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WO2025096716A1 - Conjugués anticorps anti-calréticuline (calr) mutant-médicament et leurs utilisations - Google Patents

Conjugués anticorps anti-calréticuline (calr) mutant-médicament et leurs utilisations Download PDF

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Publication number
WO2025096716A1
WO2025096716A1 PCT/US2024/053807 US2024053807W WO2025096716A1 WO 2025096716 A1 WO2025096716 A1 WO 2025096716A1 US 2024053807 W US2024053807 W US 2024053807W WO 2025096716 A1 WO2025096716 A1 WO 2025096716A1
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seq
amino acid
acid sequence
antibody
heavy chain
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Darlise DIMATTEO
Jonathan Rios-Doria
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Incyte Corp
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Incyte Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—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
    • A61K47/68035—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a pyrrolobenzodiazepine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Definitions

  • Calreticulin is a highly conserved chaperone protein that resides primarily in the endoplasmic reticulum and is involved in a variety of cellular processes including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. CALR is also found in the nucleus, suggesting that it may have a role in transcription regulation. Mutations in the gene for CALR have been identified in patients with myeloproliferative neoplasms.
  • the present disclosure is based, at least in part, on the development of antibody-drug conjugates that bind to mutant CALR (‘‘mutCALR”) on the cell surface, are internalized by the cell, and exhibit anti-proliferative effects.
  • ‘mutCALR” mutant CALR
  • aspects of the present application provide an antibody-drug conjugate comprising: an antibody that binds to human mutant calreticulin (CALR); and a pyrrolobenzodiazepine dimer, wherein the antibody comprises a heavy chain variable region (VH) comprising a VH CDR1; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1);
  • the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO: 102); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence GGX104X105X106GX107X108X109VX110 (SEQ ID NO: 103), wherein X104 is N, D, or S; wherein X105 is Y, N, or D; wherein X106 is I or T; wherein X107 is S, D, I, R, or T; wherein Xios is K, E, or I; wherein X109 is
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:26; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:53; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:69;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:70; the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 8; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR
  • VH CDR1 comprises the amino acid sequence of SEQ ID NO:27; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO:35;
  • VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:39; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO:40; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71; the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1; the VH CDR
  • the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:74;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO:1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28;
  • the VL CDR2 comprises the amino acid sequence of SEQ ID NO:54; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:75;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 7;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 118;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 93;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VL CDR1 comprises the amino acid sequence of SEQ ID NO:28;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1;
  • the VH CDR2 comprises the amino acid sequence of SEQ ID NO: 94;
  • the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18;
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:95; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71.
  • the VH is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs: 165-174 and 188-208; and the VL is at least 80% identical to the amino acid sequence of any one of SEQ ID NOs:264-292 and 315-316.
  • the VH comprises the amino acid sequence of any one of SEQ ID NOs:165-174 and 188-208; and the VL comprises the amino acid sequence of any one of SEQ ID NOs:264-292 and 315-316.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:264; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:265; the VH comprises the amino acid sequence of SEQ ID NO: 166 and the VL comprises the amino acid sequence of SEQ ID NO:266; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:266; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:267; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:269; the VH comprises the amino acid sequence of SEQ ID NO: 165 and
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:280; the VH comprises the amino acid sequence of SEQ ID NO: 172 and the VL comprises the amino acid sequence of SEQ ID NO:281; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:282; the VH comprises the amino acid sequence of SEQ ID NO: 173 and the VL comprises the amino acid sequence of SEQ ID NO:283; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:284; the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:285; the VH comprises the amino acid sequence of SEQ ID NO: 174 and the VL comprises the amino acid sequence of SEQ ID NO:286; the VH comprises the amino acid sequence of SEQ ID NO: 174 and
  • the VH comprises the amino acid sequence of SEQ ID NO: 192 and the VL comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO: 193 and the VL comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO: 194 and the VL comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO: 195 and the VL comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:316;
  • the VH comprises the amino acid sequence of SEQ ID NO: 188 and the VL comprises the amino acid sequence of SEQ ID NO:316;
  • the VH comprises
  • the VH comprises the amino acid sequence of SEQ ID NO: 199 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID N0:200 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO:201 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO: 197 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID NO: 198 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID NO: 199 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID N0:200 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID NO:315;
  • the VH comprises the amino acid sequence of SEQ ID NO:205 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO:206 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO:207 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO:208 and the VL comprises the amino acid sequence of SEQ ID NO:268; the VH comprises the amino acid sequence of SEQ ID NO: 170 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID NO:202 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ ID NO:203 and the VL comprises the amino acid sequence of SEQ ID NO:315; the VH comprises the amino acid sequence of SEQ
  • the VH comprises the amino acid sequence of SEQ ID NO:206 and the VL comprises the amino acid sequence of SEQ ID NO:316; the VH comprises the amino acid sequence of SEQ ID NO:207 and the VL comprises the amino acid sequence of SEQ ID NO:316; or the VH comprises the amino acid sequence of SEQ ID NO:208 and the VL comprises the amino acid sequence of SEQ ID NO:316.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315.
  • the antibody comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 119-128 and 142-164; and a light chain comprising the amino acid sequence of any one of SEQ ID NOs:209-237 and 260-261.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:209; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:210; the heavy chain comprises the amino acid sequence of SEQ ID NO: 120 and the light chain comprises the amino acid sequence of SEQ ID NO:211 ; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:211 ; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:212; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:214; the heavy chain comprises the amino acid sequence of SEQ ID NO: 121
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 122 and the light chain comprises the amino acid sequence of SEQ ID NO:218; the heavy chain comprises the amino acid sequence of SEQ ID NO: 123 and the light chain comprises the amino acid sequence of SEQ ID NO:219; the heavy chain comprises the amino acid sequence of SEQ ID NO: 124 and the light chain comprises the amino acid sequence of SEQ ID NO:220; the heavy chain comprises the amino acid sequence of SEQ ID NO: 125 and the light chain comprises the amino acid sequence of SEQ ID NO:221; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:222; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:223; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:224; the heavy chain comprises the amino acid sequence of SEQ ID NO: 122 and the
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:235; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:236; the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:237; the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 143 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 144 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 145 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 145 and
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 146 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 147 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 148 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 149 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 150 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:261; the heavy chain comprises the amino acid sequence of SEQ ID
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 154 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 155 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 156 and the light chain comprises the amino acid sequence of SEQ ID NO:213; the heavy chain comprises the amino acid sequence of SEQ ID NO: 152 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 153 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 154 and the light chain comprises the amino acid sequence of SEQ ID NO:260; the heavy chain comprises the amino acid sequence of SEQ ID NO: 154 and the light
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 160 and the light chain comprises the amino acid sequence of SEQ ID NO:213;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 161 and the light chain comprises the amino acid sequence of SEQ ID NO:213;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 162 and the light chain comprises the amino acid sequence of SEQ ID NO:213;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 163 and the light chain comprises the amino acid sequence of SEQ ID NO:213;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 164 and the light chain comprises the amino acid sequence of SEQ ID NO:213;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 157 and the light chain comprises the amino acid sequence of SEQ ID NO:260;
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 158 and the light chain comprises the amino acid sequence of SEQ ID NO:260;
  • the heavy chain comprises the
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 161 and the light chain comprises the amino acid sequence of SEQ ID NO:261; the heavy chain comprises the amino acid sequence of SEQ ID NO: 162 and the light chain comprises the amino acid sequence of SEQ ID NO:261; the heavy chain comprises the amino acid sequence of SEQ ID NO: 163 and the light chain comprises the amino acid sequence of SEQ ID NO:261; or the heavy chain comprises the amino acid sequence of SEQ ID NO: 164 and the light chain comprises the amino acid sequence of SEQ ID NO:261.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260.
  • the human mutant CALR is human Type 1 mutant CALR comprising the amino acid sequence of SEQ ID NO: 320. In some embodiments, the human mutant CALR is human Type 2 mutant CALR comprising the amino acid sequence of SEQ ID NO:321.
  • the antibody is a human or humanized antibody. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is an IgGl, IgG2, IgG3 or IgG4 antibody. In some embodiments, the antibody is an IgGl antibody. In some embodiments, the antibody is a bispecific antibody, a biparatopic antibody, a single chain antibody (scFv), an Fab fragment, an F(ab’)2 fragment, an Fab’ fragment, an Fsc fragment, an Fv fragment, an scFv, an sc(Fv)2, or a diabody.
  • scFv single chain antibody
  • the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD- 1882.
  • the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the pyrrolobenzodiazepine dimer is linked to a linker to form a payload.
  • the linker is cleavable.
  • the linker comprises a maleimide linker.
  • the linker comprises a peptide.
  • the linker comprises a polyethylene glycol spacer.
  • the payload is selected from the group consisting of SG3400, SG3227, SG3249, SG3710, SG3259, SG3584, SG3203, SG3231, SG3451, and SGD-1910.
  • the pyrrolobenzodiazepine dimer comprises a cap.
  • the cap is cleavable.
  • the cap comprises a glucuronide group or derivative thereof or a galactoside group or derivative thereof.
  • the payload is SG3600.
  • the antibody-drug conjugate has a drug loading of from about one to about five pyrrolobenzodiazepine dimers per antibody. In some embodiments, the antibody-drug conjugate has a drug loading of two pyrrolobenzodiazepine dimers per antibody.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:7; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ
  • SUBSTITUTE SHEET (RULE 26) ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the VH comprises the amino acid sequence of SEQ ID NO: 165 and the VL comprises the amino acid sequence of SEQ ID NO:268, and wherein the pyrrolobenzodiazepine dimer is SG3199.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 119 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is SG3199.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 142 and the light chain comprises the amino acid sequence of SEQ ID NO:213, and wherein the pyrrolobenzodiazepine dimer is SG3199.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:95; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:95; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:95; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the VH CDR1 comprises the amino acid sequence of SEQ ID NO: 1; the VH CDR2 comprises the amino acid sequence of SEQ ID NO:95; the VH CDR3 comprises the amino acid sequence of SEQ ID NO: 18; the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30; the VL CDR2 comprises the amino acid sequence of SEQ ID NO:55; and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:71, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the VH comprises the amino acid sequence of SEQ ID NO: 196 and the VL comprises the amino acid sequence of SEQ ID NO:315, and wherein the pyrrolobenzodiazepine dimer is SG3199.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and wherein the pyrrolobenzodiazepine dimer is selected from the group consisting of SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, and SGD-1882.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and wherein the pyrrolobenzodiazepine dimer is PBD-1.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and wherein the pyrrolobenzodiazepine dimer is SG2000.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and wherein the pyrrolobenzodiazepine dimer is SG3199.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, the linker is a group of formula L-l, and the pyrrolobenzodiazepine dimer is SG3199.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, the linker is a group of formula L-2, and the pyrrolobenzodiazepine dimer is PBD-1.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, the payload is PL-1.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and the payload is PL-2.
  • the heavy chain comprises the amino acid sequence of SEQ ID NO: 151 and the light chain comprises the amino acid sequence of SEQ ID NO:260, and the payload is PL-3.
  • aspects of the present disclosure provide a pharmaceutical composition comprising an antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier.
  • aspects of the present disclosure provide a method of treating a myeloproliferative neoplasm in a human subject in need thereof, the method compnsing administering to the human subject an effective amount of an antibody-drug conjugate described herein or a pharmaceutical composition described herein.
  • the myeloproliferative neoplasm is selected from the group consisting of chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.
  • methods described herein further comprise administering to the human subject an additional therapy selected from the group consisting of a Janus tyrosine kinase (JAK) inhibitor, a phosphoinositide 3-kinase (PI3K) inhibitor, a standard of care therapy, or a combination thereof.
  • a Janus tyrosine kinase (JAK) inhibitor a Janus tyrosine kinase (JAK) inhibitor
  • PI3K phosphoinositide 3-kinase
  • the JAK inhibitor is ruxolitinib and itaticinib.
  • the PI3K inhibitor is parsaclisib.
  • the standard of care therapy is selected from the group consisting of IFN-alpha, hydroxyurea, thalidomide, lenalidomide, an androgen, an erythropoietin-stimulating agent, a chemotherapeutic agent, or a combination thereof.
  • kits comprising an antibody-drug conjugate described herein, and instructions for use in treating a myeloproliferative neoplasm in a
  • SUBSTITUTE SHEET (RULE 26) human subject in need thereof, optionally with instructions for use in combination with an additional therapy.
  • FIG. 1A includes a graph showing internalization of anti-mutCALR antibody (Clone 74) in 4F2 cells or MPL-6 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. IB includes a graph showing internalization of anti-mutCALR antibody (Clone 55) in 4F2 cells or MPL-6 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. ID includes a graph showing proliferation of 4F2 or MPL-6 cells in the presence of anti-mutCALR antibody Clone 74, Clone 55, or Clone 6. Proliferation was measured in the absence or presence of dynasore for each clone.
  • FIG. IE includes a graph showing internalization of the anti-mutCALR antibody B3 in 4F2 cells or MPL-6 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. IF includes a graph showing internalization of a first isotype control antibody in 4F2 cells or MPL-6 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. 1G includes a graph showing internalization of a second isotype control antibody in 4F2 cells or MPL-6 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. 1H includes a graph showing proliferation of 4F2 or MPL-6 cells in the presence of the anti-mutCALR antibody B3 or isotype control antibodies. Proliferation was measured in the absence or presence of dynasore for each antibody.
  • FIG. 2B includes a graph showing internalization of anti-mutCALR antibody (Clone 74) or an isotype control in Ba/F3-TPOR/CALR de152 cells in the absence or presence of dynasore or DMSO (vehicle control).
  • FIG. 2C includes a graph showing proliferation of Ba/F3-TPOR/CALR deb2 cells pretreated with dynasore or DMSO (vehicle control) in the presence of anti-mutCALR antibody (Clone 74) or an isotype control.
  • FIG. 2D includes a graph showing caspase induction in Ba/F3-TPOR/CALR de152 cells pretreated with dynasore or DMSO (vehicle control) in the presence of anti-mutCALR antibody (Clone 74) or ab isotype control.
  • FIG. 2E includes a graph showing pSTAT5 inhibition in Ba/F3-TPOR/CALR de152 cells pretreated with dynasore or DMSO (vehicle control) in the presence of anti-mutCALR antibody (Clone 74) or an isotype control. Inhibition of pSTAT5 was calculated after normalization to maximal (100%) inhibition achieved by the cell treatment with 2 pM of ruxolitinib and no inhibition (0%) in isotype-treated cells. Data show a representative of 3 independent experiments.
  • FIG. 2F includes an image showing that anti-mutCALR antibody (Clone 74) is internalized upon binding mutCALR on the cell surface.
  • Arrow 1 cell membrane
  • Arrow 2 nucleus
  • Arrow 3 anti-mutCALR antibody.
  • FIG. 2G includes images showing that anti-mutCALR antibody (Clone 74) colocalizes with lysosome markers upon internalization. Light grey: LAMP-1, Dark grey: anti- mutCALR antibody.
  • FIG. 3A includes graphs from flow cytometry analysis of the surface expression of TPOR in the presence of anti-mutCALR antibody (Clone 74) or an isotype control. Graphs represent MFI mean ⁇ SD of 3 (Ba/F3-TPOR) or 5 (Ba/F3-TPOR mutC ALR de152 ) independent experiments. Significance was determined using student’s t-test: ** p ⁇ 0.01.
  • FIG. 3B includes an image from Western blot analysis of TPOR and pSTAT5 expression in the presence of anti-mutCALR antibody (Clone 74) or an isotype control. Data representative from 2 independent experiments.
  • FIG. 3C includes data from immunofluorescence analysis of internalization of TPOR/mutCALR complex upon anti-mutCALR antibody (Clone 74) binding. Areas showing both light grey (cell membrane) and dark grey (TPOR) were false colored for easier visualization of the pixels exhibiting colocalization.
  • FIG. 4A includes a graph showing in BAF3/4F2 cells internalization of anti- mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), isotype control conjugated to PBD (Isotype-PBD), or unconjugated isotype control.
  • FIG. 4B includes a graph showing caspase induction in BAF3/4F2 cells in the presence of anti-mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti- mutCALR antibody (Clone 6), isotype control conjugated to PBD (Isotype-PBD), or unconjugated isotype control.
  • FIG. 4C includes a graph showing proliferation of BAF3/4F2 cells in the presence of anti-mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to PBD (Isotype-PBD), or unconjugated isotype control.
  • FIG. 5A includes a graph showing viability of BAF3/4F2 cells in the presence of anti-mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to PBD (Isotype-PBD).
  • FIG. 5B includes a graph showing viability of BAF3/4F2 cells in the presence of anti- mutCALR antibody conjugated to MMAE (Clone 6-MMAE), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to MMAE (Isotype-MMAE).
  • FIG. 6A includes a graph showing viability of BAF3/3D3 cells in the presence of anti-mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to PBD (Isotype-PBD).
  • FIG. 6B includes a graph showing viability of BAF3/3D3 cells in the presence of anti-mutCALR antibody conjugated to MMAE (Clone 6-MMAE), unconjugated anti- mutCALR antibody (Clone 6), or isotype control conjugated to MMAE (Isotype-MMAE).
  • FIG. 7A includes a graph showing viability of BAF3/4F2 cells treated with anti- mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to PBD (Isotype-PBD) in the presence of 10 ng/mL human TPO.
  • FIG. 7B includes a graph showing viability of BAF3/4F2 cells treated with anti- mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control conjugated to PBD (Isotype-PBD) in the absence of human TPO.
  • FIG. 8A includes a graph showing tumor cell growth in mice administered anti- mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control, each at three doses (0.1 mg/kg, 0.3 mg/kg, 1 mg/kg). Untreated mice, or mice treated with ruxolitinib (90 mg/kg BID), or mice administered three doses of unconjugated Clone 6 (10 mg/kg IP 3X) were included as controls.
  • FIG. 8B includes a graph showing platelet counts in mice administered anti- mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control, each at three doses (0.1 mg/kg, 0.3 mg/kg, 1 mg/kg). Untreated mice, or mice treated with ruxolitinib (90 mg/kg BID), or mice administered three doses of unconjugated Clone 6 (10 mg/kg IP 3X) were included as controls.
  • FIG. 9 includes a graph of mouse survival after treatment with anti-mutCALR antibody conjugated to PBD (Clone 6-PBD), unconjugated anti-mutCALR antibody (Clone 6), or isotype control.
  • anti-mutCALR antibody-drug conjugates comprising a pyrrolobenzodiazepine dimer, and related pharmaceutical compositions, and kits.
  • the anti- mutCALR antibody-drug conjugates described herein are useful in the treatment or prevention of myeloproliferative neoplasms (e.g, chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts).
  • CALR is a highly conserved chaperone protein that resides primanly in the endoplasmic reticulum and is involved in a variety of cellular processes including protein folding, calcium homeostasis, cell adhesion, and integrin signaling. Mutations in the CALR gene have been identified in patients with myeloproliferative neoplasms. The two most frequent CALR mutations are a 52 base pair (bp) deletion and a 5 bp insertion, which are referred to as Type 1 and Type 2 mutations, respectively.
  • Type 1 and Type 2 mutations cause a +1 frameshift within exon 9 that generates a novel, positively-charged C-terminal amino acid sequence that lacks the KDEL domain (SEQ ID NO:347) of the WT protein, thereby enabling the mutCALR to escape the ER and activate the thrombopoietin receptor (MPL) and induce constitutive activation of Janus kinase 2 (JAK2) signaling.
  • SEQ ID NO:347 the KDEL domain
  • MPL thrombopoietin receptor
  • JK2 Janus kinase 2
  • SUBSTITUTE SHEET (RULE 26) shown below.
  • the frameshift amino acid residues in Type 1 and Type 2 mutCALR are shown in bold and the novel C-terminal sequence is marked by underlining.
  • DKKRKEEEEAEDNCRRMMRTKMRMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA (SEQ ID NO : 321 ) mutCALR C-terminal consensus mutant sequence RMRRMRRTRRKMRRKMSPARPRTSCREACLQGWTEA ( SEQ ID NO : 322 )
  • This disclosure provides anti-mutCALR antibody-drug conjugates that are useful in treating myeloproliferative neoplasms.
  • amino acid positions assigned to CDRs and frameworks in a variable region of the anti-mutCALR antibody-drug conjugates are specified according to Kabat; see EA Rabat, Sequences of Proteins of Immunological Interest, U.S. Dept, of Health and Human Sendees, Public Health Service, National Institutes of Health, 1991, (OCoLC)l 138727707.
  • the anti-mutCALR antibody-drug conjugate comprises an anti- mutCALR antibody that comprises one, two, three, four, five, and/or six CDRs of any one of the antibodies described herein.
  • an anti-mutCALR antibody-drug conjugate comprises (i) one, two, and/or three heavy chain CDRs of any one of the clones presented in Tables 1-2, and/or (ii) one, two, and/or three light chain CDRs from any one of the clones presented in Tables 1-2.
  • an anti-mutCALR antibody-drug conjugate comprises (i) three heavy chain CDRs from any one of the clones presented in Tables 4-5, and (ii) three light chain CDRs from any one of the clones presented in Tables 4-5. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises a Group
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises a heavy chain CDR1, CDR2, and CDR3 and a light chain CDR1, CDR2, and CDR3 from an antibody described herein. In some embodiments, an anti- mutCALR antibody-drug conjugate comprises a mouse version, mouse variant, human version, human variant, humanized version, humanized variant, or affinity matured variant of an antibody described herein.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain CDR1, CDR2, and CDR3 and/or a light chain variable region CDR1, CDR2, and CDR3 from any clone disclosed herein, a humanized version thereof, or variants thereof (including affinity matured variants).
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3 from any clone disclosed herein.
  • an anti- mutCALR antibody-drug conjugate comprises a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from any clone disclosed herein.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy 7 chain CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3 from antibody any clone disclosed herein.
  • an anti-mutCALR antibody-drug conjugate comprises a mouse version of any clone disclosed herein.
  • an anti-mutCALR antibody-drug conjugate comprises a mouse variant of any clone disclosed herein.
  • an anti- mutCALR antibody-drug conjugate comprises a human version of any clone disclosed herein.
  • an anti-mutCALR antibody-drug conjugate comprises a human variant of any clone disclosed herein. In some embodiments, an anti-mutCALR antibodydrug conjugate comprises a humanized version of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises a variant of any clone disclosed herein. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises an affinity matured variant of any clone disclosed herein.
  • the anti-mutCALR antibody-drug conjugate comprises a variant of an anti-mutCALR antibody described herein which comprises one to thirty conservative amino acid substitution(s), e.g., one to twenty-five, one to twenty, one to fifteen, one to ten, one to five, or one to three conservative amino acid substitution(s).
  • one to thirty conservative amino acid substitution(s) e.g., one to twenty-five, one to twenty, one to fifteen, one to ten, one to five, or one to three conservative amino acid substitution(s).
  • the conservative amino acid substitution(s) is in a CDR of the antibody. In some embodiments, the conservative amino acid substitution(s) is not in a CDR of the antibody. In some embodiments, the conservative amino acid substitution(s) is in a framework region of the antibody.
  • a CDR comprises one amino acid substitution. In some embodiments, a CDR comprises two amino acid substitutions. In some embodiments, a CDR comprises three amino acid substitutions. In some embodiments, a CDR comprises four amino acid substitutions. In some embodiments, the one or more amino acid substitutions are conservative substitutions.
  • the CDR is a heavy chain CDR1. In some embodiments, the CDR is a heavy chain variable region CDR2. In some embodiments, the CDR is a heavy chain variable region CDR3. In some embodiments, the CDR is a light chain variable region CDR1. In some embodiments, the CDR is a light chain variable region CDR2. In some embodiments, the CDR is a light chain variable region CDR3.
  • the one or more substitutions are made as part of a humanization process. In some embodiments, the one or more substitutions are made as part of a germline humanization process. In some embodiments, the one or more substitutions are made as part of an affinity maturation process. In some embodiments, the one or more substitutions are made as part of an optimization process.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, and a heavy chain variable region CDR3, each of which correspond to the heavy chain variable region CDRs set forth in Tables 1-2 for a single clone, and a light chain variable region CDR1, a light chain variable region VL CDR2, and a light chain variable region VL CDR3, each of which correspond to the VL CDRs set forth in Tables 1-2 for a single clone.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, and a light chain variable region CDR3, each of which correspond to the VH and VL CDRs set forth in Tables 1-2 for a single clone.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region (VH) comprising a VH CDR1 ; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO:1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO: 102); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is
  • VL light chain variable region
  • the VL CDR1 comprises the amino acid sequence GGX104X105X106GX107X108X109VX110 (SEQ ID NO: 103), wherein X104 is N, D, or S; wherein X105 is Y, N, or D; wherein X106 is I or T; wherein X107 is S, D, I, R, or T; wherein X108 is K, E, or I; wherein X109 is S, I, R, G, N, or A; and wherein Xno is H, F, or N;
  • the VL CDR2 comprises the amino acid sequence DDX111DRPX112 (SEQ ID NO: 104), wherein X111 is G, S, or R; and wherein X112
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region (VH) comprising a VH CDR1 ; a VH CDR2; and a VH CDR3; wherein the VH CDR1 comprises the amino acid sequence ELSMQ (SEQ ID NO: 1); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO: 102); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence X119VSX120RP
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region (VH) comprising a VH CDR1 ; a VH CDR2; and a VH CDR3; wherein: the VH CDR1 comprises the amino acid sequence GYTLTELSMQ (SEQ ID NO:329); the VH CDR2 comprises the amino acid sequence GFDPDDX101ETMYAEX102X103QG (SEQ ID NO: 102); wherein X101 is D or G; wherein X102 is K or R; and wherein X103 is F or L; the VH CDR3 is SPGYDFFDY (SEQ ID NO: 18); wherein the antibody comprises a light chain variable region (VL) comprising a VL CDR1, a VL CDR2, and a VL CDR3, wherein: the VL CDR1 comprises the amino acid sequence TGTSSDVGGYNYVS (SEQ ID NO:30); the VL CDR2 comprises the amino acid sequence
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region with the C-terminal lysine removed. In some embodiments, an anti-
  • mutCALR antibody-drug conjugate comprises a heavy chain variable region comprising an amino acid sequence that has the three VH CDRs of any anti-mutCALR clone disclosed herein and which has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VH sequences set forth in Tables 4-5 and a light chain variable region comprising an amino acid sequence that has the three VL CDRs of any anti-mutCALR clone disclosed herein and which has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of the VL sequences set forth in Tables 4-5.
  • an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region comprising any one of the VH sequences set forth in Tables 4-5. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises a light chain variable region comprising any one of the VL sequences set forth in Tables 4-5. In some embodiments, an anti-mutCALR antibody-drug conjugate comprises a heavy chain variable region comprising any one of the VH sequences set forth in Tables 4-5 and a light chain variable region comprising any one of the VL sequences set forth in Tables 4-5.
  • an anti-mutCALR antibody-drug conjugate comprises a modification which modulates (e.g.. reduces or increases) the Fc region-mediated effector function, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP).
  • CDC complement-dependent cytotoxicity
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cell phagocytosis
  • an anti-mutCALR antibody-drug conjugate has Fc effector function. In certain embodiments, an anti-mutCALR antibody-drug conjugate has enhanced Fc effector function. In certain embodiments, an anti-mutCALR antibody -drug conjugate exhibits antibody-dependent cell-mediated cytotoxicity (ADCC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • An anti-mutCALR antibody-drug conjugate can be engineered to enhance the ADCC activity (for review, see Kubota T et al. Cancer Sci. 2009; 100(9): 1566-72). For example, ADCC activity of an antibody can be improved when the antibody itself has a low ADCC activity, by slightly modifying the constant region of the antibody (Junttila TT. et al. Cancer Res.
  • ADCC activity of an antibody is by enzymatically interfering with the glycosylation pathway resulting in a reduced fucose (von Horsten HH. etal. Glycobiology. 2010;20(12): 1607-18).
  • other suitable methods can be used to achieve ADCC enhancement, for instance including glycoengineering (Kyowa
  • a binding moiety of the present disclosure exhibits enhanced antibody-dependent cell-mediated cytotoxicity (ADCC).
  • a binding moiety of the present disclosure is afucosylated.
  • an anti-mutCALR antibody-drug conjugate has reduced Fc effector function.
  • an anti-mutCALR antibody-drug conjugate exhibits reduced or substantially no complement-dependent cytotoxicity (CDC), antibodydependent cellular cytotoxicity (ADCC) or antibody -dependent cell phagocytosis (ADCP).
  • an anti-mutCALR antibody-drug conjugate exhibits reduced or substantially no antibody-dependent cell-mediated cytotoxicity (ADCC).
  • An anti-mutCALR antibody-drug conjugate can be engineered to reduce effector function, for example ADCC activity, by any suitable method including removal of glycosylation sites in the Fc region.
  • an anti-mutCALR antibody-drug conjugate that has a reduced Fc effector function comprises an N297A mutation on the heavy chain.
  • an anti-mutCALR antibody-drug conjugate comprises an IgGl isotype (e.g., IgGl, IgG2, IgG3 or IgG4).
  • an anti-mutCALR antibody - drug conjugate comprises an IgGl.
  • an IgGl, IgG2, IgG3 or IgG4 anti- mutCALR antibody-drug conjugate has Fc-effector function.
  • an IgGl, IgG2, IgG3 or IgG4 anti-mutCALR antibody-drug conjugate is Fc-effector function null.
  • an IgGl anti-mutCALR antibody-drug conjugate has Fc-effector function.
  • an IgGl anti-mutCALR antibody-drug conjugate is Fc- effector function null.
  • the anti-mutCALR antibody-drug conjugate comprises an antibody fragment.
  • Fragments of the antibodies described herein may be prepared by proteolytic digestion of intact antibodies.
  • antibody fragments can be obtained by treating the whole antibody with an enzyme such as papain, pepsin, or plasmin or the FabRICATOR® (IdeS) recombinant enzyme (Genovis AB) that digests IgG antibodies to produce a homogeneous pool of F(ab')2 and Fc/2 fragments.
  • Papain digestion of whole antibodies produces F(ab)2 or Fab fragments; pepsin digestion of whole antibodies yields F(ab')2 or Fab'; and plasmin digestion of whole antibodies yields Facb fragments.
  • antibody fragments can be produced recombinantly.
  • nucleic acids encoding the antibody fragments of interest can be constructed, introduced into an expression vector, and expressed in suitable host cells. See, e.g., Co, M.S. et al., J. Immunol., 152:2968-2976 (1994); Better, M. and Horwitz, A.H., Methods in Enzymology, 178:476-496 (1989); Plueckthun, A.
  • Antibody fragments can be expressed in and secreted from A. coll, thus allowing the facile production of large amounts of these fragments.
  • Antibody fragments can be isolated from the antibody phage libraries. Alternatively, Fab'-SH fragments can be directly recovered from A.
  • F(ab)2 fragments can be isolated directly from recombinant host cell culture.
  • Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046.
  • the anti-mutCALR antibody-drug conjugate comprises a minibody.
  • Minibodies of anti-mutCALR antibodies include diabodies, single chain (scFv), and singlechain (Fv)2 (SC(FV)2).
  • a “diabody” is a bivalent minibody constructed by gene fusion (see, e.g, Holliger, P. et al, Proc. Natl. Acad. Sci. U. S. A., 90:6444-6448 (1993); EP 404,097; WO 93/11161).
  • Diabodies are dimers composed of two polypeptide chains. The VL and VH domain of each polypeptide chain of the diabody are bound by linkers. The number of amino acid residues that constitute a linker can be between 2 to 12 residues (e.g., 3-10 residues or five or about five residues).
  • the linkers of the polypeptides in a diabody are typically too short to allow the VL and VH to bind to each other.
  • VL and VH encoded in the same polypeptide chain cannot form a single-chain variable region fragment, but instead form a dimer with a different single-chain variable region fragment.
  • a diabody has two antigenbinding sites.
  • An scFv is a single-chain polypeptide antibody obtained by linking the VH and VL with a linker (see, e.g., Huston et al., Proc. Natl. Acad. Sci. U. S. A., 85:5879-5883 (1988); and Plickthun, “The Pharmacology of Monoclonal Antibodies” Vol.113, Ed Resenburg and Moore, Springer Verlag, New York, pp.269-315, (1994)).
  • the order of VHs and VLs to be linked is not particularly limited, and they may be arranged in any order. Examples of arrangements include: [VH] linker [VL]; or [VL] linker [VH], The heavy chain variable
  • SUBSTITUTE SHEET (RULE 26) domain and light chain variable domain in an scFv may be derived from any anti-mutCALR antibody described herein.
  • An SC(FV)2 is a minibody in which two VHs and two VLs are linked by a linker to form a single chain (Hudson, et al., J. Immunol. Methods, (1999) 231: 177-189 (1999)).
  • An SC(FV)2 can be prepared, for example, by connecting scFvs with a linker.
  • the sc(Fv)2 of the present invention include antibodies preferably in which two VHs and two VLs are arranged in the order of: VH, VL, VH, and VL ([VH] linker [VL] linker [VH] linker [VL]), beginning from the N terminus of a single-chain polypeptide; however the order of the two VHs and two VLs is not limited to the above arrangement, and they may be arranged in any order.
  • the anti-mutCALR antibody-drug conjugate comprises a bispecific antibody.
  • Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of the mutCALR protein. Other such antibodies may combine a mutCALR binding site with a binding site for another antigen.
  • Bispecific antibodies can be prepared as full length antibodies or low molecular weight forms thereof (e.g., F(ab')2 bispecific antibodies, sc(Fv)2 bispecific antibodies, diabody bispecific antibodies).
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).
  • Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g, alanine or threonine).
  • SUBSTITUTE SHEET (RULE 26) provides a mechanism for increasing the yield of the heterodimer over other unwanted endproducts such as homodimers.
  • Bispecific antibodies include cross-linked or “heteroconjugate” antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods.
  • the “diabody” technology provides an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites.
  • the bispecific anti-mutCALR antibody-drug conjugate comprises a biparatopic antibody.
  • a biparatopic antibody is antibody which recognizes two non-identical epitopes (overlapping or non-overlapping epitopes) on the same target antigen (e.g. the C-terminal of mutCALR domain).
  • a biparatopic anti-mutCALR antibody-drug conjugate can comprise two immunoglobulin heavy chain-light chain pairs or one immunoglobulin heavy chain-light chain pair.
  • a biparatopic anti- mutCALR antibody-drug conjugate comprises one immunoglobulin heavy-chain-lighl chain pair.
  • a biparatopic anti-mutCALR antibody-drug conjugate comprises a full-length antibody comprising one immunoglobulin heavy -chain-light chain pair.
  • the anti-mutCALR antibody-drug conjugate comprises a multivalent antibody.
  • a multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind.
  • the antibodies describe herein can be multivalent antibodies with three or more antigen binding sites (e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • the multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • An exemplary dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • a multivalent antibody can comprise (or consist of) three to about eight (e.g, four) antigen binding sites.
  • the multivalent antibody optionally comprises at least one polypeptide chain (e.g, at least two polypeptide chains), wherein the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VDl-(Xl) n -VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of an Fc region, XI and X2 represent an amino acid or peptide spacer, and n is 0 or 1.
  • the present disclosure provides an anti-mutCALR antibody-drug conjugate comprising any one of the anti-mutCALR antibodies described herein and a pyrrolobenzodiazepine dimer, e.g., a pyrrolobenzodiazepine dimer described herein.
  • Pyrrolobenzodiazepines are sequence-selective DNA minor-groove binding agents based on the naturally occurring anthramycin family of antitumor antibiotics.
  • Pyrrolobenzodiazepine has a general structure shown below:
  • Pyrrolobenzodiazepines differ in the number, type and position of substituents, in both their aromatic A-rings and pyrrolo C-rings, and in the degree of saturation of the C-ring.
  • pyrrolobenzodiazepines can be enhanced by joining two pyrrolobenzodiazepines monomers together through their C8/C'-hydroxyl functionalities via a flexible alkylene linker to form a pyrrolobenzodiazepine dimer (see, e.g., Bose, D.S. et al., “Rational Design of a Highly Efficient Irreversible DNA Interstrand Cross-Linking Agent Based on the Pyrrolobenzodiazepine Ring System,” J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, D.E.
  • Any pyrrolobenzodiazepine dimer (e.g., a pyrrolobenzodiazepine dimer described herein) can be included in an anti-mutCALR antibody-drug conjugate described herein.
  • pyrrolobenzodiazepine dimers that can be used in anti-mutCALR antibody-drug conjugates are provided in Mantaj et al., “From Anthramycin to Pyrrolobenzodiazepine (PBD)-Containing Antibody-Drug Conjugates (ADCs),” Angew. Chem. Int. Ed.
  • Non-limiting examples of pyrrolobenzodiazepine dimers include PBD-1, SG2000,
  • the pyrrolobenzodiazepine dimer has a structure selected from the group consisting of:
  • pyrrolobenzodiazepine dimer PBD-1 further comprises a cleavable cap. In some embedments, pyrrolobenzodiazepine dimer PBD-1 further comprises a cleavable cap and is of the following formula: Non-limiting examples of pyrrolobenzodiazepine dimers include SG2000, SG2219,
  • the pyrrolobenzodiazepine dimer has a structure selected from the group consisting of:
  • the pyrrolobenzodiazepine dimer is linked to a linker to form a pay load.
  • the pyrrolobenzodiazepine dimer can be conjugated to the antibody by performing chemical and/or enzymatic modifications on the antibodies, respectively, or the lower molecular weight forms thereof described herein.
  • Methods for conjugating payloads are known in the art (e.g., lysine amide coupling, cysteine coupling, non-natural amino acid incorporation by genetic engineering, transpeptidation using sortase or microbial transglutaminase, and N-glycan engineering) and provided in, e.g., Tsuchikama and An, “Antibody-drug conjugates: recent advances in conjugation and linker chemistries,” Protein Cell, 9(l):33-46 (2018); WO2011/130613; WO2011/130616; WO2016/054315; and WO2011/130598; the contents of each of which is incorporated herein by reference in its entirety.
  • SUBSTITUTE SHEET (RULE 26) Any payload compnsing a pyrrolobenzodiazepine dimer and a linker (e.g. , a linker described herein) can be included in an anti-mutCALR antibody-drug conjugate described herein.
  • a linker e.g. , a linker described herein
  • Non-limiting examples of payloads that can be used in anti-mutCALR antibody -drug conjugates are provided in Mantaj et al, “From Anthramycin to Pyrrolobenzodiazepine (PBD)-Containing Antibody-Drug Conjugates (ADCs),” Angew. Chem. Int. Ed.
  • Non-limiting examples of payloads include PL-1, PL-2, PL-3, SG3400, SG3227, SG3249, SG3710, SG3259, SG3584, SG3203, SG3231, SG3451, and SGD-1910.
  • the payload has a structure selected from the group consisting of:
  • cyclooctynyl reaction chemistry that can be used to connect the payload and the antibody can be found, for example, in U.S. Patent Nos.: 7,807,619; 8,431,558; 8,461,298; 9,260,371; 10,434,111; 11,278,554; 7,807,619; 8,431,558; 8,461,298; 9,260,371; 10,434,111; and 11,278,554; and European Patent No.: 1812031B1, the disclosures of which are incorporated herein by reference in their entireties.
  • Non-limiting examples of payloads include SG3400, SG3227, SG3249, SG3710, SG3259, SG3584, SG3203, SG3231, SG3451, and SGD-1910.
  • the payload has a structure selected from the group consisting of:
  • SUBSTITUTE SHEET (RULE 26) wherein the wavy line (where present) represents a connection point to an antibody or wherein the terminal maleimide (when present) reacts with a thiol group in the antibody to connect the payload and the antibody.
  • Any pyrrolobenzodiazepine dimer can comprise a cap, which can also be referred to as a trigger.
  • the pyrrolobenzodiazepine dimer includes a cap at the N10 position of the pyrrolobenzodiazepine ring system.
  • a linker between the pyrrolobenzodiazepine dimer and the antibody can include a cap.
  • the cap comprises a cleavable cap, e.g., an enzyme-cleavable cap.
  • the pyrrolobenzodiazepine dimer can comprise a glucuronide group that is cleaved by P-glucuronidase, which is overexpressed in many cancers, but not present in general circulation. In such instances, the cap can mask the cytotoxic effects of the pyrrolobenzodiazepine dimer until the cap is cleaved by a factor in a cancer cell.
  • Non-limiting examples of caps include a glucuronide group or derivative thereof and a galactoside group or derivative thereof. See, also, Gregson et al., “Synthesis and evaluation of pyrrolobenzodiazepine dimer antibody-drug conjugates with dual P-glucuronide and dipeptide triggers,” European Journal of Medicinal Chemistry 179, 591-607 (2019) and Su et al., “Antibody-drug conjugates: Recent advances in linker chemistry ,” Acta Pharmaceutica Sinica B, 11 (12) : 3889-3907 (2021), the contents of each of which is incorporated herein by reference in its entirety.
  • the pyrrolobenzodiazepine dimer (e.g, PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, or SGD-1882) comprises a P-glucuronidase- cleavable cap, e.g., a glucuronide group or derivative thereof.
  • the pyrrolobenzodiazepine dimer (e.g., PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, or SGD-1882) comprises a P-galactosidase-cleavable cap, e.g., a galactoside group or derivative thereof.
  • the payload includes a pyrrolobenzodiazepine dimer (e.g., PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, or SGD-1882) comprising a cap, e.g., a glucuronide group or derivative thereof and a galactoside group or derivative thereof.
  • a pyrrolobenzodiazepine dimer e.g., PBD-1, SG2000, SG2219, SG3199, SG3312, SG3650, SG2057, or SGD-1882
  • a cap e.g., a glucuronide group or derivative thereof and a galactoside group or derivative thereof.
  • the payload has a structure of:
  • the payload has a structure of:
  • the payload has a structure of: wherein CAP is a cleavable cap as described herein.
  • the payload has a structure of:
  • a linker e.g., a linker described herein
  • linkers can be used to link the anti-mutCALR antibody and the pyrrolobenzodiazepine dimer.
  • linkers are provided in Tsuchikama and An, “Antibody-drug conjugates: recent advances in conjugation and linker chemistries.” Protein Cell, 9(1): 33-46 (2018); WO2015/052322; WO2015/095124; WO2016/054315; WO2017/137555; and WO2022/079211; the contents of each of which is incorporated herein by reference in its entirety.
  • Non-limiting examples of linkers include peptide linkers (e.g., cathepsin B-cleavable peptide linker such as valine-citrulline-p-aminobenzyloxy carbonyl (PABC) and valine- alanine-PABC), hydrozone linkers, disulfide linkers, and pyrophosphate diester linkers.
  • peptide linkers e.g., cathepsin B-cleavable peptide linker such as valine-citrulline-p-aminobenzyloxy carbonyl (PABC) and valine- alanine-PABC
  • PABC valine-citrulline-p-aminobenzyloxy carbonyl
  • hydrozone linkers e.g., disulfide linkers, and pyrophosphate diester linkers.
  • the linker can be cleavable or non-cleavable. In some embodiments, the linker is cleavable. In such instances, the linker can be cleaved upon internalization of the anti- mutCALR antibody-drug conjugate into the tumor cell expressing mutCALR, thereby releasing the pyrrolobenzodiazepine dimer, which is cytotoxic, into the tumor cell and minimizing its exposure to non-tumorigenic cells and tissues. In some embodiments, the linker is non-cleavable. In some embodiments, the non-cleavable linker comprises a maleimidocaproyl linker, a 4-maleimidomethyl cyclohexane-l-carboxylate linker, or both. In some embodiments, the anti-mutCALR antibody-drug conjugate comprises a cleavable linker, a non-cleavable linker, or both.
  • the peptide linker comprises a dipeptide linker (e.g., valinecitrulline (vc or val-cit), alanine-phenylalanie (af or ala-phe)), a tripeptide linker e.g, glycine - valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly )), a tetrapeptide linker, or a pentapeptide linker.
  • the peptide linker comprises naturally occurring amino acids, non-naturally occurring amino acids, or a combination thereof.
  • the linker comprises a functional group that reacts with a thiol group of an antibody to form an antibody-drug conjugate.
  • functional groups that are reactive with a thiol group of an antibody include bromoacetamide, disulfide, iodoacetamide, isocynate, isothiocynate, maleimide, pyridyl disulfide, and vinyl pyridine.
  • the linker comprises a terminal maleimide group that can be used to conjugate the pyrrolobenzodiazepine dimer to the anti-mutCALR antibody. In some embodiments, the linker comprises a terminal iodoacetamide group that can be used to conjugate the pyrrolobenzodiazepine dimer to the anti-mutCALR antibody.
  • the linker comprises a self-immolative moiety that generates free pyrrolobenzodiazepine dimer via an immolative elimination.
  • SUBSTITUTE SHEET (RULE 26) self-immolative moieties include 2-aminoimidazol-5-methanol derivatives, orthoaminobenzylacetals, para-aminobenzylacetals, para-amino-benzyloxy carbonyl (PAB), and thiazoles.
  • the anti-mutCALR antibody-drug conjugate comprises a spacer.
  • a spacer e.g., a spacer described herein
  • the anti-mutCALR antibody-drug conjugate comprises a polyethylene glycol spacer, a sulfamide spacer, or a combination thereof.
  • the linker provided herein is selected from a linker disclosed in any of U.S. Patent Nos.: 8,859,629, 9,636,421, 11,951,175, 11,168,085, 10,131,682, 10,456,479, 10,646,585, 10,975,112; U.S. Publication Nos.: 20230364262A1, 20230114866A1, 20230226208A1; International Publication No.: WO2024121123A1; and European Publication No.: 4450489A1, the disclosures of which are incorporated herein by reference in their entireties.
  • the linker has a structure of: wherein - indicates the bond between the linker and the pyrrolobenzodiazepine dimer.
  • linker L-l is connected to the antibody via the cyclooctynyl moiety.
  • the linker has a structure of: wherein - indicates the bond between the linker and the pyrrolobenzodiazepine dimer.
  • linker L-2 is connected to the antibody via the cyclooctynyl moiety'.
  • the anti-mutCALR antibody-drug conjugate comprises an anti- mutCALR antibody that is modified with a moiety that improves its stabilization and/or retention in circulation, e.g, in blood, serum, or other tissues, e.g, by at least 1.5, 2, 5, 10, or 50 fold.
  • the anti-mutCALR antibody can be associated with (e.g, conjugated to) a polymer, e.g, a substantially non-antigenic polymer, such as a polyalkylene oxide or a polyethylene oxide. Suitable polymers will vary substantially by weight.
  • polymers having molecular number average weights ranging from about 200 to about 35,000 Daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used.
  • the anti- mutCALR antibody can be conjugated to a water soluble polymer, e.g. , a hydrophilic polyvinyl polymer, e.g, polyvinylalcohol or polyvinylpyrrolidone.
  • a water soluble polymer e.g. , a hydrophilic polyvinyl polymer, e.g, polyvinylalcohol or polyvinylpyrrolidone.
  • examples of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene gly cols, poly oxy ethylenated polyols, copolymers thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.
  • Additional useful polymers include poly oxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene; polymethacrylates; carbomers; and branched or unbranched polysaccharides.
  • modified antibodies can be prepared by performing chemical and/or enzymatic modifications on the antibodies, respectively, or the lower molecular weight forms thereof described herein.
  • Methods for modifying antibodies are well known in the art (see, e.g., US 5,057,313 and US 5,156,840).
  • An antibody-drug conjugate described herein can include a drug loading of one or more pyrrolobenzodiazepine dimers per anti-mutCALR antibody, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrrolobenzodiazepine dimers per anti-mutCALR antibody.
  • Drug loading refers to the average number of pyrrolobenzodiazepine dimers per anti-mutCALR antibody.
  • Antibodies described herein may be produced in bacterial or eukaryotic cells. Some antibodies, e.g., Fabs, can be produced in bacterial cells, e.g., E. coli cells. Antibodies can also be produced in eukaryotic cells such as transformed cell lines (e.g, CHO, 293E, COS). In addition, antibodies (e.g, scFvs) can be expressed in a yeast cell such as Pichia (see, e.g, Powers et al., J Immunol Methods. 251: 123-35 (2001)), Hansenula, or Saccharomyces . To produce the antibody of interest, a polynucleotide encoding the antibody is constructed, introduced into an expression vector, and then expressed in suitable host cells. Standard
  • SUBSTITUTE SHEET (RULE 26) molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody.
  • the expression vector should have characteristics that permit amplification of the vector in the bacterial cells. Additionally, when E. coli such as JM109, DH5a, HB101, or XLl-Blue is used as a host, the vector must have a promoter, for example, a lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al., Science, 240: 1041-1043 (1988)), or T7 promoter that can allow efficient expression in E. coli.
  • a promoter for example, a lacZ promoter (Ward et al., 341:544-546 (1989), araB promoter (Better et al., Science, 240: 1041-1043 (1988)
  • T7 promoter that can allow efficient expression in E. coli.
  • the expression vector includes a promoter necessary for expression in these cells, for example, an SV40 promoter (Mulligan et al., Nature, 277: 108 (1979)), MMLV-LTR promoter, EFla promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or CMV promoter.
  • SV40 promoter Mulligan et al., Nature, 277: 108 (1979)
  • MMLV-LTR promoter MMLV-LTR promoter
  • EFla promoter EFla promoter
  • CMV promoter CMV promoter
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g, U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017).
  • typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.
  • examples of vectors with selectable markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
  • antibodies are produced in mammalian cells.
  • mammalian host cells for expressing an antibody include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601 621), human embryonic kidney 293 cells (e.g., 293,
  • SUBSTITUTE SHEET (RULE 26) 293E, 293T), COS cells, NIH3T3 cells, lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, and a cell from a transgenic animal, e.g., a transgenic mammal.
  • the cell is a mammary' epithelial cell.
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain of an anti-mutCALR antibody is introduced into dhfr- CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to enhancer/promoter regulatory elements e.g., derived from SV40, CMV, adenovirus and the like, such as a CMV enhancer/ AdMLP promoter regulatory element or an SV40 enhancer/ AdMLP promoter regulatory element) to drive high levels of transcription of the genes.
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplifi cation.
  • the selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains and the antibody is recovered from the culture medium.
  • Antibodies can also be produced by a transgenic animal.
  • U.S. Pat. No. 5,849,992 describes a method of expressing an antibody in the mammary gland of a transgenic mammal.
  • a transgene is constructed that includes a milk-specific promoter and nucleic acids encoding the antibody of interest and a signal sequence for secretion.
  • the milk produced by females of such transgenic mammals includes, secreted-therein, the antibody of interest.
  • the antibody can be purified from the milk, or for some applications, used directly. Animals are also provided comprising one or more of the nucleic acids described herein.
  • the antibodies of the present disclosure can be isolated from inside or outside (such as from the medium) of the host cell and purified as substantially pure and homogenous antibodies. Methods for isolation and purification commonly used for antibody purification may be used for the isolation and purification of antibodies, and are not limited to any particular method. Antibodies may be isolated and purified by appropriately selecting and combining, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS- polyacrylamide gel electrophoresis, isoelectnc focusing, dialysis, and recrystallization.
  • Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and adsorption chromatography (Strategies for Protein Purification and Charactenzation: A Laboratory Course Manual. Ed Daniel R. Marshak et al., Cold Spring Harbor Laboratory
  • Chromatography can be carried out using liquid phase chromatography such as HPLC and FPLC.
  • Columns used for affinity chromatography include protein A column and protein G column. Examples of columns using protein A column include Hyper D, POROS, and Sepharose FF (GE Healthcare Biosciences).
  • the present disclosure also includes antibodies that are highly purified using these purification methods.
  • the anti-mutCALR antibody -drug conjugates of the present disclosure can inhibit the activity of mutCALR, inhibit the activity of one or more signaling pathways downstream of MPL, increase internalization of an anti-mutCALR antibody and/or an associated cytotoxin, inhibit oncogenic cell proliferation and/or cause oncogenic cell death, increase caspase induction, increase platelet count, prolong survival, inhibit dimerization of MPL, compete with MPL for binding to mutCALR, or a combination thereof.
  • an anti-mutCALR antibody-drug conjugate that competes with MPL for binding to mutCALR means that the anti-mutCALR antibody-drug conjugate binds to mutCALR with a greater affinity than MPL, the anti-mutCALR antibody in the unconjugated form, or both.
  • the anti-mutCALR antibody-drug conjugate binds to mutCALR with about 10-fold, 50-fold, 100-fold, 500-fold or 1000-fold greater affinity than MPL, the anti-mutCALR antibody in the unconjugated form, or both.
  • the anti-mutCALR antibody-drug conjugate binds to mutCALR with an IC50 of about 10- fold, 50-fold, 100-fold, 500-fold or 1000-fold less than MPL, the anti-mutCALR antibody in the unconjugated form, or both. In some embodiments, the anti-mutCALR antibody-drug conjugate binds to mutCALR with an IC50 of between about 0.1 and 1 nM.
  • the anti-mutCALR antibody-drug conjugate binds to mutCALR with an IC50 of about 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM or 1 nM.
  • the antibody-drug conjugates or compositions described herein can be used in methods of inhibiting activity of mutCALR, inhibiting the activity of one or more signaling pathways downstream of MPL, increasing internalization of an anti-mutCALR antibody and/or an associated cytotoxin, inhibiting oncogenic cell proliferation and/or cause oncogenic cell death, increasing caspase induction, increasing platelet count, prolonging survival, inhibiting dimerization of MPL, inhibiting the binding of MPL to mutCALR, or a combination thereof in an individual/patient in need of the inhibition by administering an effective amount of an antibody-drug conjugate described herein.
  • Non-limiting examples of signaling pathways downstream of MPL include Janus tyrosine kinase (JAK) and signal transducers and activators of transcription (STAT) signaling, mitogen-activated protein kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling, serine/threonine kinase (AKT) signaling, and mammalian target of rapamycin (mTOR) signaling.
  • JAK Janus tyrosine kinase
  • STAT signal transducers and activators of transcription
  • MEK mitogen-activated protein kinase
  • ERK extracellular signal-regulated kinase
  • AKT serine/threonine kinase
  • mTOR mammalian target of rapamycin
  • Another aspect of the present disclosure pertains to methods of treating a mutCALR- associated disease or disorder in an individual (e.g, patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of one or more antibody-drug conjugates of the present disclosure or a pharmaceutical composition thereof.
  • Other aspects of the present disclosure provide an antibody-drug conjugate described herein for use in the treatment of a mutCALR-associated disease or disorder, or the use of an antibody-drug conjugate described herein for the manufacture of a medicament for the treatment of a mutCALR-associated disease or disorder.
  • a mutCALR-associated disease or disorder can include any disease, disorder or condition that is directly or indirectly linked to expression or activity of mutCALR.
  • Another aspect of the present disclosure pertains to methods of treating a myeloproliferative neoplasm in an individual (e.g., patient) by administering to the individual in need of such treatment a therapeutically effective amount or dose of one or more antibodydrug conjugates of the present disclosure or a pharmaceutical composition thereof.
  • Other aspects of the present disclosure provide an antibody-drug conjugate described herein for use in the treatment of a myeloproliferative neoplasm, or the use of an antibody-drug conjugate described herein for the manufacture of a medicament for the treatment of a myeloproliferative neoplasm.
  • Non-limiting examples of a myeloproliferative neoplasm include chronic myelogenous leukemia, polycythemia vera, primary myelofibrosis, essential thrombocythemia, chronic neutrophilic leukemia, acute myelogenus leukemia, chronic eosinophilic leukemia, chronic myelomonocytic leukemia, myeloproliferative neoplasm and myelodysplastic syndrome, including myelodysplastic syndrome with refractory' anaemia with ring sideroblasts, myelodysplastic syndrome with refractory anemia, and myelodysplastic syndrome with refractory anemia with excess blasts.
  • Anti-mutCALR antibody-drug conjugates disclosed herein can be used to treat, alone or in combination with other therapies, a myeloproliferative neoplasm, or can be used, alone or in combination with other therapies, for the manufacture of a medicament for the treatment of a myeloproliferative neoplasm.
  • Non-limiting examples of other therapies include a JAK
  • SUBSTITUTE SHEET (RULE 26) inhibitor e.g., ruxolitinib, itaticinib
  • a PI3K inhibitor e.g, parsaclisib
  • a standard of care therapy e.g, IFN-alpha, hydroxyurea, thalidomide, lenalidomide, an androgen, an erythropoietin-stimulating agent, a chemotherapeutic agent
  • JAK inhibitors for use as described herein are provided in U.S. Pat No. 7,335,667; U.S. Pat. No. 9,359,358; U.S. Pat. No. 8,691,807; U.S. Pat. No. 9,181,271; and U.S. Pat. No. 9,034,884, each of which is incorporated herein by reference in its entirety.
  • Non-limiting examples of PI3K inhibitors for use as described herein are provided in U.S. Pat. No. 9,108,984; U.S. Pat. No. 9,062,055; U.S. Pat. No. 8,759,359; and U.S. Pat. No. 9,434,746, each of which is incorporated herein by reference in its entirety.
  • mice preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans (i.e. , a human subject).
  • terapéuticaally effective amount refers to the amount of active antibodydrug conjugate that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.
  • treating refers to one or more of (1) inhibiting the disease; e.g., inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; e.g., ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.
  • the antibody-drug conjugates of the invention are useful in preventing or reducing the risk of developing any of the diseases referred to herein; e.g., preventing or reducing the risk of developing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease.
  • An anti-mutCALR antibody-drug conjugate described herein can be formulated as a pharmaceutical composition for administration to a subject, e.g., to treat a disease or disorder
  • a pharmaceutical composition typically includes a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the composition can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g, Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1-19).
  • compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g, injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories.
  • liquid solutions e.g, injectable and infusible solutions
  • dispersions or suspensions tablets, pills, powders, liposomes and suppositories.
  • the preferred form can depend on the intended mode of administration and therapeutic application.
  • compositions for the agents described herein are in the form of injectable or infusible solutions.
  • compositions may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g, injectable and infusible solutions), dispersions or suspensions, and liposomes.
  • liquid solutions e.g, injectable and infusible solutions
  • dispersions or suspensions e.g., liposomes.
  • liposomes e.g., liposomes.
  • a suitable form can depend on the intended mode of administration and therapeutic application.
  • compositions for the agents described herein are in the form of injectable or infusible solutions.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for stable storage at high concentration or as a lyophilized preparation.
  • Sterile injectable solutions can be prepared by incorporating an anti- mutCALR antibody-drug conjugate described herein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating an agent described herein into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze drying that yield a powder of an agent described herein plus any additional desired
  • SUBSTITUTE SHEET (RULE 26) ingredient from a previously sterile-filtered solution thereof.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts.
  • any of the anti-mutCALR antibody-drug conjugates described herein can also be formulated as liposomes prepared by any suitable method known in the art.
  • compositions formulated for subcutaneous administration may be suitable in some circumstances because the subject can self-administer the pharmaceutical composition.
  • Pharmaceutical formulations for subcutaneous administration can further comprise protein formulations comprising arginine-HCl, histidine, and/or polysorbate, which may confer increased potency, improved serum half-life, or enhanced solubility to the anti- mutCALR antibodies.
  • the anti-mutCALR antibody-drug conjugate can be administered to a subject, e.g, a subject in need thereof, for example, a human subject, by a variety of methods.
  • the route of administration can be intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneally (IP), or intramuscular injection.
  • the route and/or mode of administration of the antibody can also be tailored for the individual case, e.g, by monitoring the subject, e.g, using tomographic imaging, e.g, to visualize a tumor.
  • the anti-mutCALR antibody-drug conjugate can be administered as a fixed dose, or in a mg/kg patient weight dose.
  • the dose can also be chosen to reduce or avoid production of antibodies against the anti-mutCALR antibody-drug conjugate.
  • Dosage regimens are adjusted to provide the desired response, e.g. , a therapeutic response or a combinatorial therapeutic effect.
  • doses of the anti-mutCALR antibody-drug conjugate (and optionally a second agent) can be used in order to provide a subject with the agent in bioavailable quantities.
  • Dosage unit form or “fixed dose” or “flat dose” as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and optionally in association with the other agent. Single or multiple dosages may be given. Alternatively, or in addition, the antibody-drug conjugate may be administered via continuous infusion.
  • kits comprising one or more containers of an anti- mutCALR antibody or a pharmaceutical formulation thereof, optionally with one or more other prophylactic or therapeutic agents useful for the treatment of a disease or disorder, and optionally with instructions for using the anti-mutCALR antibody-drug conjugate or a pharmaceutical formulation thereof.
  • the instructions relating to the use of an anti-mutCALR antibody-drug conjugate generally include information as to dosage, dosing schedule, and route of administration for the intended treatment.
  • the containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.
  • Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert.
  • the label package insert indicates that an anti-mutCALR antibody-drug conjugate is used for treating, delaying the onset, and/or alleviating a myeloproliferative neoplasm.
  • SUBSTITUTE SHEET (RULE 26) internalized. Internalization was measured by quantifying the area of fluorescent cells as a percentage of total cell area in the well. Proliferation was measured as the area of cells in each well (confluence) Plates were scanned hourly for 24h using the IncuCyte® live-cell analysis system.
  • Clone 74 (FIG. 1A), Clone 55 (FIG. IB), and Clone 6 (FIG. 1C) were internalized at the two highest concentrations (5 pg/mL and 2 pg/mL) tested in 4F2 cells, however, no internalization was observed in MPL6 cells.
  • antibody internalization was inhibited in the presence of dynasore (FIGs. 1A-1C).
  • FIG. ID proliferation of 4F2 cells, but not MPL6 cells, was inhibited in the presence of Clone 74, Clone 55, and Clone 6.
  • No internalization or inhibition of cell proliferation was observed when cells were treated with the anti-mutCALR antibody B3 (FIG. IE and FIG. 1H) or the two isotype control antibodies (FIGs. 1F-1H).
  • Anti-mutCALR antibody (Clone 74) was conjugated with Alexa FluorTM 647 fluorophore using lysine amine chemistry. The resulting fluorophore to protein ratio was 5.53.
  • Ba/F3-TPOR/CALR de152 cells which are also referred to as 4F2 cells or BAF3/4F2 cells, were added in an amount of 500 cells/well to a retronectin-coated plate (Greiner Bio-One) and incubated at 37 °C for three days. Cells were then treated with 0.2 pg/mL Alexa Fluor- 647-Clone-74 and fixed with 8 % paraformaldehyde (Electron Microscopy Sciences) at various time points.
  • SUBSTITUTE SHEET (RULE 26) Western blot analysis of TPOR and pSTAT5 expression in the presence of anti- mutCALR antibody (Clone 74) or an isotype control was performed.
  • HAP1-JAK2 KO cells were transiently transfected with wtJAK2, TPOR and CALR de152 , treated with 10 pg/mL of anti -mutC ALR antibody (Clone 74) or isotype for 2 h or overnight and then evaluated by Western blot regarding expression of TPOR and pSTAT5.
  • P-actin staining was used as a protein loading control.
  • Western blot analysis confirmed the Clone 74- induced internalization and degradation of TPOR in HAP1 cells expressing mutCALR.
  • Immunofluorescence analysis of internalization of TPOR/mutCALR complex upon anti-mutCALR antibody (Clone 74) binding was performed.
  • CD34 + cells from a MF patient expressing CALR de152 were treated with 2 pg/mL of anti-mutCALR antibody (Clone 74) for 12 h.
  • Internalization of TPOR was assessed by a fluorescence labeled TPOR antibody after cells were fixed and permeabilized. Treatment with 10 ng/ml of TPO was used as a positive control of TPOR internalization.
  • Image analysis was performed using colocalization module in ZEN software on a pixel by pixel basis. Single label control samples were used to set up compensation crosshair. As shown in FIG. 3C, confocal microscopy analysis confirmed the Clone 74-induced internalization and degradation of TPOR in primary CD34 + cells expressing mutCALR.
  • This Example describes conjugation of an anti-mutCALR antibody (Clone 6) to monomethyl auristatin E (MMAE) or SG3249 (also known as tesirine).
  • MMAE monomethyl auristatin E
  • SG3249 also known as tesirine
  • Anti-mutCALR antibody (Clone 6) and a control antibody were labelled with MMAE using a maleimide-activated valine-citruline p-aminobenzylcarbamate (VC-PAB) linker.
  • VC-PAB maleimide-activated valine-citruline p-aminobenzylcarbamate
  • SUBSTITUTE SHEET (RULE 26) A solution of TCEP (tris(2-carboxyethyl)phosphine) was freshly prepared and 4 molar equivalents of TCEP was added to each sample to selectively reduce cysteine disulfide bridges. Samples were incubated at 37 °C for 80 minutes.
  • Concentration of antibody-drug conjugate was determined by spectroscopic absorbance at 280 nm.
  • Drug-per-antibody ratio was determined using liquid chromatography-mass spectrometry (LC/MS) and reverse-phase high-performance liquid chromatography (RP-HPLC). Characteristics of the antibody-drug conjugates are shown below:
  • Anti-mutCALR antibody (Clone 6) and a control antibody were labelled with
  • Concentration of antibody-drug conjugate was determined by spectroscopic absorbance at 280 nm.
  • Drug-per-antibody ratio was determined using liquid chromatography-mass spectrometry (LC/MS) and reverse-phase high-performance liquid chromatography (RP-HPLC). Characteristics of the antibody-drug conjugates are shown below:
  • Example 4 Improved Properties for Anti-mutCALR Antibody-Drug Conjugate Compared to Unconjugated Anti-mutCALR Antibody
  • Examples 1-2 An isotype control antibody (Isotype-PBD) and unconjugated isotype control antibody were used as controls.
  • the internalization rate of Clone 6-PBD in cells expressing Type 1 mutant CALR was significantly increased compared to unconjugated Clone 6 or isotype controls.
  • caspase induction in cells was significantly increased in the presence of Clone 6-PBD compared to unconjugated Clone 6 or isotype controls.
  • FIG. 4C increased inhibition of cell proliferation was observed in the presence of Clone 6-PBD compared to unconjugated Clone 6 or isotype controls.
  • BAF3/4F2 cells Type 1 mutant CALR or BAF3/3D3 cells (Type 2 mutant CALR) were plated at 5000 cells/well in a 96-well plate. Cells were then treated with anti-mutCALR antibody (Clone 6) conjugated to PBD or MMAE, an isotype control antibody conjugated to PBD or MMAE, or unconjugated anti-mutCALR antibody, for 72 hours. Following incubation, cells were mixed 1 : 1 with Cell Titer-Gio (Promega) and measured for luminescence via SpectraMAX plate reader. Percent viability was normalized to the viability of an IgGl control.
  • SUBSTITUTE SHEET (RULE 26) data demonstrate that cell killing with the mutCALR antibody conjugated to PBD (Clone 6) is not dependent on the presence of 10 ng/mL TPO.
  • mice Female NSG mice, aged 6 to 8 weeks, were inoculated via tail vein with 1x10 5 BA/F3 4F2 cells on Day 0. Six days following inoculation, the mice were either administered anti-mutCALR antibody (Clone 6)-PBD (single dose i.v. on Day 6), unconjugated Clone 6 (single dose i.v. on Day 6), unconjugated Clone 6 at 10 mg/kg i.p. (Day 6, 9, and 12), or ruxohtinib twice daily per orally for 7 days. Hematology analysis for tumor cells and platelet count was conducted on the Sysmex instrument.
  • mice administered anti- mutCALR antibody (Clone 6) conjugated to PBD compared to mice administered dose matched unconjugated antibody.
  • platelet count was increased in mice administered anti-mutCALR antibody (Clone 6) conjugated to PBD compared to mice administered dose matched unconjugated antibody. Platelet counts in mice administered anti- mutCALR antibody (Clone 6) were similar to mice administered ruxolitinib.
  • mice administered a single dose of Clone 6-PBD at 1 mg/kg exhibited increased survival compared to mice administered 3 doses of unconjugated antibody at 10 mg/kg.

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Abstract

L'invention concerne des conjugués anticorps-anti-calréticuline mutant (mutCALR)-médicament comprenant des dimères de pyrrolobenzodiazépine. L'invention concerne également des kits associés et des compositions pharmaceutiques. L'invention concerne également des méthodes de traitement de néoplasmes myéloprolifératifs avec les conjugués anticorps anti-mutCALR-médicament.
PCT/US2024/053807 2023-11-01 2024-10-31 Conjugués anticorps anti-calréticuline (calr) mutant-médicament et leurs utilisations Pending WO2025096716A1 (fr)

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