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of
  • the anti-CD137 antibody e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 294; and comprises a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 295, a CDR2 comprising the amino acid sequence of SEQ ID NO: 296, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 297.
  • ch-BBK2 chimeric antibody
  • humanized BBK2 antibody comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 292, a CDR2 comprising the amino acid sequence of SEQ ID NO: 293, and a CDR3 comprising the amino acid sequence of
  • BBK2, humanized BBK2, or chimeric BBK2 antibodies can be used in the anti-CD137 ADCs and methods described herein. Each of these antibodies can be conjugated to any of the cytotoxin described below using methods known in the art and those described herein.
  • compositions and methods described herein include an ADC comprising an antibody, or fragment thereof, that specifically binds to human CD5.
  • Human CD5 is also referred to as LEU or T1.
  • Human CD5 is a type-I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes and a subset of B lymphocytes. Two isoforms of human CD5 have been identified. Isoform 1 contains 438 amino acids and is described in Jones. et al. (1988) Nature 323 (6086), 346-349 and below (NCBI Reference Sequence: NP_001333385.1):
  • T cells have been shown to express CD5, which is a cell adhesion molecule and has been implicated both in the proliferative response of activated T cells and in T cell helper function. It has also been shown to function as a receptor, delivering co-stimulatory signals to T cells by interacting with CD72, a cell surface protein exclusive to B cells.
  • Antibodies, or antigen-binding fragments thereof, that bind CD5 may suppress T cell activation and T cell-mediated immune responses against hematopoietic stem cell grafts, for example, by inhibiting the interaction between CD5 and CD72.
  • Antibodies, and antigen-binding fragments thereof, that bind CD5 can also be used to kill CD5+ T cells directly, for instance, by conjugating the antibody, or antigen-binding fragment thereof, to a cytotoxin (such as a cytotoxin described herein or known in the art) or by using an unconjugated antibody, or antigen-binding fragment thereof, capable of recruiting complement proteins to the T cell.
  • a cytotoxin such as a cytotoxin described herein or known in the art
  • CD5 has also been shown to be expressed by subsets of NK cells; particularly among patients that have multiple myeloma have been shown to harbor populations of low density CD5+ (CD5LOW+) NK cells, and this surface antigen has been implicated in NK cell activation (Ishiyama et al., Anticancer Research 14:725-730 (1994), the disclosure of which is incorporated herein by reference in its entirety).
  • Antibodies, or antigen-binding fragments thereof, that specifically bind CD5 can thus be used to attenuate the activation of B cells and NK cells.
  • Antibodies, or antigen-binding fragments thereof, that bind CD5 can also be used to kill CD5+ B cells and NK cells directly, for instance, by conjugating the antibody, or antigen-binding fragment thereof, to a cytotoxin (such as a cytotoxin described herein or known in the art) or by using an unconjugated antibody, or antigen-binding fragment thereof, capable of recruiting complement proteins to the B cell or NK cell.
  • a cytotoxin such as a cytotoxin described herein or known in the art
  • the present invention encompasses ADCs comprising antibodies, and antigen-binding fragments thereof, that specifically bind to a CD5 polypeptide, e.g., a human CD5 polypeptide, and uses thereof.
  • a CD5 polypeptide e.g., a human CD5 polypeptide
  • the antibody, or antigen-binding fragment thereof, that specifically binds to a CD5 polypeptide comprises a heavy chain variable region and a light chain variable region.
  • an ADC comprises an antibody comprising the heavy chain variable region comprises one or more complementarity determining regions (CDRs).
  • the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 341.
  • the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 342.
  • the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 343.
  • the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 341, SEQ ID NO: 342, and SEQ ID NO: 343.
  • the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 341, SEQ ID NO: 342, and SEQ ID NO: 343.
  • the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO: 341, a VH CDR2 comprising SEQ ID NO: 342, and a VH CDR3 comprising SEQ ID NO: 343.
  • an ADC comprises an antibody comprising the light chain variable region comprises one or more complementarity determining regions (CDRs).
  • the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 344.
  • the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 345.
  • the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 346.
  • the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO: 344, SEQ ID NO: 345, and SEQ ID NO: 346.
  • the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO: 344, SEQ ID NO: 345, and SEQ ID NO: 346.
  • the light chain variable region comprises a VL CDR1 comprising SEQ ID NO: 344, a VL CDR2 comprising SEQ ID NO: 345, and a VL CDR3 comprising SEQ ID NO: 346.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises a VH CDR1 comprising SEQ ID NO: 341, a VH CDR2 comprising SEQ ID NO: 342, and a VH CDR3 comprising SEQ ID NO: 343, and alight chain variable region that comprises a VL CDR1 comprising SEQ ID NO: 344, a VL CDR2 comprising SEQ ID NO: 345, and a VL CDR3 comprising SEQ ID NO: 346.
  • one or more of the CDRs can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., specificity similar to an antibody, or antigen-binding fragment thereof, comprising heavy chain CDRs of SEQ ID NOs: 341 to 343, and light chain CDRs of SEQ ID NOs: 344 to 346).
  • the anti-CD5 antibody, or antigen binding fragment thereof is murine antibody 5D7, or a humanized version thereof.
  • Murine antibody 5D7 binds to human CD5 and is described in US Patent Publication No. 20008/0245027, the contents of which relating to the antibody sequences disclosed therein are incorporated by reference herein.
  • SEQ ID Nos: 353 to 358 described in the sequence summary table correspond to the CDRs of murine anti-CD5 antibody 5D7.
  • a humanized version of anti-CD5 antibody 5D7 is described in SEQ ID NO: 359 (humanized heavy chain variable region) and SEQ ID NO: 360 (humanized light chain variable region).
  • the ADCs and uses thereof described herein include an antibody comprising the CDRs set forth in SEQ ID Nos: 353 to 358. In one embodiment, the ADCs and uses thereof described herein include an antibody comprising the heavy and light chain variable regions as set forth in SEQ ID Nos: 359 and 360, respectively.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 359.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 359, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 359.
  • an antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 359, or a variant of SEQ ID NO: 359, which variant (i) differs from SEQ ID NO: 359 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 359 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 359 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 359, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region can have
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 360.
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 360, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 360.
  • an antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 360, or a variant of SEQ ID NO: 360, which variant (i) differs from SEQ ID NO: 360 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 360 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 360 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 360, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified light chain variable region can have an enhanced biological activity relative to
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 359, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 359, and a light chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 360, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 360.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises SEQ ID NO: 359, and a light chain variable region that comprises SEQ ID NO: 360.
  • the anti-CD5 antibody, or antigen-binding fragment thereof can contain a heavy chain variable region that comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 353.
  • the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 354.
  • the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 355.
  • the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 353, SEQ ID NO: 354, and SEQ ID NO: 355.
  • the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 353, SEQ ID NO: 354, and SEQ ID NO: 355.
  • the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO: 353, a VH CDR2 comprising SEQ ID NO: 354, and a VH CDR3 comprising SEQ ID NO: 355.
  • the light chain variable region comprises one or more complementarity determining regions (CDRs).
  • the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 356.
  • the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 357.
  • the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 358.
  • the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO: 356, SEQ ID NO: 357, and SEQ ID NO: 358.
  • the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO: 356, SEQ ID NO: 357, and SEQ ID NO: 358.
  • the light chain variable region comprises a VL CDR1 comprising SEQ ID NO: 356, a VL CDR2 comprising SEQ ID NO: 357, and a VL CDR3 comprising SEQ ID NO: 358.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises a VH CDR1 comprising SEQ ID NO: 353, a VH CDR2 comprising SEQ ID NO: 354, and a VH CDR3 comprising SEQ ID NO: 355, and alight chain variable region that comprises a VL CDR1 comprising SEQ ID NO: 356, a VL CDR2 comprising SEQ ID NO: 357, and a VL CDR3 comprising SEQ ID NO: 358.
  • one or more of the CDRs can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., specificity similar to an antibody, or antigen-binding fragment thereof, comprising heavy chain CDRs of SEQ ID NOs: 353 to 355, and light chain CDRs of SEQ ID NOs:356 to 358).
  • Antibodies and antigen-binding fragments thereof capable of binding CD5 antigen can be identified using techniques known in the art and described herein, such as by immunization, computational modeling techniques, and in vitro selection methods, such as the phage display and cell-based display platforms described below.
  • Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity thereto):
  • V L having the amino acid sequence (SEQ ID NO: 325) DIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQKPGKSPKTLIYR ANRLVDGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGG GTKLEIK; and a V H having the amino acid sequence (SEQ ID NO: 326) QIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLRWMGW INTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRG YDWYFDVWGQGTTVTVSS.
  • Antibodies and antigen-binding fragments thereof containing the foregoing V L and V H sequences are described, e.g., in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof, such as the he1 antibody.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the V L and V H chains of SEQ ID NO: 325 and SEQ ID NO: 326.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the CDRs contained in the V L and V H chains of SEQ ID NO: 325 and SEQ ID NO: 326.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the CDRs contained in the V L and V H chains of SEQ ID NO: 325 and SEQ ID NO: 326 and the remainder of the V L and V H sequences have at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or greater sequence identity) to the V L and V H sequences of SEQ ID NO: 325 and SEQ ID NO: 326.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the following CDRs:
  • Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99%, or more, sequence identity thereto):
  • V L having the amino acid sequence (SEQ ID NO: 333) DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIY RANRLESGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTF GGGTKLEIK; and a V H having the amino acid sequence (SEQ ID NO: 334) EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGW INTHYGEPTYADSFKGTRTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRG YDWYFDVWGQGGTTVTVSS.
  • Antibodies and antigen-binding fragments thereof containing the foregoing V L and V H sequences are described, e.g., in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof, such as the he3 antibody.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the CDRs contained in the V L and V H chains of the antibody that includes the V L and V H chains of SEQ ID NO:327 and SEQ ID NO: 328.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the CDRs contained in the V L and V H chains of SEQ ID NO:327 and SEQ ID NO: 328 and the remainder of the V L and V H sequences have at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99%, or greater sequence identity) to the V L and V H sequences of SEQ ID NO:327 and SEQ ID NO: 328.
  • the anti-CD5 antibody or antigen-binding fragment thereof includes the following CDRs:
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences are described, e.g., in U.S. Pat. No. 5,869,619, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof.
  • anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD5 antibodies that are described in U.S. Pat. Nos. 5,821,123; 5,766,886; 5,770,196; 7,153,932; 5,621,083; 6,649,742; 6,146,631; 5,756,699; 5,744,580; 6,376,217; 5,837,491; and 6,146,850, the disclosures of each of which are incorporated herein by reference as they pertain to anti-CD5 antibodies and antigen-binding fragments thereof.
  • anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, those produced by the hybridoma cell line deposited as ATCC CRL 8000 (anti-CD5 murine antibody OKT1). Such antibodies are described in U.S. Pat. Nos. 4,515,894; 4,657,760; and 4,363,799, the disclosures of each of which are incorporated herein by reference as they pertains to anti-CD5 antibodies and antigen-binding fragments thereof.
  • Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or more, or all, of the following CDRs:
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences are described, e.g., in U.S. Pat. No. 8,679,500, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof.
  • Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or more, or all, of the following CDRs:
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences are described, e.g., in U.S. Pat. No. 8,679,500.
  • Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or more, or all, of the following CDRs:
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences are described, e.g., in US Patent Application Publication No. 2008/0254027, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof.
  • anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD5 antibodies that are described in PCT Application Publication No. WO1992/014491, such as the anti-CD5 antibodies produced by hybridoma cell line deposited at the Institut Pasteur under No. 1-1025 on Jan. 10, 1991.
  • the disclosure of PCT Application Publication No. WO1992/014491 is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen-binding fragments thereof.
  • anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD5 antibodies that are described in U.S. Pat. Nos. 6,010,902 and 7,192,736, US Patent Application Publication Nos. 2011/0250203 and 2017/0129128, and PCT Application Publication Nos. WO2016/172606; WO1994/023747; and WO1996/041608; the disclosures of each of which are incorporated herein by reference as they pertain to anti-CD5 antibodies and antigen binding fragments thereof.
  • the anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those that contain a combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions set forth in the table below.
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences of the table above are described, e.g., in US Patent Application Publication No. 2011/0250203, the disclosure of which is incorporated herein by reference as it pertains to anti-CD5 antibodies and antigen binding fragments thereof.
  • compositions and methods described herein include, in certain embodiments, an antibody, or fragment thereof, that specifically binds to human CD2.
  • Human CD2 is also referred to as T-cell Surface Antigen T11/Leu-5, T11, CD2 antigen (p50), and Sheep Red Blood Cell Receptor (SRBC).
  • CD2 is expressed on T cells.
  • Two isoforms of human CD2 have been identified. Isoform 1 contains 351 amino acids is described in Seed, B. et al. (1987) 84: 3365-69 (see also Sewell et al. (1986) 83: 8718-22) and below (NCBI Reference Sequence: NP_001758.2):
  • T cells and NK cells have been shown to express CD2, which is a cell adhesion molecule and specific marker for such lymphocytes.
  • CD2 interacts with other adhesion molecules, such as lymphocyte function-associated antigen-3 (LFA-3/CD58), to potentiate T cell activation.
  • LFA-3/CD58 lymphocyte function-associated antigen-3
  • Antibodies and antigen-binding fragments thereof capable of binding CD2 may suppress T cell activation and T cell-mediated immune responses against hematopoietic stem cell grafts, for example, by inhibiting the interaction between CD2 and LFA-3.
  • Antibodies and antigen-binding fragments thereof that bind to this cell-surface antigen can be identified using techniques known in the art and described herein, including immunization, computational modeling techniques, and in vitro selection methods, such as the phage display and cell-based display platforms described below.
  • the present invention encompasses an ADC comprising an antibody, or antigen-binding fragment thereof, that specifically binds to a CD2 polypeptide, e.g., a human CD2 polypeptide, and uses thereof.
  • an ADC comprises an anti-CD2 antibody comprising a heavy chain variable region comprises one or more complementarity determining regions (CDRs).
  • the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:300.
  • the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:301.
  • the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:302.
  • the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:300, SEQ ID NO:301, and SEQ ID NO:302.
  • the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:300, SEQ ID NO:301, and SEQ ID NO:302.
  • the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:300, a VH CDR2 comprising SEQ ID NO:301, and a VH CDR3 comprising SEQ ID NO:302.
  • an ADC comprises an anti-CD2 antibody comprising light chain variable region comprises one or more complementarity determining regions (CDRs).
  • the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:303.
  • the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:304.
  • the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:305.
  • the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:303, SEQ ID NO:304, and SEQ ID NO:305.
  • the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:303, SEQ ID NO:304, and SEQ ID NO:305.
  • the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:303, a VL CDR2 comprising SEQ ID NO:304, and a VL CDR3 comprising SEQ ID NO:305.
  • the anti-CD2 antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises a VH CDR1 comprising SEQ ID NO:300, a VH CDR2 comprising SEQ ID NO:301, and a VH CDR3 comprising SEQ ID NO:302, and alight chain variable region that comprises a VL CDR1 comprising SEQ ID NO:303, a VL CDR2 comprising SEQ ID NO:304, and a VL CDR3 comprising SEQ ID NO:305.
  • one or more of the CDRs can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., specificity similar to an antibody, or antigen-binding fragment thereof, comprising heavy chain CDRs of SEQ ID NOs: 300 to 302, and light chain CDRs of SEQ ID NOs:303 to 305).
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 306.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 306, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 306.
  • an antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 306, or a variant of SEQ ID NO: 306, which variant (i) differs from SEQ ID NO: 306 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 306 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 306 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 306, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region can have an enhanced biological
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that differs from the amino acid sequence set forth in SEQ ID NO: 306 at one, two, three or four amino acids.
  • the antibody, or antigen-binding fragment thereof can comprise a heavy chain variable region that differs from the amino acid sequence set forth in SEQ ID NO: 306 at one, two, three, or four of positions 12, 13, 28, and/or 48.
  • the heavy chain variable region differs from the amino acid sequence set forth in SEQ ID NO:306 at positions 12, 13, 28, and 48.
  • the heavy chain variable region comprises one, two, three, or four of the following substitutions with respect to the sequence set forth in SEQ ID NO:306: K12Q; K13R; T28I; and M48V. In one embodiment, the heavy chain variable region comprises the substitutions K12Q; K13R; T28I; and M48V with respect to SEQ ID NO:306.
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:307.
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO:307, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:307.
  • an antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 307, or a variant of SEQ ID NO: 307, which variant (i) differs from SEQ ID NO: 307 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 307 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 307 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 307, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified light chain variable region can have
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 306, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 306, and alight chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:307, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:307.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises SEQ ID NO: 306, and a light chain variable region that comprises SEQ ID NO:307.
  • the antibody is an Ab1 antibody that comprises a heavy chain variable region comprising SEQ ID NO:306, and alight chain variable region comprising SEQ ID NO:307.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:308.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO:308, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:308.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO:308, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:308, and a light chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:309, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:309.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises SEQ ID NO:308, and a light chain variable region that comprises SEQ ID NO:309.
  • the antibody is an Ab1a antibody that comprises a heavy chain variable region comprising SEQ ID NO:308, and a light chain variable region comprising SEQ ID NO:309.
  • the heavy chain variable region comprises one or more complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:3131.
  • the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:314.
  • the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:315.
  • the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:315.
  • the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:315. In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:315.
  • the heavy chain variable region comprises one or more complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO:313.
  • the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO:314.
  • the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO:316.
  • the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316.
  • the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO:313, SEQ ID NO:314, and SEQ ID NO:316.
  • the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:316.
  • the light chain variable region comprises one or more complementarity determining regions (CDRs).
  • the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO:317.
  • the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO:318.
  • the light chain variable region comprises a VL CDR3 comprising the amino acid sequence of SEQ ID NO:319.
  • the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:317, SEQ ID NO:318, and SEQ ID NO:319.
  • the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:317, SEQ ID NO:318, and SEQ ID NO:319.
  • the light chain variable region comprises a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:315, and a light chain variable region that comprises a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises a VH CDR1 comprising SEQ ID NO:313, a VH CDR2 comprising SEQ ID NO:314, and a VH CDR3 comprising SEQ ID NO:316, and a light chain variable region that comprises a VL CDR1 comprising SEQ ID NO:317, a VL CDR2 comprising SEQ ID NO:318, and a VL CDR3 comprising SEQ ID NO:319.
  • one or more of the CDRs can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., specificity similar to an antibody, or antigen-binding fragment thereof, comprising heavy chain CDRs of SEQ ID NOs: 313 to 315, and light chain CDRs of SEQ ID NOs:18 to 20; or comprising heavy chain CDRs of SEQ ID NOs: 313, 314, 316, and light chain CDRs of SEQ ID NOs:317 to 319).
  • a conservative amino acid substitution or 2, 3, 4, or 5 amino acid substitutions
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 320.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 320, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 320.
  • an antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 320, or a variant of SEQ ID NO: 320, which variant (i) differs from SEQ ID NO: 320 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 320 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 320 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 320, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises the amino acid sequence set forth in SEQ ID NO: 321.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 321, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 321.
  • an antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 320, or a variant of SEQ ID NO: 321, which variant (i) differs from SEQ ID NO: 321 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 321 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 321 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 321, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises the amino acid sequence set forth in SEQ ID NO:322.
  • the antibody, or antigen-binding fragment thereof comprises a light chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:322, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO:322.
  • an antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 322, or a variant of SEQ ID NO: 322, which variant (i) differs from SEQ ID NO: 322 in 1, 2, 3, 4 or 5 amino acids substitutions, additions or deletions; (ii) differs from SEQ ID NO: 322 in at most 5, 4, 3, 2, or 1 amino acids substitutions, additions or deletions; (iii) differs from SEQ ID NO: 322 in 1-5, 1-3, 1-2, 2-5 or 3-5 amino acids substitutions, additions or deletions and/or (iv) comprises an amino acid sequence that is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical to SEQ ID NO: 322, wherein in any of (i)-(iv), an amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified light
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 320, e.g., at least about 95%, about 96%, about 97%, about 98% or about 99%, or 100% identity to SEQ ID NO: 320, and alight chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:322, e.g., at least about 95%, about 96%, about 97%, about 98% or about 99%, or 100% identity to SEQ ID NO:322.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises SEQ ID NO: 320, and a light chain variable region that comprises SEQ ID NO:322.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO: 321, e.g., at least about 95%, about 96%, about 97%, about 98% or about 99%, or 100% identity to SEQ ID NO: 321, and a light chain variable region that comprises an amino acid sequence having at least about 95% identity to SEQ ID NO:322, e.g., at least about 95%, about 96%, about 97%, about 98% or about 99%, or 100% identity to SEQ ID NO:322.
  • the antibody, or antigen-binding fragment thereof comprises a heavy chain variable region that comprises SEQ ID NO: 321, and a light chain variable region that comprises SEQ ID NO:322.
  • Anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include those that have one or more, or all, of the following CDRs:
  • Antibodies and antigen-binding fragments thereof containing the foregoing CDR sequences are described, e.g., in U.S. Pat. No. 6,849,258, the disclosure of which is incorporated herein by reference as it pertains to anti-CD2 antibodies and antigen-binding fragments thereof.
  • the antibodies and fragments thereof disclosed in U.S. Pat. Nos. 5,730,979; 5,817,311; 5,951,983; and 7,592,006; such as LO-CD2a, BTI-322, and antibodies produced by the hybridoma cell line deposited as ATCC Deposit No. HB 11423 e.g., antibodies or antigen-binding fragments thereof containing one or more, or all, of the CDR sequences of antibody LO-CD2a isolated from the hybridoma cell line deposited as ATCC Deposit No. HB 11423) can be used in conjunction with the compositions and methods disclosed herein.
  • Exemplary antibodies that may be used in conjunction with the compositions and methods described herein include humanized antibodies containing one or more, or all, of the CDR sequences of an antibody isolated from the hybridoma cell line deposited as ATCC Deposit No. HB 11423, such as MEDI-507.
  • MEDI-507 is a humanized anti-CD2 monoclonal antibody that contains the CDR-H and CDR-L sequences of (a) through (f) above, and is described in Branco et al., Transplantation 68:1588-1596 (1999).
  • MEDI-507 is additionally described in WO99/03502A1 and WO1994/020619A1; U.S. Pat. Nos. U.S. Pat. Nos.
  • the disclosures of each of which are incorporated herein by reference as they pertain to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody MEDI-507.
  • the anti-CD2 antibody is Siplizumab, or an antigen-binding fragment thereof.
  • anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD2 antibodies that are described in U.S. Pat. Nos. 6,541,611 and 7,250,167, the disclosures of each of which are incorporated herein by reference as they pertain to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody LO-CD2b and antibodies produced by the hybridoma cell line deposited as ATCC Deposit No. PTA-802.
  • Exemplary antibodies that may be used in conjunction with the compositions and methods described herein include humanized antibodies containing one or more, or all, of the CDR sequences of an antibody isolated from the hybridoma cell line deposited as ATCC Deposit No. PTA-802.
  • anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD2 antibodies that are described in U.S. Pat. Nos. 5,795,572 and 5,807,734, the disclosures of each of which are incorporated herein by reference as they pertains to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody produced by hybridoma cell line deposited as ATCC Deposit No. HB 69277.
  • anti-CD2 antibodies and antigen-binding fragments thereof that may be used in conjunction with the compositions and methods described herein include those that contain a hinge region having an amino acid sequence of EPKSSDKTHTSPPSP (SEQ ID NO: 316), such as scFv fragments containing a hinge region having the amino acid sequence of EPKSSDKTHTSPPSP (SEQ ID NO: 316).
  • SEQ ID NO: 316 amino acid sequence of EPKSSDKTHTSPPSP
  • scFv fragments containing a hinge region having the amino acid sequence of EPKSSDKTHTSPPSP SEQ ID NO: 316
  • the incorporation of a hinge region having the amino acid sequence of SEQ ID NO: 316 can be beneficial, as this hinge motif has been mutated relative to wild-type hinge region sequences so as to eliminate potentially reactive cysteine residues that may promote undesirable oxidative dimerization of a single-chain antibody fragment, such as a scFv fragment.
  • anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD2 antibodies that are described in U.S. Pat. No. 6,764,688, such as the anti-CD2 antibody TS2/18 and antibodies produced by hybridoma cell line deposited as ATCC Deposit No. HB-195.
  • the disclosure of U.S. Pat. No. 6,764,688 is incorporated herein by reference as it pertains to anti-CD2 antibodies and antigen-binding fragments thereof.
  • anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for instance, anti-CD2 antibodies that are described in U.S. Pat. Nos. 6,162,432, 6,558,662, 7,408,039, 7,332,157, 7,638,121, 7,939,062, and 7,115,259, US Patent Application Publication No. 2006/0084107, 2014/0369974, 2002/0051784, and 2013/0183322, and PCT Publication No. WO1992/016563, the disclosures of each of which are incorporated herein by reference as they pertain to anti-CD2 antibodies and antigen binding fragments thereof.
  • the antibodies or binding fragments described herein may also include modifications and/or mutations that alter the properties of the antibodies and/or fragments, such as those that increase half-life, increase or decrease ADCC, etc., as is known in the art.
  • an antibody, or binding fragment thereof comprises a variant Fc region, wherein said variant Fc region comprises at least one amino acid modification relative to a wild-type Fc region, such that said molecule has an altered affinity for an FcgammaR.
  • Certain amino acid positions within the Fc region are known through crystallography studies to make a direct contact with Fc ⁇ R. Specifically amino acids 234-239 (hinge region), amino acids 265-269 (B/C loop), amino acids 297-299 (C′/E loop), and amino acids 327-332 (F/G) loop. (see Sondermann et al., 2000 Nature, 406: 267-273).
  • the antibodies may comprise variant Fc regions comprising modification of at least one residue that makes a direct contact with an Fc ⁇ R based on structural and crystallographic analysis.
  • the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991), expressly incorporated herein by references.
  • the “EU index as in Kabat” refers to the numbering of the human IgG1 EU antibody.
  • the EU index or EU index as in Kabat or EU numbering scheme refers to the numbering of the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporated by reference.)
  • the Fc region comprises a D265A mutation.
  • the Fc region comprises a D265C mutation.
  • the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat.
  • the Fc region comprises a L234A mutation.
  • the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat.
  • the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises a L234A and L235A mutation. In a further embodiment, the Fc region of the antibody of an ADC described herein comprises a D265C, L234A, and L235A mutation.
  • a variant IgG Fc domain comprises one or more amino acid substitutions resulting in decreased or ablated binding affinity for an Fc.gamma.R and/or C1q as compared to the wild type Fc domain not comprising the one or more amino acid substitutions.
  • Fc binding interactions are essential for a variety of effector functions and downstream signaling events including, but not limited to, antibody dependent cell-mediated cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC).
  • ADCC antibody dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • an antibody comprising a modified Fc region e.g., comprising a L234A, L235A, and a D265C mutation
  • Affinity to an Fc region can be determined using a variety of techniques known in the art, for example but not limited to, equilibrium methods (e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or by a surface plasmon resonance assay or other mechanism of kinetics-based assay (e.g., BIACORETM. analysis or OctetTM analysis (forteBIO)), and other methods such as indirect binding assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration).
  • equilibrium methods e.g., enzyme-linked immunoabsorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay
  • binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.
  • a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
  • the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.
  • Antibodies may be further engineered to further modulate antibody half-life by introducing additional Fc mutations, such as those described for example in (Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol 176: 346-56), (Shields et al. (2001) J Biol Chem 276: 6591-604), (Petkova et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al.
  • Fc mutations such as those described for example in (Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004)
  • Exemplary mutations that may be made singularly or in combination are T250Q, M252Y, 1253A,S254T,T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.
  • the Fc region comprises a mutation resulting in a decrease in half life.
  • An antibody having a short half life may be advantageous in certain instances where the antibody is expected to function as a short-lived therapeutic, e.g., the conditioning step described herein where the antibody is administered followed by HSCs. Ideally, the antibody would be substantially cleared prior to delivery of the HSCs, which also generally express an antigen targeted by an ADC described herein, e.g., CD117, but are not the target of the ADC, unlike the endogenous stem cells.
  • the Fc regions comprise a mutation at position 435 (EU index according to Kabat).
  • the mutation is an H435A mutation.
  • the antibody described herein has a half life of equal to or less than 24 hours, a half life of equal to or less than 22 hours, a half life of equal to or less than 20 hours, a half life of equal to or less than 18 hours, a half life of equal to or less than 16 hours, a half life of equal to or less than 14 hours, equal to or less than 13 hours, equal to or less than 12 hours, or equal to or less than 11 hours.
  • the half life of the antibody is 11 hours to 24 hours; 12 hours to 22 hours; 10 hours to 20 hours; 8 hours to 18 hours; or 14 hours to 24 hours.
  • the Fc region comprises two or more mutations that confer reduced half-life and greatly diminish or completely abolish an effector function of the antibody.
  • the Fc region comprises a mutation resulting in a decrease in half-life and a mutation of at least one residue that can make direct contact with an Fc ⁇ R (e.g., as based on structural and crystallographic analysis).
  • the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation.
  • the Fc region comprises a H435A mutation and a D265C mutation.
  • the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation.
  • the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin (e.g., amatoxin) by way of a cysteine residue in the Fc domain of the antibody or antigen-binding fragment thereof.
  • the cysteine residue is introduced by way of a mutation in the Fc domain of the antibody or antigen-binding fragment thereof.
  • the cysteine residue may be selected from the group consisting of Cys118, Cys239, and Cys265.
  • the Fc region of the anti-CD117 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat.
  • the Fc region comprises a D265C mutation.
  • the Fc region comprises a D265C and H435A mutation. In one embodiment, the Fc region comprises a D265C, a L234A, and a L235A mutation. In one embodiment, the Fc region comprises a D265C, a L234A, a L235A, and a H435A mutation.
  • the cysteine residue is naturally occurring in the Fc domain of the antibody or antigen-binding fragment thereof.
  • the Fc domain may be an IgG Fc domain, such as a human IgG1 Fc domain, and the cysteine residue may be selected from the group consisting of Cys261, Csy321, Cys367, and Cys425.
  • the Fc region of Antibody 67 is modified to comprise a D265C mutation (e.g., SEQ ID NO: 111).
  • the Fc region of Antibody 67 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 112).
  • the Fc region of Antibody 67 is modified to comprise a D265C and H435A mutation (e.g., SEQ ID NO: 113).
  • the Fc region of Antibody 67 is modified to comprise a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO: 114).
  • the Fc region of Antibody 55 is modified to comprise a D265C mutation (e.g., SEQ ID NO: 117). In another embodiment, the Fc region of Antibody 55 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 118). In yet another embodiment, the Fc region of Antibody 55 is modified to comprise a D265C and H435A mutation (e.g., SEQ ID NO: 119). In a further embodiment, the Fc region of Antibody 55 is modified to comprise a D265C, L234A, L235A, and H435A mutation (e.g., SEQ ID NO: 120).
  • a D265C mutation e.g., SEQ ID NO: 117
  • the Fc region of Antibody 55 is modified to comprise a D265C, L234A, and L235A mutation (e.g., SEQ ID NO: 118).
  • the Fc region of Antibody 55 is
  • the Fc regions of any one of Antibody 54, Antibody 55, Antibody 56, Antibody 57, Antibody 58, Antibody 61, Antibody 66, Antibody 67, Antibody 68, or Antibody 69 can be modified to comprise a D265C mutation (e.g., as in SEQ ID NO: 123); a D265C, L234A, and L235A mutation (e.g., as in SEQ ID NO: 124); a D265C and H435A mutation (e.g., as in SEQ ID NO: 125); or a D265C, L234A, L235A, and H435A mutation (e.g., as in SEQ ID NO: 126).
  • a D265C mutation e.g., as in SEQ ID NO: 123
  • a D265C, L234A, and L235A mutation e.g., as in SEQ ID NO: 124
  • D265C is an Fc variant with the aspartic acid (D) at EU position 265 substituted with cysteine (C) relative to the parent Fc domain.
  • D265C/L234A/L235A defines a variant Fc variant with substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parent Fc domain.
  • a variant can also be designated according to its final amino acid composition in the mutated EU amino acid positions.
  • the L234A/L235A mutant can be referred to as LALA. It is noted that the order in which substitutions are provided is arbitrary.
  • the antibody, or antigen binding fragment thereof comprises variable regions having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the antibody, or antigen binding fragment thereof comprises CDRs comprising the SEQ ID Nos disclosed herein with framework regions of the variable regions described herein having an amino acid sequence that is at least 95%, 96%, 97% or 99% identical to the SEQ ID Nos disclosed herein.
  • the antibody, or antigen binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region having an amino acid sequence that is disclosed herein. In another embodiment, the antibody, or antigen binding fragment thereof, comprises alight chain variable region and a light chain constant region having an amino acid sequence that is disclosed herein. In yet another embodiment, the antibody, or antigen binding fragment thereof, comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region and alight chain constant region having an amino acid sequence that is disclosed herein.
  • novel ADCs that may be used, for example, in conditioning methods for stem cell transplantation.
  • other antibodies can be identified that can be used in the ADCs and methods of the invention.
  • Methods for high throughput screening of antibody, or antibody fragment libraries for molecules capable of binding a cell surface antigen can be used to identify and affinity mature antibodies useful for treating cancers, autoimmune diseases, and conditioning a patient (e.g., a human patient) in need of hematopoietic stem cell therapy as described herein.
  • a cell surface antigen e.g., CD117, CD45, CD2, CD5, CD134, CD252, CD137
  • Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others.
  • phage display to isolate ligands that bind biologically relevant molecules has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, the disclosures of each of which are incorporated herein by reference as they pertain to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select for polypeptides that bind cell surface antigens as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers.
  • Proteins such as multimeric proteins, have been successfully phage-displayed as functional molecules (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as they pertain to the use of in vitro display techniques for the discovery of antigen-binding molecules).
  • computational modeling techniques can be used to design and identify antibodies, and antibody fragments, in silico that bind a cell surface antigen (e.g., CD117, CD45, CD2, CD5, CD134, CD252, CD137).
  • a cell surface antigen e.g., CD117, CD45, CD2, CD5, CD134, CD252, CD137.
  • computational modeling techniques one of skill in the art can screen libraries of antibodies, and antibody fragments, in silico for molecules capable of binding specific epitopes, such as extracellular epitopes of this antigen.
  • the antibodies, and antigen-binding fragments thereof, identified by these computational techniques can be used in conjunction with the therapeutic methods described herein, such as the cancer and autoimmune disease treatment methods described herein and the patient conditioning procedures described herein.
  • Additional techniques can be used to identify antibodies, and antigen-binding fragments thereof, that bind a cell surface antigen (e.g., CD117) on the surface of a cell (e.g., a cancer cell, autoimmune cell, or hematopoietic stem cell) and that are internalized by the cell, for instance, by receptor-mediated endocytosis.
  • a cell surface antigen e.g., CD117
  • the in vitro display techniques described above can be adapted to screen for antibodies, and antigen-binding fragments thereof, that bind a cell surface antigen (e.g., CD117) on the surface of a cancer cell, autoimmune cell, or hematopoietic stem cell and that are subsequently internalized.
  • Phage display represents one such technique that can be used in conjunction with this screening paradigm.
  • recombinant phage libraries can be produced that encode antibodies, antibody fragments, such as scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domains, among others, or ligands that contain randomized amino acid cassettes (e.g., in one or more, or all, of the CDRs or equivalent regions thereof or an antibody or antibody fragment).
  • the framework regions, hinge, Fc domain, and other regions of the antibodies or antibody fragments may be designed such that they are non-immunogenic in humans, for instance, by virtue of having human germline antibody sequences or sequences that exhibit only minor variations relative to human germline antibodies.
  • phage libraries containing randomized antibodies, or antibody fragments, covalently bound to the phage particles can be incubated with a cell surface target antigen (e.g., CD117) antigen, for instance, by first incubating the phage library with blocking agents (such as, for instance, milk protein, bovine serum albumin, and/or IgG so as to remove phage encoding antibodies, or fragments thereof, that exhibit non-specific protein binding and phage that encode antibodies or fragments thereof that bind Fc domains, and then incubating the phage library with a population of hematopoietic stem cells.
  • blocking agents such as, for instance, milk protein, bovine serum albumin, and/or IgG
  • the phage library can be incubated with the target cells, such as cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to allow cell surface antigen specific antibodies, or antigen-binding fragments thereof, (e.g., CD117-specific antibodies, or antigen-binding fragments thereof) to bind cell-surface antigen (e.g., sell-surface CD117) antigen and to subsequently be internalized by the cancer cells, autoimmune cells, or hematopoietic stem cells (e.g., from 30 minutes to 6 hours at 4° C., such as 1 hour at 4° C.).
  • the target cells such as cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to allow cell surface antigen specific antibodies, or antigen-binding fragments thereof, (e.g., CD117-specific antibodies, or antigen-binding fragments thereof) to bind cell-surface antigen (e.g., sell-surface CD117) antigen and to subsequently be internal
  • Phage containing antibodies, or fragments thereof, that do not exhibit sufficient affinity for one or more of these antigens so as to permit binding to, and internalization by, cancer cells, autoimmune cells, or hematopoietic stem cells can subsequently be removed by washing the cells, for instance, with cold (4° C.) 0.1 M glycine buffer at pH 2.8.
  • Phage bound to antibodies, or fragments thereof, that have been internalized by the cancer cells, autoimmune cells, or hematopoietic stem cells can be identified, for instance, by lysing the cells and recovering internalized phage from the cell culture medium.
  • the phage can then be amplified in bacterial cells, for example, by incubating bacterial cells with recovered phage in 2 ⁇ YT medium using methods known in the art. Phage recovered from this medium can then be characterized, for instance, by determining the nucleic acid sequence of the gene(s) encoding the antibodies, or fragments thereof, inserted within the phage genome.
  • the encoded antibodies, or fragments thereof can subsequently be prepared de novo by chemical synthesis (for instance, of antibody fragments, such as scFv fragments) or by recombinant expression (for instance, of full-length antibodies).
  • Phage display libraries can be created by making a designed series of mutations or variations within a coding sequence for the CDRs of an antibody or the analogous regions of an antibody-like scaffold (e.g., the BC, CD, and DE loops of 10 Fn3 domains).
  • the template antibody-encoding sequence into which these mutations are introduced may be, for example, a naive human germline sequence. These mutations can be performed using standard mutagenesis techniques known in the art. Each mutant sequence thus encodes an antibody corresponding to the template save for one or more amino acid variations.
  • Retroviral and phage display vectors can be engineered using standard vector construction techniques known in the art. P3 phage display vectors along with compatible protein expression vectors can be used to generate phage display vectors for antibody diversification.
  • the mutated DNA provides sequence diversity, and each transformant phage displays one variant of the initial template amino acid sequence encoded by the DNA, leading to a phage population (library) displaying a vast number of different but structurally related amino acid sequences. Due to the well-defined structure of antibody hypervariable regions, the amino acid variations introduced in a phage display screen are expected to alter the binding properties of the binding peptide or domain without significantly altering its overall molecular structure.
  • a phage library may be contacted with and allowed to bind one of the foregoing antigens or an epitope thereof.
  • Phage bearing a cell surface-binding moiety can form a complex with the target on the solid support, whereas non-binding phage remain in solution and can be washed away with excess buffer. Bound phage can then liberated from the target by changing the buffer to an extreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding denaturants, or other known means.
  • the recovered phage can then be amplified through infection of bacterial cells, and the screening process can be repeated with the new pool that is now depleted in non-binding antibodies and enriched for antibodies that bind a target antigen (e.g., CD117).
  • a target antigen e.g., CD117
  • the recovery of even a few binding phage is sufficient to amplify the phage for a subsequent iteration of screening.
  • the gene sequences encoding the antibodies or antigen-binding fragments thereof derived from selected phage clones in the binding pool are determined by conventional methods, thus revealing the peptide sequence that imparts binding affinity of the phage to the target.
  • sequence diversity of the population diminishes with each round of selection until desirable peptide-binding antibodies remain.
  • the sequences may converge on a small number of related antibodies or antigen-binding fragments thereof.
  • An increase in the number of phage recovered at each round of selection is an indication that convergence of the library has occurred in a screen.
  • Another method for identifying antibodies includes using humanizing non-human antibodies that bind a cell surface target antigen (e.g., CD117), for instance, according to the following procedure.
  • Consensus human antibody heavy chain and light chain sequences are known in the art (see e.g., the “VBASE” human germline sequence database; Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227:776-798, 1992; and Cox et al. Eur. J. Immunol.
  • variable domain framework residues and CDRs of a consensus antibody sequence e.g., by sequence alignment.
  • This CDR exchange can be performed using gene editing techniques described herein or known in the art.
  • a cell surface target antigen e.g., CD117.
  • the affinity of the antibody for the hematopoietic stem cell antigen is determined primarily by the CDR sequences
  • the resulting humanized antibody is expected to exhibit an affinity for the hematopoietic stem cell antigen that is about the same as that of the non-human antibody from which the humanized antibody was derived.
  • Methods of determining the affinity of an antibody for a target antigen include, for instance, ELISA-based techniques described herein and known in the art, as well as surface plasmon resonance, fluorescence anisotropy, and isothermal titration calorimetry, among others.
  • the internalizing capacity of an antibody, or fragment thereof can be assessed, for instance, using radionuclide internalization assays known in the art.
  • antibodies, or fragments thereof, identified using in vitro display techniques described herein or known in the art can be functionalized by incorporation of a radioactive isotope, such as 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 Ac.
  • a radioactive isotope such as 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re,
  • radioactive halogens such as 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, can be incorporated into antibodies, or fragments thereof, using beads, such as polystyrene beads, containing electrophilic halogen reagents (e.g., Iodination Beads, Thermo Fisher Scientific, Inc., Cambridge, Mass.).
  • Radiolabeled antibodies, or fragments thereof can be incubated with cancer cells, autoimmune cells, or hematopoietic stem cells for a time sufficient to permit internalization (e.g., from 30 minutes to 6 hours at 4° C., such as 1 hour at 4° C.).
  • the cells can then be washed to remove non-internalized antibodies, or fragments thereof, (e.g., using cold (4° C.) 0.1 M glycine buffer at pH 2.8).
  • Internalized antibodies, or fragments thereof can be identified by detecting the emitted radiation (e.g., ⁇ -radiation) of the resulting cancer cells, autoimmune cells, or hematopoietic stem cells in comparison with the emitted radiation (e.g., ⁇ -radiation) of the recovered wash buffer.
  • Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567.
  • isolated nucleic acid encoding an antibody described herein is provided.
  • Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such nucleic acid is provided.
  • a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody.
  • the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0, Sp20 cell).
  • a method of making an anti-CLL-1 antibody comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).
  • nucleic acid encoding an antibody is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • For expression of antibody fragments and polypeptides in bacteria see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli .)
  • the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.
  • Vertebrate cells may also be used as hosts.
  • mammalian cell lines that are adapted to grow in suspension may be useful.
  • Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.
  • monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells.
  • Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ⁇ CHO cells (Urlaub et al., Proc. Natl. Acad. Sci.
  • ADCs described herein can be administered to a patient (e.g., a human patient suffering from an immune disease or cancer) in a variety of dosage forms.
  • ADCs described herein can be administered to a patient suffering from an immune disease or cancer in the form of an aqueous solution, such as an aqueous solution containing one or more pharmaceutically acceptable excipients.
  • Suitable pharmaceutically acceptable excipients for use with the compositions and methods described herein include viscosity-modifying agents.
  • the aqueous solution may be sterilized using techniques known in the art.
  • compositions comprising ADCs as described herein are prepared by mixing such ADC with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • ADCs described herein may be administered by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally.
  • routes such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally.
  • the most suitable route for administration in any given case will depend on the particular antibody, or antigen-binding fragment, administered, the patient, pharmaceutical formulation methods, administration methods (e.g., administration time and administration route), the patient's age, body weight, sex, severity of the diseases being treated, the patient's diet, and the patient's excretion rate.
  • the effective dose of an ADC, antibody, or antigen-binding fragment thereof, described herein can range, for example from about 0.001 to about 100 mg/kg of body weight per single (e.g., bolus) administration, multiple administrations, or continuous administration, or to achieve an optimal serum concentration (e.g., a serum concentration of 0.0001-5000 ⁇ g/mL) of the antibody, antigen-binding fragment thereof.
  • the dose may be administered one or more times (e.g., 2-10 times) per day, week, or month to a subject (e.g., a human) suffering from cancer, an autoimmune disease, or undergoing conditioning therapy in preparation for receipt of a hematopoietic stem cell transplant.
  • the ADC, antibody, or antigen-binding fragment thereof can be administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, for instance, from 1 hour to 1 week (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days) or more prior to administration of the exogenous hematopoietic stem cell transplant.
  • 1 hour to 1 week e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours
  • FIG. 1 provides the structures of three amanitin/linker conjugates.
  • FIG. 1A provides an amanitin/linker conjugate represented by Formula (IV) (also referred to as “Conjugate A” or “ADC A”), where the amanitin has a cleavable linker (BMP-Val-Ala-PAB) linker on AA1 (Asn).
  • the amanitin in Formula (IV) is based on amatoxin having thiotryptophan moiety.
  • FIG. 1B provides an amanitin/linker conjugate represented by Formula (VI) (also referred to as “Conjugate B” or “ADC B”, where the amanitin is conjugated to a non-cleavable linker on indole 6′ oxygen.
  • the amanitin described in Formula (VI) is based on amatoxin having 6-hydroxy-tryptophan sulfoxide moiety.
  • 1C provides an amanitin/linker conjugate described herein in Formula (IIa) (also referred to as “Conjugate C” or “ADC C”, including a non-cleavable linker on indole 6′ oxygen, where the amanitin is based on amatoxin having 6-hydroxy-thiotryptophan moiety.
  • Conjugates A, B and C were tested in parallel for their potency on CD117 expressing cells with or without a pre-incubation in human serum.
  • ADC A, B, and C were either pre-incubated in cell culture media without human serum or 50% human serum at 37 C for 48 hours. The titrated ADC was then added to Kasumi-1 cells and incubated for 3 days at 37 C and cell killing was determined using CellTiter-Go.
  • cleavable Conjugate A and non-cleavable Conjugate C reached similar efficacy by day 3, with non-cleavable conjugate B demonstrating reduced maximum killing at higher concentrations.
  • Each ADC conjugate demonstrated picomolar IC50 killing.
  • Kasumi-1 cells were incubated with titrations of cleavable conjugate A or non-cleavable conjugate C for 3-7 days at 37 C and cell viability was measured on day 3, 4, 5, 6, and 7 by CellTiter-Glo. As shown in FIG. 3 , significant differences were observed in maximum cell killing at high concentrations for cleavable and non-cleavable ADC. When cells were incubated for a total of 6 days at 37 C with ADC, both cleavable conjugate A and non-cleavable conjugate C were able to achieve nearly complete cell killing at high concentrations.
  • the enzyme cleavable amanitin of FIG. 1A (Conjugate A) and the non-cleavable amanitin (conjugate C in FIG. 1C ) showed long term stability in serum, while amanitin/linker B showed instability and inactivation in serum.
  • Non-cleavable conjugate C demonstrated extended kinetics of cytotoxicity as measured in an in vitro assay compared to a similar cleavable amanitin conjugate (conjugate A).
  • Example 2 Anti-CD117 ADC Demonstrates Potent Killing of AML Cells and Human CD34+ Cells In Vitro, Selectively Depletes Human CD34+ Cells in Humanized Mice, and Demonstrates Anti-AML Activity in Xenograft Models
  • An anti-CD117 antibody (Ab85 having Fc modifications L234A, L235A, D265C, and H435A) was conjugated to Conjugate C forming anti-CD117 ADC C.
  • Anti-CD117 ADC C was tested both in vitro and in vivo for its ability to target and kill cells expressing CD117 and human HSCs.
  • Anti-CD117 ADC C Two in vitro assays were performed using anti-CD117 ADC C. The first tested the ability of the ADC to kill Kasumi-1 cells (an AML cell line), which express human CD117. As described in FIG. 4A , Anti-CD117 ADC C (referred to as “Anti-CD117-AM” in FIG. 4A ) was titrated and incubated with Kasumi-1 cells for 6 days at 37 C and viability was assessed by CellTiter-Glo. The control was a non-specific isotype matched ADC C. Non-cleavable conjugate C demonstrated potent killing of Kasumi-1 cells with an IC50 of 6.4 pM. In FIG.
  • An in vivo assay was also performed to test the ability of anti-CD117 ADC C to selectively deplete human CD34+ cells.
  • a single dose of 0.1, 0.3, or 1.0 mg/kg of the anti-CD117 ADC C was administered to humanized NSG mice.
  • Controls consisted of a 1 mg/kg dose of the unconjugated antibody that comprised part of non-cleavable conjugate C and vehicle (PBS) treatment.
  • PBS vehicle
  • anti-CD117 ADC C was able to potently deplete human CD34+ cells, achieving greater than 95% depletion compared to PBS for both 0.3 and 1 mg/kg doses.
  • the unconjugated antibody demonstrated no depletion as compared to vehicle control.
  • cleavable conjugate A (1 mg/kg 1 ⁇ , 1 mg/kg QOD ⁇ 2) or 10 mg/kg isotype conjugated to cleavable amanitin
  • FIG. 1A Kasumi-1 implanted mice were treated with non-cleavable conjugate C (1 mg/kg 1 ⁇ , 3 mg/kg 1 ⁇ , 10 mg/kg 1 ⁇ ) or 10 mg/kg isotype conjugated to non-cleavable amanitin ( FIG. 1C ).
  • Kaplan-Meier curves demonstrating survival of animals treated as indicated is depicted in FIG. 6 .
  • Kasumi-1 implanted mice treated with cleavable conjugate A demonstrate median survival above 50% at day 130 for groups treated with either 1 mg/kg or 1 mg/kg QOD ⁇ 2 in comparison to isotype, ARA-C, or vehicle treated animals.
  • Mice treated with either 3 mg/kg or 10 mg/kg non-cleavable conjugate C demonstrate full survival at day 130 in comparison to isotype, ARA-C, 1 mg/kg non-cleavable conjugate C, or vehicle treated animals.
  • non-cleavable conjugate C demonstrates potent in vitro cell killing of Kasumi-1 cells, an AML cell line, and primary human CD34+ CD90+ bone marrow cells.
  • Conjugate C is capable of depleting more than 95% of human CD34+ cells from the bone marrow of humanized NSG mice. Both cleavable and non-cleavable conjugates demonstrated potent anti-tumor activity in vivo.
  • non-cleavable conjugate C male cynomologus monkeys were treated with a single dose of non-cleavable conjugate C at 0.5, 1.0, and 2.0 mg/kg.
  • the 0.5 and 1.0 mg/kg doses were with Ab85 with the following Fc modifications: D265C H435A (EU numbering) conjugated to non-cleavable amanitin C ( FIG. 1C ).
  • the 2.0 mg/kg dose comprised Ab85 with the following Fc modifications: L234A L235A D265C H435A (EU numbering) conjugated to non-cleavable amanitin C ( FIG. 1C ).
  • Bone marrow aspirates were collected from treated animals on day 7 and efficacy was assessed by quantitation of CD34+ CD90+ CD45RA ⁇ populations by flow cytometry. Significant depletion was observed at 0.5 and 1.0 mg/kg doses ( FIG. 7 ). Peripheral blood samples were collected throughout the course of the 28 day study. Reticulocyte counts of animals treated at 0.5, 1.0, and 2.0 mg/kg decreased significantly beginning on day 2 with reticulocyte rebound occurring in a dose-dependent manner ( FIG. 7 ). This is expected on-target pharmacology as reticulocytes are short-lived and progenitor populations in this hematopoietic lineage strongly express CD117. Neutrophil depletion was observed beginning on day 18 for all treatment groups ( FIG. 7 ). This is also expected on-target pharmacology, as neutrophils are dependent on hematopoietic stem cell differentiation for regeneration.
  • Pharmacokinetic characteristics were also studied in anti-CD117 ADC A vs. C. As described in FIG. 9 , well tolerated doses of ADC conjugate C achieved saturating explosures in NHPs compared to ADC Conjugate A. Pharmacokinetic data was also analyzed for ADC conjugate C with various Fc mutations. For both anti-CD117 ADC A and anti-CD117 ADC C, ex vivo cytotoxicity assays revealed that for boths ADCs there was a loss of efficacy at plasma concentrations below 100 ng/ml (data not shown).
  • anti-CD117 ADC C demonstrated efficacy at doses as low as 0.5 mg/kg and was tolerated at a single dose of 2.0 mg/kg in cynomologus monkeys while the cleavable conjugate A was efficacious at a dose as low as 0.3 mg/kg but not tolerated at a single dose of 0.6 mg/kg supporting the finding that non-cleavable amanitin ( FIG. 1C ) is better tolerated.
  • Anti-CD2 ADCs A and C included anti-CD2 antibody RPA with FC modifications D265C/H435A. Naked RPA was used as a control.
  • Anti-CD5 ADCs A and C included anti-CD5 antibody 5D7 with Fc modifications D265/H435A. Naked 5D7 was used as a control.
  • Conjugate C demonstrated T cell depletion comparable to Conjugate A. Effiicacy results are also provide in the table below.
  • FIG. 11 provides results showing that CD2 is saturated by day 5, whereas some cells still express CD5.
  • Conjugate C was tested in comparison to Conjugate A in the context of anti-CD45 antibodies.
  • a chimeric anti-CD45 antibody 3D6 was used in the below experiments (murine 3D6 variable regions and a human IgG1 framework with Fc modifications D265C and H435A).
  • An in vitro cell killing assay was performed comparing anti-CD45 ADC A and anti-CD45 ADC C. As described in FIG. 12A , both ADCs A and C were able to kill human bone marrow CD34+ cells, as well as PBMC. An isotype negative control was also tested. As described in the Table presented in FIG. 12A , both anti-CD45 ADCs A and C were effective at killing CD34+ bone marrow and PBMC cells. This assay was repeated with anti-CD45 ADC A and B, As described in FIG. 12B , anti-CD45 ADC A showed a higher efficiency for killing CD34+ bone marrow cells vs. anti-CD45 ADC B. Both molecules were effective in a similar manner at killing human and cyno PBMCs.
  • a second in vitro cell killing assay was also performed using anti-CD45 ADCs A and C looking at the ability of each ADC to kill cells expressing either SKNO-1 or REH.
  • the results are provided in FIG. 13 (left panel are the number of live SKNO-1 cells following exposure to anti-CD45 ADC A or C; right panel are the number of live REH cells following exposure to anti-CD45 ADC A or C). The efficiency of each molecule in these assays is described in the table in FIG. 13 .
  • Anti-CD45 ADCs A and C were further tested for the ability of each ADC to deplete CD45+ cells in vivo. Both ADC A (cleavable) and ADC C (non-cleavable) were able to deplete CD45+ cells efficiently in a humanized NSG mouse model. The results of the study are provided in FIG. 14 . As described in FIG. 14 , both ADCs were able to deplete peripheral CD45+ cells as well as bone marrow (BM) CD34+ cells. The maximum total dose in the mice for anti-CD45 ADC A was 3 mg/kg, whereas a much higher dose of ADC C was tolerated for anti-CD45 ADC C at more than 51 mg/kg. Thus, efficacy was observed for both anti-CD45 ADCs, but the ADC C was tolerated by the mice at higher doses.
  • BM bone marrow
  • Anti-CD45 ADCs A and C were also tested for their ability to deplete peripheral lymphocytes, bone marrow (BM) HSCs and lymphocytes in humanized NSG mice. Anti-CD45 ADCs A and C were each delivered at a dose of 1 mg/kg to the mice. The results from these studies are described in FIG. 15 and show that anti-CD45 ADCs A and C had comparable depletion of peripheral lymphocytes, HSCs, and BM lymphocytes at day 7 post injection at 1 mg/kg. Anti-CD45 ADC A showed mild, transient reversible liver enzyme elevation at 1 mg/kg, while little to no liver enzyme elevation was observed with anti-CD45 ADC C with the same 1 mg/kg dose.
  • Anti-CD137 ADC A and ADC C were tested for their ability to kill T cells, as well as for serum stability.
  • Anti-CD137 antibody BBK2 was used in this example.
  • An in vitro T cell killing assay was performed, where the results shown in FIG. 16 demonstrated both anti-CD137 ADC A and C were able to kill activated T cells in comparison to the isotype ADC A and C control.
  • cell line serum stability was tested for both anti-CD137 ADC A and C over 48 hours, as described in FIG. 17 .
  • the ADCs of formula (IIa) as disclosed herein were prepared from the corresponding linker-amatoxin conjugates according to standard methods known to one of skill in the art.
  • the penultimate amatoxin-linker conjugate to the ADCs of formula (IIa) (compound 14; Scheme 3) may be prepared from amatoxin derivative 11 following the general procedure for O-alkylation of the related amatoxin ( ⁇ -amanitin) disclosed in U.S. Patent Application Publication No. 2018/0043033, the disclosure of which is incorporated by reference herein in its entirety.
  • Amatoxin derivative 11 may be prepared according to the procedures disclosed in International Patent Application Publication No. WO2019/030173, the disclosure of which is incorporated by reference herein in its entirety.
  • Compound 7 (Scheme 1) may be prepared according to the methods reported in International Patent Application Publication No. WO2014/009025, the disclosure of which is incorporated herein in its entirety.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11572411B2 (en) 2017-10-24 2023-02-07 Magenta Therapeutics, Inc. Anti-CD117 antibodies and conjugates

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2016228415B2 (en) * 2015-03-09 2021-04-01 Heidelberg Pharma Gmbh Amatoxin-antibody conjugates
BR112020010816A2 (pt) * 2017-11-29 2020-11-10 Magenta Therapeutics, Inc. composições e métodos para a depleção de células de cd2+
CA3208117A1 (en) * 2021-03-19 2022-09-22 Torsten HECHLER B-lymphocyte specific amatoxin antibody conjugates
US20240034791A1 (en) * 2021-03-31 2024-02-01 Emergence Therapeutics Ag Anti-nectin-4-antibodies and uses thereof
US20230355792A1 (en) 2022-04-07 2023-11-09 Heidelberg Pharma Research Gmbh Methods of improving the therapeutic index

Family Cites Families (147)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4363799A (en) 1979-03-20 1982-12-14 Ortho Pharmaceutical Corporation Monoclonal antibody to human T cells, and methods for preparing same
US4657760A (en) 1979-03-20 1987-04-14 Ortho Pharmaceutical Corporation Methods and compositions using monoclonal antibody to human T cells
US4515894A (en) 1979-03-20 1985-05-07 Ortho Pharmaceutical Corporation Hybrid cell line for producing monoclonal antibody to human T cells
US4816567A (en) 1983-04-08 1989-03-28 Genentech, Inc. Recombinant immunoglobin preparations
US4681581A (en) 1983-12-05 1987-07-21 Coates Fredrica V Adjustable size diaper and folding method therefor
US5776093A (en) 1985-07-05 1998-07-07 Immunomedics, Inc. Method for imaging and treating organs and tissues
US4735210A (en) 1985-07-05 1988-04-05 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US5101827A (en) 1985-07-05 1992-04-07 Immunomedics, Inc. Lymphographic and organ imaging method and kit
US4880935A (en) 1986-07-11 1989-11-14 Icrf (Patents) Limited Heterobifunctional linking agents derived from N-succinimido-dithio-alpha methyl-methylene-benzoates
WO1988006630A1 (en) 1987-03-02 1988-09-07 Genex Corporation Method for the preparation of binding molecules
US5648471A (en) 1987-12-03 1997-07-15 Centocor, Inc. One vial method for labeling antibodies with Technetium-99m
IL89220A (en) 1988-02-11 1994-02-27 Bristol Myers Squibb Co Immunoconjugates of anthracycline, their production and pharmaceutical preparations containing them
US6010902A (en) 1988-04-04 2000-01-04 Bristol-Meyers Squibb Company Antibody heteroconjugates and bispecific antibodies for use in regulation of lymphocyte activity
US5530101A (en) 1988-12-28 1996-06-25 Protein Design Labs, Inc. Humanized immunoglobulins
US5413923A (en) 1989-07-25 1995-05-09 Cell Genesys, Inc. Homologous recombination for universal donor cells and chimeric mammalian hosts
WO1991001754A1 (en) 1989-08-09 1991-02-21 Rhodes Buck A Direct radiolabeling of antibodies and other proteins with technetium or rhenium
US5208020A (en) 1989-10-25 1993-05-04 Immunogen Inc. Cytotoxic agents comprising maytansinoids and their therapeutic use
ATE356869T1 (de) 1990-01-12 2007-04-15 Amgen Fremont Inc Bildung von xenogenen antikörpern
GB9015198D0 (en) 1990-07-10 1990-08-29 Brien Caroline J O Binding substance
US5661016A (en) 1990-08-29 1997-08-26 Genpharm International Inc. Transgenic non-human animals capable of producing heterologous antibodies of various isotypes
US5814318A (en) 1990-08-29 1998-09-29 Genpharm International Inc. Transgenic non-human animals for producing heterologous antibodies
ES2108048T3 (es) 1990-08-29 1997-12-16 Genpharm Int Produccion y utilizacion de animales inferiores transgenicos capaces de producir anticuerpos heterologos.
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5633425A (en) 1990-08-29 1997-05-27 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5625126A (en) 1990-08-29 1997-04-29 Genpharm International, Inc. Transgenic non-human animals for producing heterologous antibodies
IT1249051B (it) 1991-02-26 1995-02-11 Italfarmaco Spa Immunotossina da anticorpi monoclonali anti-cd5
WO1992016563A1 (en) 1991-03-12 1992-10-01 Biogen, Inc. Monoclonal antibodies recognizing lymphocyte function associated antigen-3
US7018809B1 (en) 1991-09-19 2006-03-28 Genentech, Inc. Expression of functional antibody fragments
US6764681B2 (en) 1991-10-07 2004-07-20 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
US6162432A (en) 1991-10-07 2000-12-19 Biogen, Inc. Method of prophylaxis or treatment of antigen presenting cell driven skin conditions using inhibitors of the CD2/LFA-3 interaction
US5621083A (en) 1991-11-04 1997-04-15 Xoma Corporation Immunotoxins comprising ribosome-inactivating proteins
US6146850A (en) 1991-11-04 2000-11-14 Xoma Corporation Proteins encoding gelonin sequences
US5837491A (en) 1991-11-04 1998-11-17 Xoma Corporation Polynucleotides encoding gelonin sequences
WO1993011236A1 (en) 1991-12-02 1993-06-10 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5869619A (en) 1991-12-13 1999-02-09 Xoma Corporation Modified antibody variable domains
JP4157160B2 (ja) 1991-12-13 2008-09-24 ゾーマ テクノロジー リミテッド 改変抗体可変領域の調製のための方法
US5622929A (en) 1992-01-23 1997-04-22 Bristol-Myers Squibb Company Thioether conjugates
US5730979A (en) 1993-03-05 1998-03-24 Universite Catholique Delouvain LO-CD2a antibody and uses thereof for inhibiting T cell activation and proliferation
US5951983A (en) 1993-03-05 1999-09-14 Universite Catholique De Louvain Methods of inhibiting T cell mediated immune responses with humanized LO-CD2A-specific antibodies
JP3711143B2 (ja) 1993-03-05 2005-10-26 ユニヴェルシテ カトリク ドゥ ルーヴェン Ｔ細胞の活性化と増殖を阻害するＬＯ−ＣＤ２ａ抗体およびその用途
US7592006B1 (en) 1993-03-05 2009-09-22 Université Catholique de Louvain Composition comprising the LO-CD2a antibody
US5817311A (en) 1993-03-05 1998-10-06 Universite Catholique De Louvain Methods of inhibiting T-cell medicated immune responses with LO-CD2a-specific antibodies
DE4312916C2 (de) 1993-04-14 1995-03-23 Fresenius Ag Arzneimittel zur Behandlung von Immunreaktionen
US6214345B1 (en) 1993-05-14 2001-04-10 Bristol-Myers Squibb Co. Lysosomal enzyme-cleavable antitumor drug conjugates
US5795572A (en) 1993-05-25 1998-08-18 Bristol-Myers Squibb Company Monoclonal antibodies and FV specific for CD2 antigen
EP0647450A1 (en) 1993-09-09 1995-04-12 BEHRINGWERKE Aktiengesellschaft Improved prodrugs for enzyme mediated activation
EP1025856A3 (en) 1994-03-08 2002-10-16 Dana-Farber Cancer Institute Methods for modulating T Cell unresponsiveness
US5789199A (en) 1994-11-03 1998-08-04 Genentech, Inc. Process for bacterial production of polypeptides
US5840523A (en) 1995-03-01 1998-11-24 Genetech, Inc. Methods and compositions for secretion of heterologous polypeptides
AU5632296A (en) 1995-04-27 1996-11-18 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996034096A1 (en) 1995-04-28 1996-10-31 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO1996041608A2 (en) 1995-06-07 1996-12-27 Thera Pro Pyrularia thionin containing immunotoxins and immunotoxin-like conjugates
ES2195036T3 (es) 1995-12-22 2003-12-01 Bristol Myers Squibb Co Conectores de hidrazona ramificados.
US6218519B1 (en) 1996-04-12 2001-04-17 Pro-Neuron, Inc. Compounds and methods for the selective treatment of cancer and bacterial infections
US6764688B2 (en) 1996-09-03 2004-07-20 Kaneka Corporation Method for inducing immunosuppressive cells and a culture device to be used therefor
AU739028B2 (en) 1996-09-27 2001-10-04 Bristol-Myers Squibb Company Hydrolyzable prodrugs for delivery of anticancer drugs to metastatic cells
US5916771A (en) 1996-10-11 1999-06-29 Abgenix, Inc. Production of a multimeric protein by cell fusion method
US6759509B1 (en) 1996-11-05 2004-07-06 Bristol-Myers Squibb Company Branched peptide linkers
WO1998024893A2 (en) 1996-12-03 1998-06-11 Abgenix, Inc. TRANSGENIC MAMMALS HAVING HUMAN IG LOCI INCLUDING PLURAL VH AND Vλ REGIONS AND ANTIBODIES PRODUCED THEREFROM
US6849258B1 (en) 1997-07-18 2005-02-01 Universite Catholique De Louvain LO-CD2a antibody and uses thereof for inhibiting T cell activation and proliferation
US6358506B1 (en) 1997-11-05 2002-03-19 University Of Southern California Use of cytokines and mitogens to inhibit pathological immune responses
CA2309919A1 (en) 1997-11-14 1999-05-27 The General Hospital Corporation Treatment of hematologic disorders
US6835807B1 (en) 1998-05-22 2004-12-28 Daiichi Pharmaceuticals Co., Ltd. Drug complex and drug delivery system
US7157418B1 (en) 1998-07-22 2007-01-02 Osprey Pharmaceuticals, Ltd. Methods and compositions for treating secondary tissue damage and other inflammatory conditions and disorders
WO2000045842A2 (en) 1999-02-04 2000-08-10 The General Hospital Corporation Methods for human allografting
US6268488B1 (en) 1999-05-25 2001-07-31 Barbas, Iii Carlos F. Prodrug activation using catalytic antibodies
US6541611B1 (en) 1999-06-18 2003-04-01 Universite Catholique De Louvain LO-CD2b antibody
US20040052793A1 (en) 2001-02-22 2004-03-18 Carter Paul J. Caspase activivated prodrugs therapy
US20040018194A1 (en) 2000-11-28 2004-01-29 Francisco Joseph A. Recombinant anti-CD30 antibodies and uses thereof
US20050069538A1 (en) 2003-09-18 2005-03-31 Gregorio Aversa Therapeutic binding molecules
AU2002322478A1 (en) 2001-03-02 2002-12-16 Medimmune, Inc. Methods of administering/dosing cd2 antagonists for the prevention and treatment of autoimmune disorders or inflammatory disorders
EP1243276A1 (en) 2001-03-23 2002-09-25 Franciscus Marinus Hendrikus De Groot Elongated and multiple spacers containing activatible prodrugs
US6441163B1 (en) 2001-05-31 2002-08-27 Immunogen, Inc. Methods for preparation of cytotoxic conjugates of maytansinoids and cell binding agents
US7091186B2 (en) 2001-09-24 2006-08-15 Seattle Genetics, Inc. p-Amidobenzylethers in drug delivery agents
CA2467597A1 (en) 2001-12-03 2003-06-12 Abgenix, Inc. Anti-cd45rb antibodies for use in treating autoimmune disease and transplant rejection
US20080254027A1 (en) 2002-03-01 2008-10-16 Bernett Matthew J Optimized CD5 antibodies and methods of using the same
US6887673B2 (en) 2002-07-30 2005-05-03 Bristol-Myers Squibb Company Humanized antibodies against human 4-1BB
WO2004032828A2 (en) 2002-07-31 2004-04-22 Seattle Genetics, Inc. Anti-cd20 antibody-drug conjugates for the treatment of cancer and immune disorders
CA2497628A1 (en) 2002-09-05 2004-03-18 Medimmune, Inc. Methods of preventing or treating cell malignancies by administering cd2 antagonists
US8088387B2 (en) 2003-10-10 2012-01-03 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
US7288638B2 (en) 2003-10-10 2007-10-30 Bristol-Myers Squibb Company Fully human antibodies against human 4-1BB
SG195524A1 (en) 2003-11-06 2013-12-30 Seattle Genetics Inc Monomethylvaline compounds capable of conjugation to ligands
WO2005082023A2 (en) 2004-02-23 2005-09-09 Genentech, Inc. Heterocyclic self-immolative linkers and conjugates
SI1791565T1 (sl) 2004-09-23 2016-08-31 Genentech, Inc. Cisteinsko konstruirana protitelesa in konjugati
KR100694508B1 (ko) 2005-05-24 2007-03-13 울산대학교 산학협력단 Ｈｂｂｋ４항체를 포함하는 암 질환 치료용 약학조성물및 이를 이용한 암의 면역치료 방법
KR100745488B1 (ko) 2006-07-04 2007-08-02 학교법인 울산공업학원 항-4-1ｂｂ 항체 및 화학 항암제를 포함하는 암 질환 예방및 치료용 약학 조성물
ITBO20070242A1 (it) 2007-04-03 2008-10-04 Gsg Int Spa Profilato per serramenti scorrevoli, metodo per la realizzazione del profilato, e serramento ottenuto con il profilato stesso.
KR20080107050A (ko) 2007-06-05 2008-12-10 울산대학교 산학협력단 항-ｃｄ１３７ 단일클론 항체를 포함하는 만성이식편대숙주 질환의 예방 또는 치료용 약학적 조성물
WO2009134389A2 (en) 2008-05-01 2009-11-05 Gtc Biotherapeutics, Inc. An anti-cd137 antibody as an agent in the treatment of inflammatory conditions
MX2011000970A (es) 2008-08-29 2011-03-15 Symphogen As Anticuerpos anti-cd5.
WO2010042433A1 (en) 2008-10-06 2010-04-15 Bristol-Myers Squibb Company Combination of cd137 antibody and ctla-4 antibody for the treatment of proliferative diseases
AU2010234334B2 (en) 2009-04-08 2014-10-09 Deutsches Krebsforschungszentrum Amatoxin-armed therapeutic cell surface binding components designed for tumour therapy
PT2430051T (pt) 2009-05-14 2016-07-11 Inst Nat De La Santé Et De La Rech Medicale Composições contendo anticorpos para tratar doenças relacionadas com células b ou t cd5+ hla- dr+
WO2010132389A2 (en) 2009-05-14 2010-11-18 University Of Maryland, Baltimore Methods for treating cancers and diseases associated with 4-1bb (cd137) expression
WO2011031063A2 (ko) 2009-09-09 2011-03-17 울산대학교 산학협력단 항 4-1bb 항체를 포함하는 대사성 질환의 예방 또는 치료용 조성물
JP5950824B2 (ja) 2009-12-07 2016-07-13 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 抗腫瘍抗体療法を増強するための方法
KR20110085038A (ko) 2010-01-19 2011-07-27 울산대학교 산학협력단 항 ｃｄ137-항체 및 독소 결합물을 이용한 ｃｄ137 양성세포의 제거방법
WO2012032525A2 (en) 2010-09-08 2012-03-15 Yeda Research And Development Co. Ltd. An immunosuppressive drug combination for a stable and long term engraftment
SG10201506906VA (en) 2010-09-09 2015-10-29 Pfizer 4-1bb binding molecules
ES2402254T3 (es) * 2010-09-30 2013-04-30 Heidelberg Pharma Ag Conjugados de amatoxinas con ligadores mejorados
EP2497499A1 (en) 2011-03-10 2012-09-12 Heidelberg Pharma GmbH Amatoxin-conjugates with improved linkages
DK2699598T3 (en) 2011-04-19 2019-04-23 Pfizer COMBINATIONS OF ANTI-4-1BB ANTIBODIES AND ADCC-INducing ANTIBODIES FOR TREATMENT OF CANCER
JP2015504047A (ja) 2011-12-22 2015-02-05 イェダ リサーチ アンド デベロップメント カンパニー リミテッド 安定かつ長期の生着のための併用療法
US20130280282A1 (en) 2012-04-24 2013-10-24 Daiichi Sankyo Co., Ltd. Dr5 ligand drug conjugates
EP2684865A1 (en) 2012-07-13 2014-01-15 Heidelberg Pharma GmbH Methods for synthesizing amatoxin building block and amatoxins
WO2014043403A1 (en) 2012-09-12 2014-03-20 Agensys, Inc. Amatoxin derivatives and cell-permeable conjugates thereof as inhibitors of rna polymerase
EP2774624A1 (en) 2013-03-04 2014-09-10 Heidelberg Pharma GmbH Amatoxin derivatives
CN105007950B (zh) * 2013-03-15 2019-01-15 诺华股份有限公司 抗体药物缀合物
KR20220127364A (ko) 2013-12-19 2022-09-19 씨젠 인크. 표적화된-약물 컨쥬게이트와 함께 사용되는 메틸렌 카바메이트 링커
US10899840B2 (en) 2014-02-04 2021-01-26 Pfizer Inc. Combination of a PD-1 antagonist and a 4-1BB agonist for treating cancer
AU2015264528A1 (en) 2014-05-21 2016-11-03 Kyowa Hakko Kirin Co., Ltd. Combination of an anti-CCR4 antibody and a 4-1BB agonist for treating cancer
WO2015188047A1 (en) 2014-06-06 2015-12-10 University Of Maryland, Baltimore ANTI-CD-137 MONOCLONAL ANTIBODIES WITH DISTINCT FcγR BINDING ABILITIES FOR TREATMENT OF CANCER OR AUTOIMMUNITY
WO2016016442A1 (en) 2014-08-01 2016-02-04 INSERM (Institut National de la Santé et de la Recherche Médicale) An anti-cd45rc antibody for use as drug
WO2016029073A2 (en) 2014-08-22 2016-02-25 Bristol-Myers Squibb Company Treatment of cancer using a combination of an anti-pd-1 antibody and an anti-cd137 antibody
CN107106701B (zh) 2014-10-03 2020-11-06 西纳福克斯股份有限公司 磺酰胺接头、其缀合物及制备方法
EP3215519A1 (en) 2014-11-06 2017-09-13 Novartis AG Amatoxin derivatives and conjugates thereof as inhibitors of rna polymerase
EP3026060A1 (en) 2014-11-26 2016-06-01 Miltenyi Biotec GmbH Humanized antibody or fragment thereof specific for CD45R0
PL3623386T3 (pl) 2015-01-08 2022-08-08 BioNTech SE Agonistyczne środki wiążące receptor tnf
CN107921104A (zh) 2015-02-22 2018-04-17 索伦托治疗有限公司 结合cd137的抗体疗法
AU2016228415B2 (en) * 2015-03-09 2021-04-01 Heidelberg Pharma Gmbh Amatoxin-antibody conjugates
WO2016172606A1 (en) 2015-04-23 2016-10-27 Baylor College Of Medicine Cd5 chimeric antigen receptor for adoptive t cell therapy
RU2017142008A (ru) 2015-05-21 2019-06-24 Эллигейтор Биосайенс Аб Новые полипептиды
CN108026169B (zh) 2015-09-22 2021-05-28 苏州丁孚靶点生物技术有限公司 抗人cd137的完全人抗体及其应用
GB201519481D0 (en) 2015-11-04 2015-12-16 Cancer Rec Tech Ltd Immunomodulatory antibodies
US20170129128A1 (en) 2015-11-05 2017-05-11 Black & Decker Inc. Saw
CA3009494C (en) 2015-12-22 2019-03-05 3M Innovative Properties Company Health management system with multidimensional performance representation
EP3408294A1 (en) 2016-01-25 2018-12-05 Pfizer Inc. Combination of an ox40 agonist and a 4-1bb agonist monoclonal antibody for treating cancer
EP3222292A1 (en) 2016-03-03 2017-09-27 Heidelberg Pharma GmbH Amanitin conjugates
WO2017155937A1 (en) 2016-03-07 2017-09-14 Actinium Pharmaceuticals, Inc. Stabilized radiolabeled anti-cd45 immunoglobulin compositions
WO2017181034A1 (en) 2016-04-14 2017-10-19 Bristol-Myers Squibb Company Combination therapy using an anti-fucosyl-gm1 antibody and an anti-cd137 antibody
WO2017046658A1 (en) 2016-04-20 2017-03-23 Hangzhou Dac Biotech Co, Ltd Derivatives of amanita toxins and their conjugation to a cell binding molecule
CN115645544A (zh) * 2016-05-31 2023-01-31 索伦托药业有限公司 具有鹅膏毒肽的衍生物作为药物的抗体药物缀合物
WO2018017761A1 (en) 2016-07-20 2018-01-25 Igm Biosciences, Inc. Multimeric cd137/4-1bb binding molecules and uses thereof
GB201619648D0 (en) 2016-11-21 2017-01-04 Alligator Bioscience Ab Novel antibodies and uses thereof
JP7274417B2 (ja) 2016-11-23 2023-05-16 イミュノア・セラピューティクス・インコーポレイテッド ４－１ｂｂ結合タンパク質及びその使用
CA3044508A1 (en) 2016-12-23 2018-06-28 Heidelberg Pharma Research Gmbh Novel amanitin conjugate
BR112019007714B1 (pt) 2017-01-06 2022-01-18 Eutilex Co., Ltd Anticorpos anti-4-1bb, uso do mesmo, composição farmacêutica, métodos para determinar uma dose, para aumentar a secreção de ifn-gama por uma célula in vitro e para proliferação ex vivo ou isolamento de células t ativadas e uso das mesmas
SG10202102897PA (en) 2017-01-20 2021-04-29 Magenta Therapeutics Inc Compositions and methods for the depletion of cd137+ cells
SG11201907753TA (en) 2017-02-24 2019-09-27 Macrogenics Inc Bispecific binding molecules that are capable of binding cd137 and tumor antigens, and uses thereof
WO2018191502A2 (en) 2017-04-13 2018-10-18 Agenus Inc. Anti-cd137 antibodies and methods of use thereof
JP7402521B2 (ja) 2017-07-11 2023-12-21 コンパス セラピューティクス リミテッド ライアビリティ カンパニー ヒトｃｄ１３７に結合するアゴニスト性抗体およびその使用
GB201712032D0 (en) 2017-07-26 2017-09-06 Bioinvent Int Ab Antibodies and uses thereof
KR20210158420A (ko) 2017-08-01 2021-12-30 일라이 릴리 앤드 캄파니 항-cd137 항체
CN111051310B (zh) * 2017-08-07 2022-11-08 海德堡医药研究有限责任公司 合成鹅膏蕈碱的新方法
WO2019036855A1 (en) 2017-08-21 2019-02-28 Adagene Inc. ANTI-CD137 MOLECULES AND THEIR USE
CN111989122A (zh) 2018-04-13 2020-11-24 海德堡医药研究有限责任公司 用于治疗实体瘤的靶向鹅膏毒素缀合物
SI3830132T1 (sl) 2018-07-31 2023-03-31 Heidelberg Pharma Research Gmbh Humanizirana protitelesa proti PSMA

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11572411B2 (en) 2017-10-24 2023-02-07 Magenta Therapeutics, Inc. Anti-CD117 antibodies and conjugates

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