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WO2025040820A1 - Conjugués d'anticorps anti-cd203c et leurs utilisations - Google Patents

Conjugués d'anticorps anti-cd203c et leurs utilisations Download PDF

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Publication number
WO2025040820A1
WO2025040820A1 PCT/EP2024/073759 EP2024073759W WO2025040820A1 WO 2025040820 A1 WO2025040820 A1 WO 2025040820A1 EP 2024073759 W EP2024073759 W EP 2024073759W WO 2025040820 A1 WO2025040820 A1 WO 2025040820A1
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seq
amino acid
acid sequence
sequence comprises
domain amino
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William James Jonathan Finlay
Orla Margaret Cunningham
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Granular Therapeutics Ltd
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Granular Therapeutics Ltd
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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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the disclosure is related to antibody-drug conjugates and uses thereof for treatment of proliferative and inflammatory disorders.
  • mast cells are central drivers of pathology in many chronic diseases, including systemic mastocytosis and mast cell leukemia, which are driven by aberrant mast cell proliferation.
  • systemic mastocytosis and mast cell leukemia are driven by aberrant mast cell proliferation.
  • mast cell-selective drugs which leaves a significant unmet medical need for patients suffering from mast cell driven disease.
  • c-Kit signaling may be an important survival signal.
  • Kinase inhibitors with broader activities beyond c-Kit have suffered from even greater off-target systemic toxicity and can also show limited therapeutic benefit due to sub-optimal pharmacokinetics.
  • Drug designs that enable more specific, top-down targeting of mast cells and basophils would have the potential to control aberrant activation and/or proliferation of those cells while protecting cell populations that are not diseasemediating. There remains a significant unmet medical need to develop therapies with more effective and longer duration benefit that combine potency and specificity of targeting with minimal side effects.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:
  • the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;
  • VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;
  • VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;
  • VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;
  • VH domain amino acid sequence comprises SEQ ID NO: 55, and the VL domain amino acid sequence comprises SEQ ID NO: 58;
  • VH domain amino acid sequence comprises SEQ ID NO: 61
  • VL domain amino acid sequence comprises SEQ ID NO: 65
  • VH domain amino acid sequence comprises SEQ ID NO: 68
  • VL domain amino acid sequence comprises SEQ ID NO: 70
  • VH domain amino acid sequence comprises SEQ ID NO: 78
  • VL domain amino acid sequence comprises SEQ ID NO: 80
  • the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;
  • VH domain amino acid sequence comprises SEQ ID NO: 92
  • VL domain amino acid sequence comprises SEQ ID NO: 96
  • VH domain amino acid sequence comprises SEQ ID NO: 100
  • VL domain amino acid sequence comprises SEQ ID NO: 104
  • the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;
  • VH domain amino acid sequence comprises SEQ ID NO: 116
  • VL domain amino acid sequence comprises SEQ ID NO: 117
  • the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;
  • VH domain amino acid sequence comprises SEQ ID NO: 125
  • VL domain amino acid sequence comprises SEQ ID NO: 129
  • VH domain amino acid sequence comprises SEQ ID NO: 133
  • VL domain amino acid sequence comprises SEQ ID NO: 134
  • VH domain amino acid sequence comprises SEQ ID NO: 138
  • VL domain amino acid sequence comprises SEQ ID NO: 142
  • VH domain amino acid sequence comprises SEQ ID NO: 144
  • VL domain amino acid sequence comprises SEQ ID NO: 146
  • the antibody or antigen-binding portion comprises the c- Kit inhibitor and a linker comprising the structure:
  • the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a hydrazone linker. In some embodiments, the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a non-cleavable linker.
  • the drug-to-antibody ratio (DAR) of the antibody-drug conjugate is about 2 to about 5. In some embodiments, the DAR is about 4.
  • composition comprising an antibody-drug conjugate disclosed herein, and a pharmaceutically acceptable carrier.
  • a method for treating an inflammatory disorder in a subject in need thereof comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein.
  • a method for ameliorating a symptom of an inflammatory disorder in a subject in need thereof comprising administering to the subject an antibody-drug conjugate or a pharmaceutical composition provided herein.
  • the inflammatory disorder is a mast cell inflammatory disorder.
  • the inflammatory disorder is c-Kit-reliant.
  • the inflammatory disorder is a chronic inflammatory disorder.
  • the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.
  • FIG. 1 shows a schematic of an anti-CD203c-c-Kit inhibitor conjugate mechanism of action.
  • Anti-CD203c conjugates are internalized on CD203c + granulocytes and trafficked to the lysosome where they are proteolytically processed, releasing free c-Kit inhibitor which potently inhibits c-Kit receptor activity, leading to leading to the suppression of c-Kit related granulocyte activation and degranulation, and/or cell death via apoptosis.
  • FIG. 2 shows a schematic of the targeting modality disclosed herein.
  • a granulocyte (or CD203c) targeting antibody is conjugated via a linker to dasatinib, a dasatinib derivative, or another c-Kit inhibitor.
  • FIG. 3 shows results from a direct binding ELISA of anti-CD203c antibodies against human and rhesus CD203c.
  • Purified anti-CD203c IgG-null antibodies were titrated (in nM) in a direct binding ELISA against purified recombinant human and rhesus CD203c proteins.
  • FIG. 4A - FIG. 4B show results from an analysis of binding of anti-CD203c antibodies to cell-surface expressed CD203c on stimulated KU812 cells.
  • Purified anti- CD203c IgG-null antibodies were titrated (in nM) in a flow-based assay against the basophilic cell line KU812 which expresses CD203c.
  • FIG. 5A - FIG. 5B show results from an analysis of internalization of anti- CD203c antibodies by TF1 (CD2O3c 10 ) and KU812 (CD203c high ) cells.
  • Purified anti- CD203c antibodies were tested for their ability to internalize using Fab-ZAP-mediated killing of cells as an indirect measure of internalization capacity. Internalization efficiency was dependent on level of CD203c expression.
  • FIG. 6A - FIG. 6B show results from biophysical characterization of anti- CD203c antibodies.
  • Antibody aggregation propensity was measured using an affinitycapture self-interaction nanoparticle spectroscopy (AC-SINS) assay (FIG. 6A).
  • the clinical antibodies alirocumab and bococizumab were used as negative and positive controls, respectively.
  • antibody specificity was measured by direct ELISA against double-stranded DNA and insulin using bococizumab and briakinumab as positive controls (non-specific) and bevacizumab and ustekinumab as negative controls (antigen-specific) (FIG. 6B).
  • FIG. 7A - FIG. 7C show results from an analysis of internalization of 2.23- AF594 conjugate on KU812 cells. Internalization of fluorescently conjugated antibody 2.23 in KU812 cells was visualized using the Amnis® ImageStream® imaging flow cytometer platform (FIG. 7A). Internalization rate was measured at 4°C and 37°C (FIG. 7B), in addition to co-localization with LAMP2 as a marker of lysosomal uptake (FIG. 7C).
  • FIG. 8A - FIG. 8B show structures of the BTP-Glu(AA-cKit-inhibitor 1)- PEG(24u) (FIG. 8A) and BTP-Glu(AA-cKit-inhibitor 2)-PEG(24u) linker-payloads (FIG. 8B)
  • FIG. 9 shows synthesis of the BTP-Glu(AA-cKit-inhibitor l)-PEG(24u) linkerpayload.
  • the linker-payload was prepared by adding a mixture of c-Kit inhibitor 1.2HC1 salt and BTP-Bl-Glu(OH)-PEG(24u) to HATU reagent prepared in DMF and N-MethylMorpholine (NMM).
  • FIG. 10 shows a RPLC (reversed-phase liquid chromatography) chromatogram of the BTP-Glu(AA-c-Kit inhibitor l)-PEG(24u) linker-payload. Detection was performed at 214 nm and 254 nm.
  • FIG. 11 shows a LC-MS ((liquid chromatography-mass spectrometry) spectrum of the BTP-Glu(AA-c-Kit inhibitor l)-PEG(24u) linker-payload.
  • the purified linker-payload was diluted to 0.05 mg/mL and 10 pl was analyzed by LC-MS using a flow rate of 0.4 mL/min. The expected mass was confirmed via doubly and triply charged species.
  • FIG. 12 shows synthesis of the BTP-Glu(AA-cKit-inhibitor 2)-PEG(24u) linker-payload.
  • the linker-payload was prepared by adding a mixture of c-Kit inhibitor 2.2HC1 salt and BTP-Bl-Glu(OH)-PEG(24u) to HATU reagent prepared in DMF and N-MethylMorpholine (NMM).
  • FIG. 13 shows a RPLC chromatogram of the BTP-Glu(AA-c-Kit inhibitor 2)- PEG(24u) linker-payload. Detection was performed at 214 nm and 254 nm.
  • FIG. 14 shows a LC-MS spectrum of the BTP-Glu(AA-c-Kit inhibitor 2)- PEG(24u) linker-payload.
  • the purified linker-payload was diluted to 0.05 mg/mL and 10 pl was analyzed by LC-MS using a flow rate of 0.4 mL/min. The expected mass was confirmed via doubly and triply charged species.
  • FIG. 15 shows a deconvoluted LC-MS spectrum for 2.23-BTP c-Kit inhibitor
  • FIG. 16A - FIG. 16B show results from analytical characterization of 2.23- BTP c-Kit inhibitor 1 ADC.
  • FIG. 16A shows a hydrophobic interaction chromatogram at 280 nm comparing naked 2.23 antibody (2.23-IgG) and 2.23-ADC.
  • FIG. 16A shows a size exclusion chromatogram at 280 nm for the 2.23-ADC compared to PBS buffer alone.
  • FIG. 17 shows a deconvoluted LC-MS spectrum for 2.23-BTP c-Kit inhibitor
  • FIG. 19 shows an analytical summary for 2.23-c-Kitl-ADC.
  • FIG. 20 shows an analytical summary for 2.23-c-Kit2-ADC.
  • FIG. 21A - FIG. 21B show results from an assessment of ADC potency in cell killing and c-Kit receptor inhibition assays.
  • 2.23-cKitl-ADC, 2.23-cKit2- ADC, 2.23-IgG and free payload were assessed in a KU812 cell killing assay using CellTiterGLO to measure viability.
  • potency of inhibition of 2.23-cKitl- ADC, 2.23-cKit2-ADC, 2.23-IgG and free payload were compared in a c-Kit receptor phosphorylation assay.
  • FIG. 22 shows results from a functional characterization of 2.23-cKit2-ADC. Potency of 2.23-cKit2-ADC, 2.23-IgG and free payload were compared in a KU812 cell killing assay. Both cell viability and cytotoxicity were measured at 24, 48 and 72h using CellTiterGLO and CellToxGreen, respectively.
  • FIG. 23 shows results from an analysis of specificity of 2.23-cKit2-ADC. Specificity of 2.23-cKit2-ADC targeting was demonstrated by comparing potency against a relevant isotype control-cKit2-ADC conjugate in KU812 cells after 72h incubation.
  • FIG. 24A - FIG. 24B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing.
  • the effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free pay load treatment were measured in RKO cells.
  • FIG. 24 A shows that RKO cells do not express CD203c or c-Kit.
  • FIG. 24B shows that 2.23-cKit-ADC does not affect RKO cell viability.
  • FIG. 25A - FIG. 25B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing.
  • the effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free payload treatment were measured in RXF393 cells.
  • FIG. 25A shows that RXF393 cells express low levels of CD203c but not c-Kit.
  • FIG. 25B shows that 2.23- cKit-ADC does not affect RXF393 cell viability.
  • FIG. 26A - FIG. 26B show results from an analysis of selectivity of 2.23-cKit2- ADC cell killing.
  • the effects of 2.23-cKit2-ADC, 2.23-IgG, isotype control-cKit2- ADC and free payload treatment were measured in HepG2 cells.
  • FIG. 26A shows that HepG2 cells express CD203c but not c-Kit.
  • FIG. 26B shows that 2.23-cKit-ADC does not affect HepG2 cell viability.
  • FIG. 27A - FIG. 27D show linker variants for cKit2 payload testing in KU812 cells and results from viability and cytotoxicity studies.
  • “dAla” refers to “delta-Ala”, a structure where one alanine in the linker has been removed.
  • “dAlaAla” refers to a structure where both alanines in the linker have been removed.
  • FIG. 28A - FIG. 28B show structures and results from viability and cytotoxicity studies in KU812 cells testing anlotinib as a potential payload for a 2.23 ADC.
  • FIG. 29A - FIG. 29B show structures and results from viability and cytotoxicity studies in KU812 cells testing imatinib as a potential payload for a 2.23 ADC.
  • FIG. 30A - FIG. 30B show structures and results from viability and cytotoxicity studies in KU812 cells testing crenolanib as a potential payload for a 2.23 ADC.
  • FIG. 31A - FIG. 31B show structures and results from viability and cytotoxicity studies in KU812 cells testing ponatinib as a potential payload for a 2.23 ADC.
  • FIG. 32A - FIG. 32B show structures and results from viability and cytotoxicity studies in KU812 cells testing avapritinib as a potential payload for a 2.23 ADC.
  • FIG. 33A - FIG. 33B show structures and results from viability and cytotoxicity studies in KU812 cells testing elenestinib as a potential payload for a 2.23 ADC.
  • FIG. 34A - FIG. 34B show structures and results from viability and cytotoxicity studies in KU812 cells testing bezuclastinib as a potential payload for a 2.23 ADC.
  • FIG. 35A - FIG. 35B show structures and results from viability and cytotoxicity studies in KU812 cells testing midostaurin as a potential payload for a 2.23 ADC.
  • FIG. 36A - FIG. 36B show results from a pharmacokinetic analysis in rats of naked 2.23 vs 2.23-cKit 2 ADC.
  • FIG. 37 shows results from an in-vivo non-tumor bearing tolerability study of 2.23-cKit2 ADC using female NOD SCID gamma (NSG) mice.
  • FIG. 38A - FIG. 38B show results from an in-vivo efficacy study of 2.23-cKit2 ADC in a KU812 tumor model in NSG mice. Results are reported in both linear (FIG. 38A) and log (FIG. 38B) scales.
  • FIG 39 shows results from a dose-dependent in-vivo efficacy study of 2.23- cKit2 ADC in a KU812 tumor model in NSG mice.
  • FIG. 40 shows results from a multiple dose pharmacokinetics study of 2.23- cKit2 ADC in cynomolgus monkeys.
  • FIG. 41A - FIG. 411 show results from blood parameters measured in cynomolgus monkey after treatment with 2.23-cKit2 ADC.
  • This panel included White Blood cells (FIG. 41A), Red blood cells (FIG. 41B), Hemoglobin (FIG. 41C), Platelets (FIG. 41D), Neutrophils (FIG. 41E), Lymphocytes (FIG. 41F), Monocytes (FIG. 41G), Eosinophils (FIG. 41H) and Basophils (FIG. 411).
  • FIG. 42A - FIG. 42D show results from a direct filter-binding radiometric kinase activity assay measuring the potency of cKit2 free payload in inhibiting tyrosine kinases including Bruton’s Tyrosine Kinase (BTK; FIG. 42A), wild-type c-Kit (FIG. 42B) and variant c-Kit (D816V; FIG. 42C). Calculated IC50 values are summarized in FIG. 42D
  • FIG. 43 shows results from a KINOMEscan competition assay (Eurofins) used to assess the selectivity of the cKit2 free payload compared to other benchmark tyrosine kinase inhibitors.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen-binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).
  • an antibody or an antigen-binding portion is a triabody, a tetrabody, a bis-scFv or a tandem scFv.
  • an antibody or an antigen-binding portion is a dual affinity re-targeting protein.
  • CDR sequences are underlined in variable region sequences.
  • CDR sequences are underlined in variable region sequences.
  • CDR sequences are underlined in variable region sequences.
  • CDR sequences are underlined in variable region sequences.
  • CDR sequences are underlined in variable region sequences.
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion cross-competes for binding to CD203c with antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23, or an antibody that comprises one or more amino acid sequences of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23 (see Tables 1-23).
  • cross-compete means the ability of an antibody or an antigen-binding portion thereof to interfere with the binding directly or indirectly through allosteric modulation of the anti-CD203c antibodies of the disclosure to the target CD203c (e.g., human CD203c).
  • target CD203c e.g., human CD203c
  • the extent to which an antibody or portion thereof is able to interfere with the binding of another to the target, and therefore whether it can be said to cross-block or cross-compete can be determined using competition binding assays.
  • HTRF Homogeneous Time Resolved Fluorescence
  • One particularly suitable quantitative cross-competition assay uses a FACS- or an Alphascreen-based approach to measure competition between the labelled (e.g., His-tagged, biotinylated or radioactive labelled) antibody or portion thereof and the other antibody or portion thereof in terms of their binding to the target.
  • a cross-competing antibody or portion thereof is, for example, one which will bind to the target in the crosscompetition assay such that, during the assay and in the presence of a second antibody or portion thereof, the recorded displacement of the immunoglobulin single variable domain or polypeptide according to the invention is up to 100% (e.g. in a FACS-based competition assay) of the maximum theoretical displacement (e.g.
  • cross-competing antibodies or portions thereof have a recorded displacement that is between 10% and 100%, or between 50% and 100%.
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein:
  • the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ ID NO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 63; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 66; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 76; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 77;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 79; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 81; a LCDR2 comprising SEQ ID NO: 82; and a LCDR3 comprising SEQ ID NO: 83;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 85; a HCDR2 comprising SEQ ID NO: 86; and a HCDR3 comprising SEQ ID NO: 87; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 89; a LCDR2 comprising SEQ ID NO: 90; and a LCDR3 comprising SEQ ID NO: 91;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 145; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein:
  • VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;
  • VH domain amino acid sequence comprises SEQ ID NO: 55
  • VL domain amino acid sequence comprises SEQ ID NO: 58
  • VH domain amino acid sequence comprises SEQ ID NO: 133
  • VL domain amino acid sequence comprises SEQ ID NO: 134
  • VH domain amino acid sequence comprises SEQ ID NO: 138
  • VL domain amino acid sequence comprises SEQ ID NO: 142
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein: the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain wherein: the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.
  • an anti-CD203c antibody or an antigen-binding portion thereof wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH and VL domain sequences are those of antibody 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22, or 2.23, with 1, 2 or 3 conservative amino acid substitutions in the VH domain sequence, the VL domain sequence, or both the VH domain and the VL domain sequences. In some embodiments, conservative amino acid substitutions are made only in the FR sequences and not in the CDR sequences of an antibody or antigen-binding portion.
  • terminal insertions include an antibody molecule with an N-terminal methionyl residue or the antibody molecule fused to an epitope tag.
  • Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the half-life of the antibody in the blood circulation.
  • the anti-CD203c antibody or antigen-binding portion provided herein may include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation.
  • the antibody or antigen-binding portion may be mutated to alter such post-translational modifications, for example by adding, removing or replacing one or more amino acid residues to form or remove a glycosylation site.
  • the anti-CD203c antibody or antigen-binding portion provided herein may be modified for example by amino acid substitution to remove potential proteolytic sites in the antibody or portion.
  • an anti-CD203c antibody or an antigen-binding portion thereof comprises an immunoglobulin constant region.
  • the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.
  • the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.
  • the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region.
  • the immunoglobulin constant region comprises one or more mutations to increase FcyR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP) activity, and/or complement-dependent cytotoxicity (CDC) activity.
  • the immunoglobulin constant region is immunologically inert.
  • the immunoglobulin constant region comprises one or more mutations to reduce or prevent FcyR binding, ADCC activity, ADCP activity and/or CDC activity.
  • the immunoglobulin constant region is a wild-type human IgGl constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A, a human IgGl constant region comprising the amino acid substitutions L234A, L235A, G237A and P331S or a human IgG4 constant region comprising the amino acid substitution S228P, wherein numbering is according to the EU index as in Kabat.
  • an anti-CD203c antibody or an antigen-binding portion thereof may comprise an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.
  • an anti-CD203c antibody may comprise an immunoglobulin constant region comprising any one of the amino acid sequences in Table 30. The Fc region sequences in Table 30 begin at the CHI domain.
  • an anti-CD203c antibody may comprise an immunoglobulin constant region comprising an amino acid sequence of an Fc region of human IgG4 or human IgGl.
  • an anti-CD203c antibody may comprise an immunoglobulin constant region comprising an amino acid sequence of an Fc region of human IgG4, human IgG4(S228P), human IgG2, human IgGl, human IgGl effector null.
  • the human IgG4(S228P) Fc region comprises the following substitution compared to the wild-type human IgG4 Fc region: S228P.
  • the human IgGl effector null Fc region comprises the following substitutions compared to the wild-type human IgGl Fc region: L234A, L235A and G237A.
  • an anti-CD203c antibody may comprise an immunoglobulin constant region comprising the amino acid sequence of any one of SEQ ID NOS: 155-160.
  • an anti-CD203c antibody may comprise the six CDR amino acid sequences of any one of the clones in Tables 1-23 and any one of the Fc region amino acid sequences in Table 24.
  • an anti-CD203c antibody may comprise an immunoglobulin heavy chain constant region comprising any one of the Fc region amino acid sequences in Table 24 and an immunoglobulin light chain constant region that is a kappa light chain constant region or a lambda light chain constant region.
  • nucleic acid molecule e.g., an isolated nucleic acid molecule
  • a nucleic acid molecule encoding an amino acid sequence of an anti-CD203c antibody or anti- CD203c antigen-binding portion described herein (or an amino acid sequence of a (i) VH domain, (ii) a VL domain, or (iii) both a VH domain and a VL domain of an antibody or antigen-binding portion).
  • nucleic acid molecule e.g., an isolated nucleic acid molecule
  • a nucleic acid molecule encoding (i) a heavy chain, (ii) a light chain, or (iii) both a heavy chain and a light chain of an anti-CD203c antibody or anti- CD203c antigen-binding portion described herein.
  • a nucleic acid molecule encoding a VH domain, a VL domain, a heavy chain or a light chain comprises a signal sequence.
  • a nucleic acid molecule encoding a VH domain, a VL domain, a heavy chain or a light chain does not comprise a signal sequence.
  • an expression vector comprising a nucleic acid molecule described herein.
  • a nucleic acid molecule is operatively linked to one or more regulatory sequences suitable for expression of the nucleic acid segment in a host cell.
  • an expression vector comprises sequences that mediate replication and comprises one or more selectable markers.
  • vector means a construct that is capable of delivering, and, preferably, expressing, one or more gene(s) or sequence(s) of interest in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
  • a recombinant host cell comprising an expression vector or a nucleic acid molecule disclosed herein.
  • a “host cell” includes an individual cell, a cell line or cell culture that can be or has been a recipient for vector(s) for incorporation of polynucleotide inserts.
  • Host cells include progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • An expression vector can be transfected into a host cell by standard techniques. Non-limiting examples include electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • a recombinant host cell comprises a single vector or a single nucleic acid molecule encoding both a VH domain and a VL domain of an anti-CD203c antibody or an antigen-binding portion thereof. In some embodiments, a recombinant host cell comprises (i) a first vector or a first nucleic acid molecule encoding a VH domain of an anti-CD203c antibody or an antigen-binding portion thereof and (ii) a second vector or a second nucleic acid molecule encoding a VL domain of an anti-CD203c antibody or an antigen-binding portion thereof.
  • Antibody molecules of the invention can be produced using techniques well known in the art, for example, recombinant technologies, phage display technologies, synthetic technologies, computational technologies or combinations of such technologies or other technologies readily known in the art.
  • an anti-CD203c antibody or an antigen-binding portion thereof comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid segment is expressed, thereby producing the anti-CD203c antibody or antigen-binding portion. The antibody or antigen-binding portion may then be isolated from the host cell or culture.
  • Anti-CD203c antibodies and antigen-binding portions thereof can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, anti-CD203c antibodies and antigen-binding portions thereof can be produced recombinantly.
  • nucleic acid sequences encoding one or more of SEQ ID NOS: 1-154, or portions thereof may be introduced into a bacterial cell (e.g., E. coli, B. subtilis) or a eukaryotic cell (e.g., a yeast such as S. cerevisiae, or a mammalian cell such as a CHO cell line, various Cos cell lines, a HeLa cell, a HEK293 cell, various myeloma cell lines, or a transformed B-cell or hybridoma), or into an in vitro translation system, and the translated polypeptide may be isolated.
  • a bacterial cell e.g., E. coli, B. subtilis
  • a eukaryotic cell e.g., a yeast such as S. cerevisiae, or a mammalian cell such as a CHO cell line, various Cos cell lines, a HeLa cell, a HEK293 cell, various myeloma
  • ADCs provided herein comprise small molecule c-Kit inhibitors.
  • a c-Kit inhibitor may be referred to as a “payload” in this disclosure.
  • c-Kit inhibitors that may be components of ADCs are described in more detail below.
  • a c-Kit inhibitor inhibits multiple tyrosine kinases, including c-Kit.
  • a c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.
  • a c-Kit inhibitor is dasatinib. Dasatinib is described, for example, in US 6,596,746. Polymorphic forms of dasatinib are described in, for example, US 11,059,813.
  • the c-Kit inhibitor can include all stereoisomeric forms, mixtures of stereoisomeric forms, diastereomeric forms, and pharmaceutically acceptable salts thereof.
  • pharmaceutically acceptable salts refers to derivatives of the disclosed inhibitors wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the non-toxic salts of the parent compound formed, e.g., from nontoxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • a linker is a bifunctional or multifunctional moiety that can be used to link one or more c-Kit inhibitor drug moi eties to an anti-CD203c antibody or antigen-binding portion to form an ADC.
  • an ADC can be prepared using a linker having reactive functionalities for covalently attaching to the drug and to the antibody or antigen-binding portion.
  • a cysteine thiol of an antibody or antigen-binding portion can form a bond with a reactive functional group of a linker or a drug-linker intermediate to make an ADC.
  • a linker is divalent, meaning it can link only one drug molecules to an antibody, or it can be trivalent (able to link two drug molecules to an antibody), or it can be polyvalent.
  • Trivalent, tetravalent, and polyvalent linkers can be used to increase the loading of drug on an antibody, increasing the drug-to-antibody ratio (DAR) without requiring additional sites on the antibody for attaching linkers.
  • DAR drug-to-antibody ratio
  • Such linkers are known in the art, see e.g., US 6,638,499; US 10,214,560.
  • a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond.
  • reactive functionalities include maleimide, haloacetamides, a- haloacetyl, pyridyl disulfide, activated esters such as succinimide esters, N- hydroxysuccinimide, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. See, e.g., the conjugation method described in Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773.
  • a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody.
  • electrophilic groups include aldehyde and ketone carbonyl groups.
  • a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit.
  • reactive functionalities include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • a linker may comprise one or more linker components, including but not limited to, a stretcher unit, a peptidomimetic unit, a peptide unit, and a spacer unit.
  • an ADC provided herein comprises a polyethylene glycol (PEG) spacer.
  • Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP”), valine-citrulline (“val-cit” or “vc”), alaninephenylalanine (“ala-phe”), phenylalanine-lysine (phe-lys), p-aminobenzyloxycarbonyl (“PAB”), N-Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP”), and 4-(N- maleimidomethyl) cyclohexane- 1 carboxylate (“MCC”).
  • MC 6-maleimidocaproyl
  • MP maleimidopropanoyl
  • val-cit valine-citrulline
  • vc valine-citrulline
  • alaninephenylalanine ala-phe
  • phenylalanine-lysine phe-lys
  • PAB p-aminobenz
  • a linker may be a “cleavable linker,” facilitating release of a drug.
  • cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52: 127-131 (1992); US 5,208,020).
  • Acid-labile linkers are linkers cleavable at acidic pH.
  • certain intracellular compartments such as endosomes and lysosomes, have an acidic pH (pH 4-5), and provide conditions suitable to cleave acid- labile linkers.
  • photolabile linkers are linkers that are useful at the body surface and in body cavities that are accessible to light. Photolabile linkers may also be cleaved by infrared light that can penetrate tissue.
  • a linker is an acid cleavable linker. Cleavage of these linkers is induced by low pH of the tumor acidic microenvironment or lysosomes. Nonlimiting examples include hydrazone, carbonate, and silyl ether linkers.
  • a linker is a glutathione (GSH) cleavable linker. Cleavage of these linkers is induced by high level of GSH in the cytoplasm.
  • GSH glutathione
  • a linker is a Fe(II) cleavable linker. Cleavage of these linkers is induced by elevating levels of ferrous iron.
  • Nonlimiting examples include a 1, 2, 4-tri oxolane linker.
  • a linker is a cathepsin cleavable linker. Cleavage of these linkers is induced by cathepsin in lysosomes.
  • Nonlimiting examples include dipeptide, triglycyl (CX), and cyclobutane- 1,1 -dicarboxamide (cBu)-Cit linkers.
  • a linker is a glycosidase cleavable linker. Cleavage of these linkers is induced by P-glucuronidase or P-galactosidase in lysosomes. Nonlimiting examples include P-glucuronide and P-galactoside linkers.
  • a linker is a phosphatase cleavable linker. Cleavage of these linkers is induced by phosphatase and pyrophosphates in lysosomes. Nonlimiting examples include a pyrophosphate linker.
  • a linker is a sulfatase cleavable linker. Cleavage of these linkers is induced by sulfatase in lysosomes.
  • Nonlimiting examples include an aryl sulfate linker.
  • NIR near-infrared
  • Nonlimiting examples include heptamethine cyanine fluorophore, O-nitrobenzyl, and PC4AP linkers.
  • a linker is a bioorthogonal cleavable linker. Cleavage of these linkers is induced by the bioorthogonal cleavage pair: Cu(I)-BTTAA/dsProc. Nonlimiting examples include a dsProc linker.
  • a linker may be a “non-cleavable linker”.
  • Non-cleavable linkers are any chemical moiety capable of linking a drug to an antibody in a stable, covalent manner that does not fall off under the categories listed above for cleavable linkers.
  • non-cleavable linkers are substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage and disulfide bond cleavage.
  • non-cleavable refers to the ability of the chemical bond in the linker or adjoining to the linker to withstand cleavage induced by an acid, photolabile-cleaving agent, a peptidase, an esterase, or a chemical or physiological compound that cleaves a disulfide bond, at conditions under which the drug or the antibody does not lose its activity.
  • the active part of an ADC with a non-cleavable linker is composed of an amino acid appendage, a linker and a cytotoxic payload.
  • Nonlimiting examples include 2-(maleimidom ethyl)- 1,3- dioxane (MD) linkers, Mal-PAB linkers, and PEG linkers with intermediates of alkyne, triazole and piperazine.
  • a linker is a peptide linker.
  • a peptide linker comprises two amino acid residues independently selected from glycine (Gly), alanine (Ala), phenylalanine (Phe), lysine (Lys), arginine (Arg), valine (Vai), and citrulline (Cit).
  • a peptide linker comprises at least one unnatural amino acid.
  • Chromatography can include, for example, reverse-phase and normal phase; size exclusion; ion exchange; high, medium and low pressure liquid chromatography methods and apparatus; small scale analytical; simulated moving bed (SMB) and preparative thin or thick layer chromatography, as well as techniques of small scale thin layer and flash chromatography.
  • the completed reaction mixture may be evaporated to dryness followed by re-dissolution in a polar aprotic solvent.
  • the solution may be filtered and then precipitated by combining the solution with a nonpolar antisolvent such as, for instance, hexane or cyclohexane.
  • the precipitate may then be collected by filtration, optionally washed, and then dried.
  • an antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • an antibody-drug conjugate comprises dasatinib (or a dasatinib derivative), a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • BTP-Glu-(AA-c-kit inhibitor 2)-PEG(24u) linker-payload in the Examples and Drawings.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a c-Kit inhibitor, wherein the antibody-drug conjugate comprises the c-Kit inhibitor and a linker comprising the structure:
  • Antibody conjugation to linker-payload intermediate compounds may be performed by methods provided in the Examples, or other methods known in the art.
  • site-specific conjugation technology through antibody engineering is used for antibody conjugation to the linker.
  • chemical structural modification of the linker-antibody attachment is used for antibody conjugation to the linker.
  • the c-Kit inhibitor is linked to the immunoglobulin constant region of the anti-CD203c antibody or antigen-binding portion.
  • conjugation is performed via maleimide structure conjugation, bis(vinylsulfonyl)piperazine conjugation, A -methyl -A - phenylvinylsulfonamide conjugation, or Pt(II)-based conjugation.
  • An ADC composition generally comprises a mixture of antibody-drug conjugate compounds, with varied numbers of drug molecules conjugated per antibody.
  • the average drug-to-antibody ratio (DAR) in the mixture of antibody-drug conjugate compounds is about 2 to about 5. In some embodiments, the average DAR is about 4.
  • the number of drug moieties that can be conjugated to an antibody is limited by the number of free cysteine residues.
  • the average DAR in preparations of antibody-drug conjugates from conjugation reactions may be characterized by conventional means such as mass spectroscopy, ELISA assay, and HPLC.
  • ADCs may be purified and separated from unconjugated reactants and/or conjugate aggregates by purification methods known in the art such as, for example and not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, chromatofocusing, ultrafiltration, centrifugal ultrafiltration, and combinations thereof.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and aLCDR3 comprising SEQ ID NO: 83.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and aLCDR3 comprising SEQ ID NO: 83; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor; wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.
  • an antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibodydrug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise an ADC, and a pharmaceutically acceptable carrier, diluent or excipient.
  • a pharmaceutically acceptable carrier diluent or excipient.
  • Such materials should be non-toxic and should not interfere with the efficacy of the ADC.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be by injection, bolus, infusion, or any other suitable route, as discussed below.
  • the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83; and wherein the antibodydrug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • the antibodydrug conjugate comprises a peptide linker
  • the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • the term “pharmaceutically acceptable” refers to molecular entities and compositions that do not generally produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions approved by a regulatory agency of the U.S. federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans are considered to be “pharmaceutically acceptable.”
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutically acceptable carrier, diluent or excipient may be a compound or a combination of compounds that does not provoke secondary reactions and that allows, for example, facilitation of the administration of the ADC, an increase in its lifespan and/or in its efficacy in the body or an increase in its solubility in solution.
  • a pharmaceutical composition disclosed herein may be formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (z.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition is sterile and fluid to the extent that easy syringeability exists.
  • the composition is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound 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 the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or com starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or com starch
  • a lubricant such as magnesium stearate
  • a glidant such as colloidal silicon dioxide
  • a sweetening agent such
  • the compounds may be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the pharmaceutical agents can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the ADC may be provided in a lyophilized form for reconstitution prior to administration.
  • lyophilized antibody molecules may be reconstituted in sterile water and mixed with saline prior to administration to an individual.
  • compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.
  • ADCs and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subject with a proliferative disorder.
  • methods and uses of ADCs and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subj ect with an inflammatory disorder are provided herein.
  • the therapeutic ADCs provided herein combine potency and specificity of targeting with minimal side effects. This is particularly important in the case of indolent systemic mastocytosis, which represents more than 90% of cases, where most patients have a normal life span. In these cases, adverse events associated with treatment often outweigh the burden of chronic disease symptoms, from a quality-of-life perspective.
  • An ADC or pharmaceutical composition disclosed herein may be used in a method of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treatment before the onset of a condition in a subject to reduce the risk of the condition occurring in the subject; delay its onset; or reduce its severity after onset).
  • the method of treatment may comprise administering the ADC or pharmaceutical composition to a subject in need thereof.
  • a method for treating a proliferative disorder in a subject comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein.
  • a method for ameliorating a symptom of a proliferative disorder in a subject comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein.
  • an ADC or a pharmaceutical composition disclosed herein for use in the treatment of a proliferative disorder is provided herein.
  • the proliferative disorder is a mast cell proliferative disorder. In some embodiments, the proliferative disorder is c-Kit-reliant. In some embodiments, the proliferative disorder is a mast cell proliferative disorder and is c- Kit-reliant.
  • the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.
  • a method for treating an inflammatory disorder in a subject comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein.
  • a method for ameliorating a symptom of an inflammatory disorder in a subject comprising administering to the subject an ADC or a pharmaceutical composition disclosed herein.
  • an ADC or a pharmaceutical composition disclosed herein for use in the treatment of an inflammatory disorder is provided herein.
  • the proliferative disorder is a mast cell inflammatory disorder.
  • the inflammatory disorder is c-Kit-reliant.
  • the inflammatory disorder is a mast cell inflammatory disorder and is c- Kit-reliant.
  • the inflammatory disease is a chronic inflammatory disease.
  • the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.
  • the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer
  • the ADC is an ADC comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor, wherein the c-Kit inhibitor is dasatinib (or a dasatinib derivative); wherein the antibody or antigen-binding portion comprises a VH domain and a VL domain, wherein the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153; and wherein the antibody-drug conjugate comprises a peptide linker comprising L-Ala-L-Ala, and a PEG spacer.
  • the term “effective amount” or “therapeutically effective amount” refers to the amount of a pharmaceutical agent, e.g., an ADC or a pharmaceutical composition disclosed herein, which is sufficient to reduce or ameliorate the severity and/or duration of a proliferative disorder or an inflammatory disorder, or one or more symptoms thereof, prevent the advancement of a disease, cause regression of a disease, prevent the recurrence, development, onset or progression of one or more symptoms associated with a disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related therapy (e.g., prophylactic or therapeutic agent) for a proliferative disorder or an inflammatory disorder.
  • a pharmaceutical agent e.g., an ADC or a pharmaceutical composition disclosed herein
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antibody -based protein molecules are well known in the art (Ledermann J. A. et al., 1991, Int. J. Cancer 659-664; Bagshawe K.D.
  • a therapeutically effective amount or suitable dose of an antibody-based protein molecule may be determined by comparing its in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the antibody-based protein is for prevention or for treatment, the size and location of the area to be treated, the precise nature of the antibody-based protein, and the nature of any detectable label or other molecule attached to the antibody -based protein.
  • a typical ADC dose will be in the range 100 pg to 1 g for systemic applications, and 1 pg to 1 mg for intradermal injection.
  • An initial higher loading dose, followed by one or more lower doses, may be administered.
  • a dose for a single treatment of an adult subject may be proportionally adjusted for children and infants. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician.
  • the treatment schedule for a subject may be dependent on the pharmacokinetic and pharmacodynamic properties of the ADC composition, the route of administration and the nature of the condition being treated.
  • Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g., about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Treatment may be given before, and/or after surgery, and/or may be administered or applied directly at the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.
  • an ADC or a pharmaceutical composition disclosed herein may be administered as a subcutaneous injection.
  • Subcutaneous injections may be administered using an auto-injector, for example for long term prophylaxis/treatment.
  • the therapeutic effect of an ADC or a pharmaceutical composition disclosed herein may persist for several half-lives, depending on the dose.
  • the therapeutic effect of a single dose of an ADC or a pharmaceutical composition disclosed herein may persist in a subject for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more.
  • a subject may be treated with an ADC or a pharmaceutical composition disclosed herein and an additional therapeutic agent or therapy that is used to treat a cancer or a symptom or complication of a proliferative disorder or an inflammatory disorder.
  • the ADC or pharmaceutical composition disclosed herein and the additional therapeutic agent or therapy may be administered simultaneously or sequentially.
  • a subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse or a rat.
  • a subject is an adult human.
  • a subject is a pediatric human.
  • an ADC or a pharmaceutical composition disclosed herein, for use in the treatment of a disease or a disorder is provided herein.
  • an ADC or a pharmaceutical composition disclosed herein for use as a medicament.
  • the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured. Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.
  • amino-terminal N-terminus
  • carboxyl-terminal C- terminus
  • amino-terminal N-terminus
  • carboxyl-terminal C- terminus
  • these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position.
  • a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl-terminus of the reference sequence but is not necessarily at the carboxyl-terminus of the complete polypeptide.
  • CD203c (also known as ENPP3, B10, NPP3, PDNP3, PD-IBETA, and ectonucleotide pyrophosphatase/phosphodiesterase 3) is a type II transmembrane protein that belongs to the ectonucleotide pyrophosphatase / phosphosdiesterase 3 (E- NPP3) family of enzymes involved in hydrolysis of oligonucleotides and nucleoside phosphates. CD203c is specific to the mast cell/basophil lineage of hematopoietic effector cells.
  • E- NPP3 ectonucleotide pyrophosphatase / phosphosdiesterase 3
  • c-Kit also known as KIT, Cluster of Differentiation 117 (CD117), PBT, SCFR, KIT proto-oncogene receptor tyrosine kinase
  • SCF stem cell factor
  • c-Kit is a receptor tyrosine kinase type III that is highly expressed by hematopoietic stem cells as well as multiple other cell types, such as mature mast cells.
  • conservative substitution refers to replacement of an amino acid with another amino acid which does not significantly deleteriously change the functional activity.
  • a preferred example of a “conservative substitution” is the replacement of one amino acid with another amino acid which has a value > 0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919):
  • HCDR refers to a heavy chain complementarity determining region.
  • LCDR refers to a light chain complementarity determining region.
  • isolated molecule (where the molecule is, for example, a protein, a polypeptide, a polynucleotide, an antibody or an antigen-binding molecule) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature.
  • a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art.
  • Molecule purity or homogeneity may be assayed by several methods known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide. For certain purposes, higher resolution may be provided by using high-performance liquid chromatography (HPLC) or other means known in the art for purification.
  • HPLC high-performance liquid chromatography
  • sequence identity refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of residues, e.g., nucleotides or amino acids.
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical residues which are shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, z.e., the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100.
  • Percentage identity can be calculated using the alignment program Clustal Omega, available at ebi.ac.uk/Tools/msa/clustalo using default parameters. See, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega” (2011 October 11) Molecular Systems Biology 7:539. For the purposes of calculating identity to the sequence, extensions, such as tags, are not included.
  • the terms “treat,” “treating” or “treatment of’ mean that the severity of the subject's condition is reduced, at least partially improved or stabilized and/or that some alleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder.
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the use of the alternative e.g., “or” should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the terms “include” and “comprise” are used synonymously.
  • Embodiment 1 An antibody-drug conjugate comprising an anti-CD203c antibody or an antigen-binding portion thereof linked to a small molecule c-Kit inhibitor.
  • Embodiment 2 The antibody-drug conjugate of embodiment 1, wherein the antibody or antigen-binding portion comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain, wherein:
  • the VH domain amino acid sequence comprises a heavy chain complementarity determining region 1 (HCDR1) comprising SEQ ID NO: 2; a heavy chain complementarity determining region 2 (HCDR2) comprising SEQ ID NO: 3; and a heavy chain complementarity determining region 3 (HCDR3) comprising SEQ ID NO: 4; and the VL domain amino acid sequence comprises a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 6; a light chain complementarity determining region 2 (LCDR2) comprising SEQ ID NO: 7; and a light chain complementarity determining region 3 (LCDR3) comprising SEQ ID NO: 8;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 10; a HCDR2 comprising SEQ ID NO: 11; and a HCDR3 comprising SEQ ID NO: 12; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 14; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 16;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 18; a HCDR2 comprising SEQ ID NO: 19; and a HCDR3 comprising SEQ ID NO: 20; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 22; a LCDR2 comprising SEQ ID NO: 15; and a LCDR3 comprising SEQ ID NO: 23;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 26; and a HCDR3 comprising SEQ ID NO: 27; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 29; a LCDR2 comprising SEQ ID NO: 30; and a LCDR3 comprising SEQ ID NO: 31;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 34; and a HCDR3 comprising SEQ ID NO: 35; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 37; a LCDR2 comprising SEQ ID NO: 38; and a LCDR3 comprising SEQ ID NO: 39;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 41; a HCDR2 comprising SEQ ID NO: 42; and a HCDR3 comprising SEQ ID NO: 43; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 45; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 46;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 48; a HCDR2 comprising SEQ ID NO: 49; and a HCDR3 comprising SEQ ID NO: 50; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 52; a LCDR2 comprising SEQ ID NO: 53; and a LCDR3 comprising SEQ ID NO: 54;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 56; and a HCDR3 comprising SEQ ID NO: 57; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 59; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 60;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 62; a HCDR2 comprising SEQ ID NO: 69; and a HCDR3 comprising SEQ ID NO: 64; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 71; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 67;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 93; a HCDR2 comprising SEQ ID NO: 94; and a HCDR3 comprising SEQ ID NO: 95; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 97; a LCDR2 comprising SEQ ID NO: 98; and a LCDR3 comprising SEQ ID NO: 99;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 101; a HCDR2 comprising SEQ ID NO: 102; and a HCDR3 comprising SEQ ID NO: 103; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 105; a LCDR2 comprising SEQ ID NO: 106; and a LCDR3 comprising SEQ ID NO: 107;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 114; and a LCDR3 comprising SEQ ID NO: 115;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 25; a HCDR2 comprising SEQ ID NO: 73; and a HCDR3 comprising SEQ ID NO: 74; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 6; a LCDR2 comprising SEQ ID NO: 118; and a LCDR3 comprising SEQ ID NO: 119;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 121; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 113; a LCDR2 comprising SEQ ID NO: 123; and a LCDR3 comprising SEQ ID NO: 124;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 126; a HCDR2 comprising SEQ ID NO: 127; and a HCDR3 comprising SEQ ID NO: 128; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 132;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 111; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 135; a LCDR2 comprising SEQ ID NO: 136; and a LCDR3 comprising SEQ ID NO: 137;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 139; a HCDR2 comprising SEQ ID NO: 140; and a HCDR3 comprising SEQ ID NO: 141; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 130; a LCDR2 comprising SEQ ID NO: 131; and a LCDR3 comprising SEQ ID NO: 143;
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 109; a HCDR2 comprising SEQ ID NO: 110; and a HCDR3 comprising SEQ ID NO: 145; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 147; a LCDR2 comprising SEQ ID NO: 148; and a LCDR3 comprising SEQ ID NO: 149; or
  • the VH domain amino acid sequence comprises a HCDR1 comprising SEQ ID NO: 33; a HCDR2 comprising SEQ ID NO: 151; and a HCDR3 comprising SEQ ID NO: 152; and the VL domain amino acid sequence comprises a LCDR1 comprising SEQ ID NO: 154; a LCDR2 comprising SEQ ID NO: 7; and a LCDR3 comprising SEQ ID NO: 83.
  • Embodiment 3 The antibody-drug conjugate of embodiment 2, wherein:
  • VH domain amino acid sequence comprises SEQ ID NO: 1, and the VL domain amino acid sequence comprises SEQ ID NO: 5;
  • VH domain amino acid sequence comprises SEQ ID NO: 9
  • VL domain amino acid sequence comprises SEQ ID NO: 13;
  • VH domain amino acid sequence comprises SEQ ID NO: 17, and the VL domain amino acid sequence comprises SEQ ID NO: 21;
  • VH domain amino acid sequence comprises SEQ ID NO: 24, and the VL domain amino acid sequence comprises SEQ ID NO: 28;
  • VH domain amino acid sequence comprises SEQ ID NO: 32, and the VL domain amino acid sequence comprises SEQ ID NO: 36;
  • VH domain amino acid sequence comprises SEQ ID NO: 40, and the VL domain amino acid sequence comprises SEQ ID NO: 44;
  • VH domain amino acid sequence comprises SEQ ID NO: 47, and the VL domain amino acid sequence comprises SEQ ID NO: 51;
  • VH domain amino acid sequence comprises SEQ ID NO: 55
  • VL domain amino acid sequence comprises SEQ ID NO: 58
  • VH domain amino acid sequence comprises SEQ ID NO: 61
  • VL domain amino acid sequence comprises SEQ ID NO: 65
  • VH domain amino acid sequence comprises SEQ ID NO: 68
  • VL domain amino acid sequence comprises SEQ ID NO: 70
  • VH domain amino acid sequence comprises SEQ ID NO: 72, and the VL domain amino acid sequence comprises SEQ ID NO: 75;
  • VH domain amino acid sequence comprises SEQ ID NO: 78
  • VL domain amino acid sequence comprises SEQ ID NO: 80
  • the VH domain amino acid sequence comprises SEQ ID NO: 84, and the VL domain amino acid sequence comprises SEQ ID NO: 88;
  • VH domain amino acid sequence comprises SEQ ID NO: 92
  • VL domain amino acid sequence comprises SEQ ID NO: 96
  • VH domain amino acid sequence comprises SEQ ID NO: 100
  • VL domain amino acid sequence comprises SEQ ID NO: 104
  • the VH domain amino acid sequence comprises SEQ ID NO: 108, and the VL domain amino acid sequence comprises SEQ ID NO: 112;
  • VH domain amino acid sequence comprises SEQ ID NO: 116
  • VL domain amino acid sequence comprises SEQ ID NO: 117
  • the VH domain amino acid sequence comprises SEQ ID NO: 120, and the VL domain amino acid sequence comprises SEQ ID NO: 122;
  • VH domain amino acid sequence comprises SEQ ID NO: 125
  • VL domain amino acid sequence comprises SEQ ID NO: 129
  • VH domain amino acid sequence comprises SEQ ID NO: 133
  • VL domain amino acid sequence comprises SEQ ID NO: 134
  • VH domain amino acid sequence comprises SEQ ID NO: 138
  • VL domain amino acid sequence comprises SEQ ID NO: 142
  • the VH domain amino acid sequence comprises SEQ ID NO: 144, and the VL domain amino acid sequence comprises SEQ ID NO: 146; or (w) the VH domain amino acid sequence comprises SEQ ID NO: 150, and the VL domain amino acid sequence comprises SEQ ID NO: 153.
  • Embodiment 4 The antibody-drug conjugate of embodiment 1, wherein the antibody or antigen-binding portion is human, humanized, or chimeric.
  • Embodiment 5 The antibody-drug conjugate of any one of embodiments 1-4, wherein the antibody or antigen-binding portion comprises an immunoglobulin constant region.
  • Embodiment 6 The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region is IgG, IgE, IgM, IgD, IgA or IgY.
  • Embodiment 7 The antibody-drug conjugate of embodiment 6, wherein the immunoglobulin constant region is IgGl, IgG2, IgG3, IgG4, IgAl or IgA2.
  • Embodiment 8 The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region comprises one or more mutations to reduce or abrogate immune effector functions of the immunoglobulin constant region.
  • Embodiment 9 The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region is a wild-type human IgG4 constant region, a human IgG4 constant region comprising the amino acid substitution S228P, a wild-type human IgGl constant region, a human IgGl constant region comprising the amino acid substitutions L234A, L235A and G237A or a wild-type human IgG2 constant region, wherein numbering is according to the EU index as in Kabat.
  • Embodiment 10 The antibody-drug conjugate of embodiment 5, wherein the immunoglobulin constant region comprises the amino acid sequence of any one of SEQ ID NOs: 155-160.
  • Embodiment 11 The antibody-drug conjugate of any one of embodiments 1-10, wherein the antibody or antigen-binding portion is an Fab, an Fab', an F(ab')2, an Fv, an scFv, a maxibody, a minibody, a diabody, a triabody, a tetrabody, or a bis-scFv.
  • Embodiment 12 The antibody-drug conjugate of any one of embodiments 1-10, wherein the antibody or antigen-binding portion is tetrameric, tetravalent, or multispecific.
  • Embodiment 13 The antibody-drug conjugate of any one of embodiments 1-12, wherein the c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.
  • the c-Kit inhibitor is dasatinib, anlotinib, imatinib, crenolanib, ponatinib, avapritinib, elenestinib, bezuclastinib, midostaurin, or radotinib.
  • Embodiment 14 The antibody-drug conjugate of any one of embodiments 1-12, wherein the c-Kit inhibitor is dasatinib.
  • Embodiment 15 The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a peptide linker.
  • Embodiment 16 The antibody-drug conjugate of embodiment 15, wherein the peptide linker comprises two amino acid residues independently selected from glycine, alanine, phenylalanine, lysine, arginine, valine, and citrulline.
  • Embodiment 17 The antibody-drug conjugate of embodiment 15, wherein the peptide linker is an alanine-alanine (Ala-Ala) linker.
  • Embodiment 18 The antibody-drug conjugate of embodiment 15, wherein the peptide linker is L-Ala-L-Ala, D-Ala-D-Ala, D-Ala-L-Ala, or L-Ala-D-Ala.
  • Embodiment 19 The antibody-drug conjugate of embodiment 15, wherein the peptide linker is L-Ala-L-Ala.
  • Embodiment 20 The antibody-drug conjugate of any one of embodiments 1-19, wherein the peptide linker is L-Ala-L-Ala; and wherein the antibody-drug conjugate comprises a PEG spacer.
  • Embodiment 21 The antibody-drug conjugate of any one of embodiments 1-14, wherein the antibody or antigen-binding portion comprises the c-Kit inhibitor and a linker comprising the structure:
  • Embodiment 22 The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via an acid cleavable linker, a glutathione (GSH) cleavable linker, a Fe(II) cleavable linker, a cathepsin cleavable linker, a glycosidase cleavable linker, a phosphatase cleavable linker, a sulfatase cleavable linker, a photo-responsive cleavable linker, or a bioorthogonal cleavable linker.
  • GSH glutathione
  • Fe(II) cleavable linker a cathepsin cleavable linker
  • a glycosidase cleavable linker a phosphatase cleavable linker
  • Embodiment 23 The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a hydrazone linker.
  • Embodiment 24 The antibody-drug conjugate of any one of embodiments 1-14, wherein the c-Kit inhibitor is linked to the antibody or antigen-binding portion via a non-cleavable linker.
  • Embodiment 25 The antibody-drug conjugate of any one of embodiments 1-19 and 21-24, further comprising a PEG spacer.
  • Embodiment 26 The antibody-drug conjugate of any one of embodiments 1-25, wherein the drug-to-antibody ratio (DAR) is about 2 to about 5.
  • DAR drug-to-antibody ratio
  • Embodiment 27 The antibody-drug conjugate of any one of embodiments 1-26, wherein the DAR is about 4.
  • Embodiment 28 A pharmaceutical composition comprising the antibodydrug conjugate of any one of embodiments 1-27, and a pharmaceutically acceptable carrier.
  • Embodiment 29 A method for treating a proliferative disorder in a subj ect in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.
  • Embodiment 30 A method for ameliorating a symptom of a proliferative disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.
  • Embodiment 31 The method of embodiment 29 or 30, wherein the proliferative disorder is a mast cell proliferative disorder.
  • Embodiment 32 The method of any one of embodiments 29-31, wherein the proliferative disorder is c-Kit-reliant.
  • Embodiment 33 The method of any one of embodiments 29-32, wherein the proliferative disorder is systemic mastocytosis, mast cell leukemia, or basophil leukemia.
  • Embodiment 34 A method for treating an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28.
  • Embodiment 35 A method for ameliorating a symptom of an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject the antibody-drug conjugate of any one of embodiments 1- 27 or the pharmaceutical composition of embodiment 28.
  • Embodiment 36 The method of embodiment 34 or 35, wherein the inflammatory disorder is a mast cell inflammatory disorder.
  • Embodiment 37 The method of any one of embodiments 34-36, wherein the inflammatory disorder is c-Kit-reliant.
  • Embodiment 38 The method of any one of embodiments 34-37, wherein the inflammatory disorder is a chronic inflammatory disorder.
  • Embodiment 39 The method of any one of embodiments 34-38, wherein the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.
  • the inflammatory disorder is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.
  • Embodiment 40 Use of the antibody-drug conjugate of any one of embodiments 1-27 or the pharmaceutical composition of embodiment 28 in the preparation of a medicament.
  • the disclosure will be further clarified by the following example, which is intended to be purely exemplary of the disclosure and in no way limiting.
  • Mammalian codon-optimized synthetic genes encoding the heavy and light chain variable domains of the anti-CD203c antibodies were cloned into mammalian expression vectors comprising effector function null human IgGl (“IgGlnull”; human IgGl containing L234A, L235A, G237A mutations in the lower hinge that abrogate normal immunoglobulin ADCC, ADCP and CDC functions) and human CK domains, respectively.
  • IgGlnull effector function null human IgGl
  • human IgGlnull human IgGl containing L234A, L235A, G237A mutations in the lower hinge that abrogate normal immunoglobulin ADCC, ADCP and CDC functions
  • Binding and cross-reactivity of the antibodies to the human and rhesus CD203c was initially assessed by binding ELISA.
  • the human CD203c His-tagged recombinant protein and the rhesus monkey CD203c His-tagged recombinant protein were coated to the surface of MaxiSorpTM flat-bottom 96 well plates at a concentration of 1 pg/ml.
  • the purified IgG samples were titrated in 5-fold serial dilutions starting from 100 nM and allowed to bind to the coated antigens.
  • the IgGs were detected using mouse anti-human IgG conjugated to horseradish peroxidase. Binding signals were visualized with 3,3',5,5'-Tetramethylbenzidine Substrate Solution (TMB) and the absorbance measured at 450 nm.
  • TMB 3,3',5,5'-Tetramethylbenzidine Substrate Solution
  • the assay was performed according to manufacturer’ s instructions.
  • KU812 cells were revived and cultured in suspension in RPMI 1640 media supplemented with 10% FBS, 100 units/ml penicillin, 100 pg/ml streptomycin and 80 ng/ml SCF.
  • TF-1 cells were revived and cultured in suspension in RPMI 1640 media supplemented with 10% FBS, 100 units/ml penicillin, 100 pg/ml streptomycin and 2 ng/ml GM-CSF.
  • Targeting antibodies were serially diluted and combined with Fab-ZAP reagent 1 : 1 so that both reagents were at 4x in cell appropriate growth media.
  • Antib ody/Fab -ZAP solutions were incubated for 30 minutes at 37 °C to allow complex formation.
  • Cells were loaded at a final seeding density of 2500 cells/well in appropriate growth media.
  • Fab-ZAP complexes were loaded in an 8-point 4.5-fold serial dilution at final assay concentrations of 0.00013 to 5 nM.
  • Final Fab-ZAP concentrations were 5 nM for TF-1 cells and 10 nM for KU812 cells.
  • Fab-ZAP only, media only and no cell controls were included on every plate.
  • Cells were incubated with complexes for 72 hours (TF-1) or 96 hours (KU812). At the end of the incubation, CellTiterGlo reagent was added to all wells. Plates were incubated for 10 minutes before luminescence signal was measured using a FLUOstar Omega plate reader (BMG Labtec).
  • AC-SINS affinity-capture self-interaction nanoparticle spectroscopy
  • ELISA plates were coated overnight at 4°C with DNA (10 pg/ml) and insulin (5 pg/ml) in PBS pH 7.5. Wells were washed with water, blocked with 50 pl of Polyreactivity ELISA Buffer (PEB; PBS containing 0.05% Tween-20, 1 mM EDTA) for 1 hour at room temperature, and rinsed three times with water. Serially-diluted antibodies were added in quadruplicate to the wells and incubated for 1 h at room temperature. Plates were washed three times with water, and goat anti-human IgG-HRP (Fc specific) was added to each well.
  • PEB Polyreactivity ELISA Buffer
  • KU812 cells were revived and cultured in suspension in RPMH640 media supplemented with 10% FBS, 100 units/ml penicillin, 100 pg/ml streptomycin and 80 ng/ml SCF. Two days prior to the start of the experiment, KU812 cells were cultured in SCF-free media. KU812 cells (passage 23) were resuspended at 4xl0 7 cells/ml. For the main staining protocol, KU812 cells were plated into 96 well plates (50 pl per well). Duplicate wells were prepared for all variables.
  • Cells were stained with anti-LAMP2 (1/50) diluted in BD Perm/Wash for 30 mins at 4°C. Cells were washed once with BD Perm/Wash then once with PBS. Cells were kept at 4°C until analysis. Immediately before analysis, cells were filtered through a 30 pm cell strainer (Celltrics) and transferred to 1.5ml tubes. Cells were analyzed on an Amnis® ImageStream® imaging flow cytometer, and 1000 events collected per sample.
  • the reduction mixture was diluted to 4.4 mg/mL with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool to 22°C.
  • a 5.0 mg/mL (2.21 mM) solution of BTP-Glu-(AA-c-kit inhibitor 1)- PEG(24u) was prepared by dissolving 26 mg (11464 nmol) of linker-payload into 5.2 mL of DMF.
  • a portion of the BTP linker-payload solution (343.8 pL, 758.4 nmol, 6.6 eq) was added to the reduced antibody solution, resulting in a final co-solvent concentration of 10% DMF.
  • the conjugation reaction was allowed to proceed at 22°C for 16 h.
  • HIC loading buffer 50 mM sodium phosphate, 4 M NaCl, pH 7.0
  • the mobile phase buffers used were Buffer A: 50 mM sodium phosphate, 2 M NaCl, pH 7.0
  • Buffer B 50 mM sodium phosphate, 20% isopropanol, pH 7.0.
  • a linear gradient from 0 to 100% buffer B was ran at a flow rate of 2 mL/min over 35 column volumes. Fractions were collected and analyzed by HIC and LC-MS.
  • the reduction mixture was diluted to 4.4 mg/mL with Dulbecco’s PBS, pH 7.5, 5 mM EDTA and allowed to cool to 22°C.
  • a 5.0 mg/mL (2.16 mM) solution of BTP-Glu-(AA-c-kit inhibitor 2)-PEG(24u) was prepared by dissolving 26 mg (11251 nmol) of linker-payload into 5.2 mL of DMF.
  • a portion of the BTP linker-payload solution (309.2 pL, 668 nmol, 6.0 eq) was added to the reduced antibody solution, resulting in a final co-solvent concentration of 10% DMF.
  • the conjugation reaction was allowed to proceed at 22°C for 16 h.
  • HIC loading buffer 50 mM sodium phosphate, 4 M NaCl, pH 7.0
  • the mobile phase buffers used were Buffer A: 50 mM sodium phosphate, 2 M NaCl, pH 7.0
  • Buffer B 50 mM sodium phosphate, 20% isopropanol, pH 7.0.
  • a linear gradient from 0 to 100% buffer B was ran at a flow rate of 2 mL/min over 35 column volumes. Fractions were collected and analyzed by HIC and LC-MS.
  • LC-MS liquid chromatography-mass spectrometry analysis was carried out using a Waters XEVO G2S TOF mass spectrometer and a POROSHELL 300SB C3 column (2.1 x 12.5 mm, 5 pm) connected to a Waters Acquity H Class UPLC system.
  • the mobile phase was buffer A (0.1 % formic acid in water).
  • a gradient (2.5 min 10% B, 10-80% B gradient in 3.5 min) was applied using Buffer B (acetonitrile, 0.1 % formic acid) at a flow rate of 0.4 mL/min.
  • Buffer B acetonitrile, 0.1 % formic acid
  • Average DAR drug-to-antibody ratio
  • SI main glycoform signal intensity
  • MW ADC MW mAb + DAR x MW LP
  • Analytical SEC was carried out using an ACQUITY UPLC BEH 200 SEC column (4.6 mm x 15 cm, 200 A, 1.7 pm) and guard column (4.6 mm x 3 cm), connected to a Dionex Ultimate 3000 UPLC system.
  • the mobile phase was 0.2 M potassium phosphate buffer, pH 6.8, 0.2 M potassium chloride, 15% (v/v) isopropanol.
  • the flow rate was kept constant at 0.35 mL/min.
  • the column was maintained at 30°C throughout the analysis.
  • the analysis was carried out in a 10 min isocratic elution with UV detection at 248, 280 nm and 330 or 365 nm. 10 pg of ADC were injected for analysis.
  • the percentage of high molecular weight (HMW) species was calculated by comparing the peak area corresponding to HMW species at 280 nm to total peak area corresponding to HMW and monomeric species at 280 nm.
  • the percentage of free drug related species was calculated by comparing the peak area corresponding to free drug related species at a wavelength specific for the drug to total peak area corresponding to ADC species at the same wavelength.
  • the concentration of the conjugates was determined by UV absorbance at 280 nm (A280) using a Nanodrop 2000 spectrophotometer. Measurements were taken in triplicate and the average values used for calculations. Molar extinction coefficients used for antibodies and linker-payloads were determined experimentally (Table 25 and Table 26).
  • MW LP linder-payload MW (g.mol -1 )
  • EndoSafe®-PTSTM platform (Charles River) was used to determine the level of endotoxin.
  • EndoSafe®-PTSTM is a chromogenic kinetic test system aligned with USP ⁇ 85> and Pharm Eur 2.6.14 that provides quantitative Limulus Amebocyte Lysate (LAL) results.
  • the EndoSafe®-PTSTM utilizes LAL reagents in FDA-licensed disposable test cartridges which are pre-loaded with all the reagents required to perform a LAL test.
  • EndoSafe®-PTSTM mimics licensed LAL kinetic chromogenic methodology by measuring colour intensity directly related to the endotoxin concentration in a sample; the concentrations are calculated against an internal standard curve (0.01-1.00 EU/mL) associated with the lot number of the cartridges.
  • KU812 cells (passage 23) were seeded at a density of 4.0 x 10 cells per ml in complete phenol red-free RPMI media without SCF in a volume of 50 pl per well in 3 black walled 96-well tissue culture plates. Cells were incubated at 37°C in a humidified atmosphere for 2 h to allow for the SCF starvation to take effect. Test agents were added to cells at concentrations between 0.0015 nM and 100 nM (9-point, 4-fold dilution series). Vehicle (RPMI), DMSO, and cells only wells were included as controls for the assay. Test antibodies were added in a 50 pl volume at a 2X concentration.
  • Cells were incubated at 37°C, 5% CO2 in a humidified atmosphere for 24, 48 or 72 h (one plate per timepoint).
  • CellTox Green assay components were thawed by immersing in a 37°C water bath. Once thawed, each component was vortexed and equilibrated to room temperature.
  • CellTiter-GLO buffer was thawed and equilibrated to room temperature overnight. 30 min prior to each timepoint, the plate was incubated at room temperature. Lysis solution (25X) was added to the cells (4 pl per well). 20 pl CellTox Green dye was added to 2 ml of Assay buffer to form CellTox Green reagent. 20 pl of CellTox Green reagent was added to each well.
  • the plate was then placed on an orbital shaker at 700 rpm for 1 min. The plate was incubated for 15 minutes at room temperature, protected from light. Fluorescence was measured on a FLUOStar plate reader at 485 nm excitation and 520 nm emission with a gain of 1000. 10 ml CellTiter-GLO buffer was used to reconstitute a vial of CellTiter-GLO substrate to form CellTiter-GLO Reagent. 100 pl of CellTiter-GLO reagent was added to each well. The plate was then placed on an orbital shaker at 700 rpm for 10 min to facilitate cell lysis and ATP extraction from cells. Luminescence was measured on a FLUOStar plate reader. CellTox Green and CellTiter-GLO data were expressed as a percentage of untreated control cells (Cells alone).
  • KU812 cells were cultured in SCF-free RPMI +10% FCS for 48 h. Cells were seeded in 24-well plates at 1 x 10 6 /mL in a total volume of 1.5 mL for 1 h, followed by treatment with isotype control or test agents for 2 h. Cells were stimulated with SCF at 40 ng/mL for 5 min, washed in PBS and cell lysates were prepared using lysis buffer according to manufacturer’s instructions (Biotechne). Total and phospho-c-kit levels were detected using 6.25 pg and 50 pg total protein, respectively, using ELISA kits from Biotechne.
  • Rat pharmacokinetics (PK) of naked 2.23 vs 2.23-cKit 2 ADC Test agents were administered to Sprague-Dawley rats at 3 mg/kg via an intravenous route (IC) on Day 0 (3 animals per group), with blood collected at nine time points post-dosing (5 min, 3, 6, 24, 48, 72, 168, 336 and 504 hours). Samples were diluted 1/2000 and antibody levels were quantified using a human IgG ELISA kit (Mabtech) (FIG. 36A). PK parameters shown in the table (FIG. 36B) were calculated using PKsolver.
  • NSG mice were implanted with 5xl0 6 KU812-luciferin cells IV via the lateral tail vein. Tumors were allowed to progress to exponential growth phase and dosing with PBS control, 2, 10 & 50 mg/kg 2.23-cKit 2 ADC (10 mice/group, Q4D IP for 4 weeks) or 10 mg/kg dasatinib (QD PO 5 days on, 2 days off for 4 weeks) was initiated in therapeutic mode on Day 25. Tumor growth was monitored by bioluminescent imaging twice a week from Day 7 and is reported in log scale.
  • Test agents were administered to adult male cynomolgus monkeys at 10 or 30 mg/kg (3 animals/group) via an intravenous route (IV) on Day 0 and again on Day 21 with necropsy at Day 42. Blood was collected at multiple time points post-dosing (for Day 0 dose: pre-dose and 0.5, 2, 6, 24, 48, 72, 168, 556 & 504 h post-dose; Day 21 samples were taken at the same time points post-dose). Antibody levels in serum were quantified using a human IgG bioanalytical ELISA method. The data were subjected to a non-compartmental pharmacokinetic analysis using the Phoenix WinNonlin software (version 8.1, Pharsight, Mountain View, CA).
  • Test agents were administered to adult male cynomolgus monkeys at 10 or 30 mg/kg (3 animals/group) via an intravenous route (IV) on Day 0 and again on Day 21 with necropsy at Day 42. Blood was collected at multiple time points - Day 0 pre-dose, Day 7, Day 14, Day 21 (pre-dose), Day 28 & Day 42 prior to terminal necropsy. At each time point, 1 mL of whole blood was collected into a Potassium -EDT A tube and mixed. Samples were kept on ice and subjected to complete blood count tests on the same day as collection. This panel included white blood cells, red blood cells, hemoglobin, platelets, neutrophils, lymphocytes, monocytes, eosinophils, and basophils.
  • a direct filter-binding radiometric kinase activity assay (Eurofins) was used to measure the potency of cKit2 free payload in inhibiting a number of tyrosine kinases including Bruton’s Tyrosine Kinase (BTK; FIG. 42A), wild-type c-Kit (FIG. 42B) and variant c-Kit (D816V; FIG. 42C). Each test agent was assayed across a concentration range from 10 mM to 1 nM in triplicate. Calculated IC50 values are summarized in FIG. 42D.
  • a KINOMEscan competition assay (Eurofins) was used to assess the selectivity of the cKit2 free payload compared to other benchmark tyrosine kinase inhibitors. This assay format measures the ability of test agent to complete with an immobilized active site directed ligand. Each test agent was assayed at 12 concentrations ranging from 1 mM to 0.1 nM and dose response curves were used to determine relative binding Kd values.
  • Anti-CD203c antibodies (described in Tables 1-23) were readily expressed and purified from CHO cells using Protein-A affinity chromatography. Antibodies are referred to by the number in the table title providing the sequence of that antibody (2.1 through 2.23). Analytical SEC demonstrated that all clones were >95% monomeric and preparations had a low endotoxin content ⁇ 1 EU/mg. [0262] All 23 purified IgGs were initially tested for binding to His-tagged recombinant versions of human and rhesus CD203c (FIG. 3) and all clones (18) that demonstrated measurable binding to both orthologs were brought forward for binding analysis using flow cytometry on KU812 cells, which endogenously express human CD203c.
  • the AC-SINS assay was used to look at propensity of each IgG to self-interact compared to the well-understood comparators Alirocumab, which is well behaved, and Bococizumab, which displays high aggregation via self-association.
  • antibodies were assessed for binding to DNA and insulin, which are highly negatively charged molecules and predict polyreactivity and potential for off-target binding.
  • Bevacizumab and Ustekinumab were used as well-behaved comparators and Bococizumab and Briakinumab as benchmarks for antibodies with known polyreactivity.
  • FIG. 8A and FIG. 8B illustrate the linker-payload design elements.
  • a bis-thiol alkylating reagent (BTP) is first conjugated via a stable amino acid linker to dasatinib and ultimately links to both cysteine thiols from the reduced disulfide of 2.23 resulting in a controlled and homogenous DAR of 4.
  • Incorporation of a hydrophilic 24u PEG spacer aids in conjugate solubility, linker masking and half-life extension.
  • Two versions of the 2.23 conjugate are shown cKitl (FIG. 8A) and cKit2 (FIG. 8B), which has a slightly longer amino acid linker.
  • the cKit2 version has an additional amino ethyl group between the piperazine and the alanine moieties.
  • the piperazine therefore no longer forms the amide bond directly with the alanine amino acid (as it does in the cKitl version), but the amino group within the amino ethyl moiety does this instead.
  • FIG. 9 - FIG. 11 describe the synthesis and analytical characterization of the BTP-cKitl-PEG linker payload by RPLC and LC/MS.
  • the RPLC analysis shows a single peak with LC/MS confirming the expected mass. This is also true for BTP-cKit2- PEG, the data for which is summarized in FIG. 12 - FIG. 14.
  • FIG. 15 shows the Yi mAb analysis by LC/MS confirming a DAR of 3.9 for 2.23-cKitl ADC.
  • FIG. 16A estimates an average DAR of 3.9 by HIC, and a single peak corresponding to >99.1% monomer by SEC (FIG. 16B). The same data was generated for 2.23-cKit2 ADC, summarized in FIG. 17 and FIG. 18A - FIG. 18B. A summary for both ADC molecules is presented in Table format in FIG. 19 (2.23-cKitl-ADC) and FIG. 20 (2.23-cKit2-ADC).
  • a series of modified cKit2 linker-payloads were synthesized to explore the impact of using unnatural amino acids in the linker (shown in FIG. 27A). Instead of the parental L-Ala-L-Ala linker, D-Ala-D-Ala, D-Ala-L-Ala and L-Ala-D-Ala variants were compared to the parental and its cleavage products, cKit2-dAla (with one Ala cleaved from the linker) and cKit2-dAlaAla (with both Ala now cleaved from the linker to release the free cKit2 payload) in viability and cytoxicity assays in KU812 cells (FIG. 27C and FIG. 27D, respectively). The comparative potencies are described in FIG. 27B and show that incorporation of unnatural amino acids into the linker have a significant negative impact on potency.
  • a number of alternative tyrosine kinase inhibitors were tested as potential payloads for the CD203c-ADC. These were all synthesized as free payload, a dAla version (with a single L-Ala linker) and an L-Ala-L-Ala version to mimic the parental cKit2 molecule and its respective cleavage products. These potential payload variants were subsequently compared to cKit2dAlaAla in cytoxicity and viability assays at 24h, 48h and 72h time points. Anlotinib variants (FIG. 28A) had no potency in KU812 cell killing (FIG. 28B) compared to cKit2dAlaAla.
  • Crenolanib (FIG. 30), Avapritinib (Fig 32), Elenestinib (FIG. 33) and Midostaurin (FIG. 35).
  • Imatinib (FIG. 29) showed some potency at the highest concentration tested, 100 nM, and this became apparent after 48h of treatment.
  • Ponatinib free payload (FIG. 31) showed almost equivalent potency to cKit2, with Bezuclastinib (FIG. 34) showing minimal toxicity at concentrations > 100 nM.
  • Rats were administered with a single dose of 3 mg/kg 2.23 antibody or 2.23- cKit2 ADC to explore the impact of payload conjugation on the antibody PK.
  • a standard human IgG ELISA was used to measure antibody levels in blood samples taken from rats at a series of time points post-administration. Analysis of PK samples shows a two-phase decay of both antibody and ADC over time, with half lives of 326 and 285 hours, respectively (FIG. 36A and FIG. 36B).
  • ADCs often suffer with poor solubility and hydrophobicity which can have negative impacts on subsequent biodistribution and PK.
  • 2.23-cKit2 ADC behaves very similarly to the naked antibody, suggesting a well-behaved molecule with a standard half-life.
  • the 2.23-cKit2 ADC is well tolerated in mice [0275]
  • the 2.23-cKit2 ADC was dosed in non-tumor bearing NSG mice every 4 days for a total of 6 doses at 3 and 10 mg/kg to explore any potential safety concerns. Animals were monitored for body weight before dosing and body weight compared over time after dosing for the duration of the 28-day study.
  • FIG. 37 shows that there was no impact on body weight at either dose with animals feeding normally and experiencing no weight loss. There was no impact on behavior over the course of the study, and no overt clinical signs. No follow up histopathological analysis was required. 10 mg/kg dosing was selected as a safe dose for the subsequent KU812 in vivo efficacy model.
  • a luciferin variant of the KU812 cell line was generated to enable the use of bioluminescent imaging (BLI) to dynamically monitor tumor growth in vivo.
  • NSG mice were implanted with KU812 cells and animals monitored until a stage of exponential tumor growth was reached.
  • therapeutic dosing with 2.23-cKit2 ADC was initiated (Day 19) at 10 mg/kg, with dosing repeated every 4 days over a 4-week period.
  • BLI measurements were taken twice-weekly over the course of the study and data is summarized in FIG 38.
  • 2.23-cKit2 ADC significantly ablates tumor growth compared to the PBS control (FIG. 38A), despite the fact that dosing was only initiated after tumors were already established and in exponential growth phase (FIG. 38B).
  • a luciferin variant of the KU812 cell line was generated to enable the use of bioluminescent imaging (BLI) to dynamically monitor tumor growth in vivo.
  • NSG mice were implanted with KU812 cells and animals monitored until a stage of exponential tumor growth was reached.
  • therapeutic dosing with 2.23-cKit2 ADC was initiated (Day 25) across a range of concentrations from 2-50 mg/kg, with dosing repeated every 4 days over a 4-week period.
  • BLI measurements were taken twice- weekly over the course of the study, and data are summarized in FIG. 39.
  • 2.23-cKit2 ADC significantly ablates tumor growth compared to the PBS control in a dosedependent manner, with 98% inhibition observed at the highest dose of 50 mg/kg compared to 96% at 10 mg/kg and 83% at 2 mg/kg by study end.
  • 2.23-cKit2 ADC shows similar potency to the benchmark small molecule dasatinib, which was also included in the study (10 mg/kg daily dosing by the oral route).
  • 2.23-cKit2 ADC demonstrates standard antibody-like PK in cynomolgus monkeys [0278]
  • the 2.23-cKit2 ADC was dosed in cynomolgus monkeys at 10 or 30 mg/kg on Days 0 & 21 with monitoring to study end at Day 42.
  • the serum antibody concentrations are shown in FIG. 40.
  • the antibody has a half-life of 8-11 days with no evidence of TMDD and a Ctrough of > 20 pg/mL, providing strong support for an infrequent dosing regimen in patients.
  • C-kit targeting antibodies and small molecules suffer from hematological adverse events due to the broad global expression of c-Kit across a number of cell types, including hematopoietic stem cells (HSCs) and myeloid/erythroid precursors.
  • 2.23- cKit ADC aims to avoid these off-target cell events through selective delivery of the tyrosine kinase inhibitor to activated mast cells, avoiding HSCs.
  • 2.23-cKit2 ADC had no significant impact on blood parameters after 6- week dosing at 10 and 30 mg/kg in cynomolgus monkeys. Blood samples taken from dosed animals across multiple time points were subjected to complete blood cell counts and the results are summarized in FIG. 41A - FIG. 411. All counts remained in the normal range with no significant or dose-dependent decreases observed over time.
  • cKit2 free payload exhibits potent inhibition across multiple tyrosine kinases
  • the potency of the cKit2 free payload was measured using a radiometric kinase activity assay across a broad concentration range (1 nM - 10 pM). While the cKit2 payload effectively inhibited the kinase activity of wild-type cKit (270 nM), it showed higher potency against the D816V variant form (71 nM). This mutant form of c-Kit is particularly prevalent in patients with systemic mastocytosis. In addition, cKit2 also potently inhibited Bruton’ s Tyrosine Kinase (4 nM), which is critically involved in mast cell activation and signaling. Representative dose-response inhibition curves are shown in FIG.
  • FIG. 42A 42A - FIG. 42C with associated IC50 values summarized in FIG. 42D.
  • a high throughput competition assay format was also used to compare cKit2 potency against c-Kit D816V to other well-described c-Kit inhibitors which are currently in clinical use or clinical testing, including avapritinib and bezuclastinib.
  • cKit2 potency against the disease relevant D816V compared favorably to benchmarks and was the only inhibitor tested that demonstrated dual activity against c-Kit and BTK, which are both critical for mast cell function and represent validated clinical targets for mast cell modulation.
  • cKit2 had similar sub-nM potency to Remibrutinib in binding to BTK, in this assay format. Comparative Kd values are summarized in FIG. 43. Combined with the data in FIG. 42A - FIG. 42D, these observations suggest that cKit2 has the potential to mediate multi-pathway inhibition of mast cell activation.

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Abstract

L'invention concerne des anticorps anti-CD203c et des parties de liaison à l'antigène liées à des inhibiteurs de c-Kit. De tels conjugués anticorps-médicament peuvent être utilisés pour le ciblage spécifique de mastocytes et/ou de basophiles dans des troubles prolifératifs et inflammatoires.
PCT/EP2024/073759 2023-08-23 2024-08-23 Conjugués d'anticorps anti-cd203c et leurs utilisations Pending WO2025040820A1 (fr)

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