[go: up one dir, main page]

WO2025047993A1 - Conjugués anticorps-médicament anti-doppel - Google Patents

Conjugués anticorps-médicament anti-doppel Download PDF

Info

Publication number
WO2025047993A1
WO2025047993A1 PCT/KR2023/012720 KR2023012720W WO2025047993A1 WO 2025047993 A1 WO2025047993 A1 WO 2025047993A1 KR 2023012720 W KR2023012720 W KR 2023012720W WO 2025047993 A1 WO2025047993 A1 WO 2025047993A1
Authority
WO
WIPO (PCT)
Prior art keywords
doppel
seq
adc
antibody
mmae
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2023/012720
Other languages
English (en)
Inventor
Youngro Byun
Ha Kyeong Lee
Seungwoo CHUNG
Byoung Mo Kim
So-Young Choi
Se-Ra Lee
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pharosgen Co Ltd
Original Assignee
Pharosgen Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pharosgen Co Ltd filed Critical Pharosgen Co Ltd
Priority to PCT/KR2023/012720 priority Critical patent/WO2025047993A1/fr
Priority to KR1020247029994A priority patent/KR20250034273A/ko
Publication of WO2025047993A1 publication Critical patent/WO2025047993A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68037—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a camptothecin [CPT] or derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6843—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 material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A61P31/06—Antibacterial agents for tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • anti-doppel antibody-drug conjugates Described are anti-doppel antibody-drug conjugates, compositions comprising them, and related methods of treating doppel-associated diseases and conditions, including cancer.
  • ADCs Antibody-drug conjugates
  • ADCs may comprise a potent cytotoxic drug and a target-specific antibody (e.g., an antibody that selectively binds a target-specific biomarker, such as a cancer-specific biomarker).
  • ADCs may be described as a multistage rocket that consist of parts within parts that bring the payload (the cytotoxic drug) closer to its target (cancer cells).
  • the primary targeting moiety of an ADC is the antibody. Once the antibody reaches and binds its target binding partner, effectively binding the ADC to the target binding partner, the ADC must be able to be internalized into the cell to exert its intended effect.
  • the cytotoxic drug must have a sufficient cytotoxicity to neutralize target cells in doses capable of delivery by the ADC.
  • ADCs The concept behind ADCs is deceptively simple, but it has proven extremely difficult to achieve sufficient efficacy of the approach in vivo .
  • ADC-based therapeutics still face challenges such as in vivo toxicity, suboptimal target biomarkers, unpredictable clinical value in combination therapies, and poorly understood pathways of drug resistance.
  • Cancer is no longer considered a single disease. Because a cancer may be the result of accumulated mutations, a single cancer may be divided into multiple subtypes, each with its own set of identifying mutations. Conversely, tumors from completely different parts of the body can have similar mutations and respond to the same medications. See, e.g., Sun et al., Bioconjugate Chem. 2020, 31 (4): 1012-1024. Therefore, it has been increasingly important in ADC-based chemotherapies to identify and quantify tumor-specific antigens for use as target-specific biomarkers.
  • tumor-specific and tumor-associated antigens to be safely and effectively used in ADC biomarkers should meet several requirements, such as being highly expressed on the tumor surface, conducive to internalization of the ADC, and having limited expression on normal tissues (to prevent off-target toxicities).
  • tumor-associated antigens for solid tumors such as HER2, TROP2 and nectin 4 have been used to develop monoclonal antibodies for therapeutic and chemotherapeutic use, these tumor-associated antigens also are expressed in large amounts in normal tissues, which can lead to undesired targeting of healthy tissue (off-target toxicity).
  • ADCs that selectively bind to their target (e.g., tumor cells), and do not harm non-target cells (e.g., normal tissue) but are effective against their target cells (e.g., tumor cells).
  • ADCs anti-doppel antibody drug conjugates
  • ADCs comprising: (i) a doppel-targeting moiety, (ii) a cleavable linker, and (iii) a therapeutic agent.
  • anti-doppel ADCs comprising: (i) a doppel-targeting moiety, joined directly or through a linker to (ii) a cleavable linker, joined directly or through a linker to (iii) a therapeutic agent.
  • the doppel-targeting moiety is selected from a doppel-binding monoclonal antibody, a doppel-binding polyclonal antibody, a doppel-binding single chain antibody, a doppel-binding chimeric antibody, a doppel-binding humanized antibody, a doppel-binding veneered antibody, and doppel-binding fragments of any thereof.
  • the doppel-targeting moiety is a doppel-binding antibody selected from human monoclonal antibody A12 disclosed herein; human monoclonal antibody B2 disclosed herein; human monoclonal antibody E9 disclosed herein; human monoclonal antibody 3D5 disclosed herein; human monoclonal antibody 3D1 disclosed herein; human monoclonal antibody 4D1 disclosed herein; human monoclonal antibody 3H9 disclosed herein, and doppel-binding fragments of any thereof.
  • the cleavable linker is cleavable by an intracellular protease.
  • the cleavable linker is selected from a dipeptide cleavable linker such as valine-citrulline, valine-alanine, and phenylalanine-lysine; a hydrazone linker hydrolyzed at a pH of less than 5.5, and a disulfide linker cleaved in a reduced environment.
  • the cleavable peptide linker is a caspase-cleavable peptide linker.
  • the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Xaa-Xaa-Asp, Leu-Xaa-Xaa-Asp, and Val-Xaa-Xaa-Asp, where Xaa represents any amino acid residue.
  • the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Glu-Val-Asp (SEQ ID NO:4), Asp-Leu-Val-Asp (SEQ ID NO:5) Asp-Glu-Ile-Asp (SEQ ID NO:6), and Leu-Glu-His-Asp (SEQ ID NO:7).
  • the six C-terminal amino acid residues of the caspase-cleavable peptide linker consist of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO:8).
  • the therapeutic agent comprises a chemotherapeutic agent. In some aspects, the therapeutic agent comprises a chemotherapeutic agent that induces apoptosis of tumor cells. In some aspects the therapeutic agent is selected from 5-FU, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, exatecan, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, COX-2 inhibitors, irinotecan, SN-38, cladribine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib
  • the chemotherapeutic agent is selected from anthracyclines, antibiotics, alkylating agents, platinum-based agents, antimetabolites, topoisomerase inhibitors, and mitotic inhibitors.
  • the chemotherapeutic agent is selected from the group consisting of doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, and derivatives thereof.
  • the chemotherapeutic agent is selected from actinomycin-D, bleomycin, mitomycin-C, calicheamicin, and derivatives thereof.
  • the chemotherapeutic agent is selected from cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, 5-fluorouracil, 6-mercaptopurine, capecitabine, cladribine, clofarabine, cystarbine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, pentostatin, thioguanine, exatecan, camptothecin, topotecan, irinotecan, etoposide,
  • the therapeutic agent comprises an immunomodulatory agent.
  • the immunomodulatory agent is selected from cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interrerons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF-alpha, TGF-beta, insulin-like growth factor (IGF), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), TNF-alpha, TNF-beta, mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin,
  • TGF
  • the therapeutic agent comprises a toxin.
  • the toxin is selected from ricin, abrin, ribonuclease (RNase), DNase 1, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • the therapeutic agent comprises a radionuclide.
  • radionuclide is selected from the 11C, 13N, 15O, 32P, 33P, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 62Cu, 67Cu, 67Ga, 75Br, 75Se, 76Br, 77As, 77Br, 80mBr, 89Sr, 90Y, 95Ru, 97Ru, 99Mo, 99mTc, 103mRh, 103Ru, 105Rh, 105Ru, 107Hg, 109Pd, 109Pt, 111Ag, 111In, 113mIn, 119Sb, 121mTe, 122mTe, 125I, 125mTe, 126I, 131I, 133I, 142Pr, 143Pr, 149Pm, 152Dy, 153Sm, 161Ho, 161Tb, 165Tm
  • the therapeutic agent is an DNA cross-linking agent, such as one or more selected from indolionobenzodiazepine dimer (IGN), pyrrolobenzodiazepine (PBD), and derivatives thereof.
  • IGN indolionobenzodiazepine dimer
  • PBD pyrrolobenzodiazepine
  • the doppel-targeting moiety is a doppel-targeting antibody
  • the linker is a caspase-cleavable peptide linker having an amino acid sequence consisting of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO:8)
  • the chemotherapeutic agent is MMAE.
  • the doppel-targeting moiety is human monoclonal antibody 3H9
  • the linker is a caspase-cleavable peptide linker having an amino acid sequence consisting of Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO:8)
  • the chemotherapeutic agent is MMAE.
  • compositions comprising an ADC as described herein, and a pharmaceutically acceptable carrier.
  • the composition is formulated for intravenous administration.
  • the doppel-associated disease or condition is selected from asthma, tuberculosis, atherosclerosis, and pulmonary arterial hypertension (PAH).
  • the doppel-associated disease or condition is cancer.
  • the cells of the cancer express doppel.
  • kits comprising an ADC as described herein contained in a suitable container, and optionally further comprising instructions for use.
  • FIG. 2 illustrates the internalization of 3H9 monoclonal antibody (mAb) into doppel-expressing cells (HCTEC) observed by confocal microscopy.
  • mAb monoclonal antibody
  • HCTEC doppel-expressing cells
  • FIG. 3 illustrates the lysosomal colocalization of 3H9 mAb in doppel-expressing cells (HCTEC) observed by confocal microscopy.
  • FIG. 4 illustrates the in vivo efficacy of anti-doppel ADCs as described herein (3D1-vc-MMAE; 3D5-vc-MMAE; 3H9-vc-MMAE; and 4D1-vc-MMAE).
  • FIG. 5 illustrates the dose-dependency of anti-doppel ADCs as described herein (3D1-vc-MMAE; 3D5-vc-MMAE; and 3H9-vc-MMAE).
  • tumor cell(s) refers to cells of any type of tumor tissue, benign or malignant.
  • cancer refers to cancer originating from any part of the body or any cell type. This includes, but is not limited to, carcinoma, sarcoma, lymphoma, germ cell tumors, and blastoma. The cancer may be associated with a specific location in the body or a specific disease.
  • carcinoma a cell that is associated with a specific location in the body or a specific disease.
  • sarcoma a cell that is associated with a specific location in the body or a specific disease.
  • tumor generally are used interchangeably.
  • cell includes cells in animals (e.g., in vivo cells) and cultured cells.
  • doppel refers to any doppel protein, including doppel protein of mouse or human origin, including doppel protein in glycosylated or aglycosylated (not glycosylated) form, including doppel in monomeric or dimeric form.
  • doppel refers to any doppel protein, including doppel protein of mouse or human origin, including doppel protein in glycosylated or aglycosylated (not glycosylated) form, including doppel in monomeric or dimeric form.
  • the terms “doppel,” “PRND,” and “doppel protein” generally are used interchangeably herein.
  • the term "subject" refers to any animal in need of treatment by any one or more of the methods described herein, including humans and other mammals, such as dogs, cats, rabbit, horses, and cows.
  • a subject may be suffering from or at risk of developing a disease or condition associated with doppel, including a doppel-associated cancer.
  • the subject is a human diagnosed with a doppel-associated tumor or a doppel-associated cancer.
  • the phrase “disease or condition associated with doppel” refers to a disease or condition in which the subject's endothelial cells express doppel, including asthma, tuberculosis, atherosclerosis, pulmonary arterial hypertension (PAH), and neoplasms and neoplasm-related conditions, including doppel-associated cancers and doppel-associated tumors.
  • the terms "doppel-associated” tumor and “doppel-associated” cancer refer to a tumor or cancer, the cells of which express doppel.
  • ADCs anti-doppel antibody-drug conjugates
  • ADCs comprising: (i) a doppel-targeting moiety, (ii) a cleavable linker, and (iii) a therapeutic agent.
  • nomenclature such as [antibody name]-cleavable linker-[therapeutic agent name] is used to designate ADCs comprising the named antibody, cleavable linker, and therapeutic agent.
  • the other moieties may be present, such as one or more other chemical linking agents, as discussed in more detail below and illustrated in the examples.
  • doppel is a tumor endothelial cell (TEC) surface marker that plays a role in pathological angiogenesis, and that inhibiting doppel angiogenetic activity, such as by inhibiting the interaction of doppel with a tyrosine kinase receptor (e.g.
  • TEC tumor endothelial cell
  • VEGFR2 VEGFR2
  • doppel expression on endothelial cells may be increased during pathological angiogenesis relative to its expression during normal or physiological angiogenesis conditions.
  • doppel expression on TECs may be associated with pathological tumor-associated angiogenesis or tumorigenesis.
  • Doppel is a prion-like protein encoded by a gene, PRND, which is located near the PRNP (Prion protein coding gene) locus.
  • PRNP Primary protein coding gene
  • Full-length human doppel is a 179 amino acid residue protein (UniProtKB_Q9UKY0; NCBI Ref._NP_036541.2) with a molecular weight of 14 kDa for the non-glycosylated form.
  • Doppel undergoes a C-terminal glycosylphosphatidylinositol (GPI) modification and is expressed on the cell surface anchored to a lipid raft by GPI.
  • GPI glycosylphosphatidylinositol
  • Doppel is transiently expressed in the brain endothelium of neonates, but in adults, it is expressed only in testicular cells.
  • PRND is nearly nonexistent outside the gonads in normal human adults.
  • anti-doppel ADCs as described herein offer a highly target-specific approach to ADC targeting that is not possible with other tumor biomarkers. Therefore, the methods described herein will have limited off-target toxicities, offering a significant improvement over current approaches.
  • doppel is associated with various diseases and conditions for which safe and effective therapies are still needed.
  • doppel has been implicated in neurodegeneration and angiogenesis.
  • doppel is highly expressed in tumors. The most extensive study on the relationship between malignant tissue and doppel expression was done by Comincini et al. (Anticancer Research, 2004, 24:1507-1518) in astrocytoma, who studied PRND expression in glioblastoma-derived cell lines and non-glial tumor specimens, and found PRND expression to be directly related to tumor malignancy.
  • doppel as a potential therapeutic target for tumor angiogenesis, and found doppel expression in tumor endothelial cells (TECs), but not normal endothelial cells (ECs), and reported that blocking doppel could potentially selectively inhibit tumor angiogenesis.
  • TECs tumor endothelial cells
  • ECs normal endothelial cells
  • the localization of doppel on the surface of TECs make it a superior target for ADC compared to targets on the tumor itself for several reasons.
  • TECs are localized in the blood vessel and constitute the first cell layer encountered by therapeutics administered intravenously.
  • anti-doppel ADCs are, in effect, vascular-targeting ADCs (VT-ADCs).
  • VT-ADCs vascular-targeting ADCs
  • One advantage of VT-ADCs over ADCs that target other biomarkers is the proximity of the target to the blood vessel.
  • the chemotherapeutic agent has to overcome multiple barriers in order to reach its target. For example, even before it reaches the cell, it has to pass through the interstitial exocellular matrix (ECM) of the tumor, which presents diverse barriers to the chemotherapeutic agent.
  • ECMs are hypertensive by nature due to leaky vessels and blocked lymph nodes.
  • This interstitial hypertension suppresses extravasation and intestinal transport of macromolecules. Further complicating target localization is the dense network of collagen fiber saturating the ECM, as well as the increased distance between the tumor cells. In this regard, the relative volume of interstitial space is about 3-5 time larger than normal cells, extending the length the chemotherapeutic agent has to travel. (Kratz, F. et al., Drug delivery in oncology, (2011) John Wiley & Sons, Ltd. pp. 40-44) Furthermore, even if the chemotherapeutic agent reaches the center of the tumor, its efficacy may be limited by the nature of its design. Most chemotherapeutic agents target the rapid proliferation of tumor cells.
  • anti-doppel ADCs as described herein can be effective against the majority of the tumor mass (e.g., including tumor cells distant from blood vessels) due to loss of vasculature induced by the chemotherapeutic agent carried by the anti-doppel ADCs described herein.
  • TECs are of normal hematopoietic origins and are less likely to undergo somatic mutation.
  • doppel is not only selectively expressed on tumor cells but also on endothelial cells under pathological conditions, such as atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH).
  • pathological conditions such as atherosclerosis, tuberculosis, asthma, and pulmonary arterial hypertension (PAH).
  • PAH pulmonary arterial hypertension
  • the anti-doppel ADCs described herein comprise a doppel-targeting moiety.
  • the doppel-targeting moiety is an anti-doppel antibody that binds doppel, or a related species, such as a doppel-binding antibody fragment, including, but not limited to, an antibody fragment or peptide that binds to doppel.
  • the binding inhibits interaction of doppel with a tyrosine kinase receptor, such as one or more of VEGFR2, VEGFR1, VEGFR3, bFGFR, and PDGFR.
  • the doppel-targeting moiety binds to doppel and thereby inhibits the interaction of doppel with VEGFR2.
  • the anti-doppel antibody is directed against the extracellular domain (ECD) of doppel.
  • antibody or "anti-doppel antibody” as used herein includes a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody (see, e.g., Jones et al., Nature 321 (1986), 522-525; Riechmann et al., Nature 332 (1988), 323-329; Presta, Curr. Op. Struct. Biol. 2 (1992), 593-596), a chimeric antibody (see, e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A.
  • a human antibody a fully humanized antibody (see, e.g., Tomizuka et al., Nature Genetics (1997) 16, 133-143; Kuroiwa, et al., Nucl. Acids Res. (1998) 26, 3447-3448; Yoshida, H. et al., Animal Cell Technology: Basic and Applied Aspects Vol. 10, 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.) (Kluwer Academic Publishers, 1999); Tomizuka, et al., Proc. Natl. Acad. Sci. USA (2000) 97, 722-727, WO 2007/077028), a multispecific antibody (e.g., a bispecific antibody) formed from at least two antibodies, or an antibody fragment of any thereof.
  • a multispecific antibody e.g., a bispecific antibody formed from at least two antibodies, or an antibody fragment of any thereof.
  • doppel-binding antibody fragment refers to any portion of the afore-mentioned types antibodies capable of binding to doppel, typically comprising an antigen-binding region or variable regions.
  • antibody fragments include Fab fragments, Fab ' fragments, F(ab')2 fragments, Fv fragments, diabodies (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. U.S.A.
  • an antibody fragment useful as a doppel-targeting moiety of the ADCs described herein may comprise a portion of a full-length antibody, such as its antigen-binding domain or variable region domain.
  • Suitable antibody fragments include Fab, F(ab')2, Fv, and single-chain Fv (scFv) constructs (in which Fv fragments from the heavy and light chains are ligated by an appropriate linker) (see, e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883 (1988); diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL) by a linker.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the linker is too short to allow pairing between the two domains on the same chain so that the domains are forced to pair with complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404 097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993).
  • antibody or "anti-doppel antibody” as used herein may include antibody-like molecules that contain engineered sub-domains of antibodies or naturally occurring antibody variants.
  • These antibody-like molecules may be single-domain antibodies such as VH-only or VL-only domains derived either from natural sources such as camelids (see, e.g., Muyldermans et al., J. Biotech. 2001, 74, 277-302) or through in vitro display of libraries from humans, camelids or other species (see, e.g., Holt et al., Trends Biotechnol., 2003, 21, 484-90).
  • monoclonal antibody refers to an antibody obtained by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include multiple antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant (epitope) on the antigen.
  • the doppel-targeting moiety of an ADC as described herein may be any antibody or antibody-like molecule, including, but not limited to, a polyclonal antibody or a monoclonal antibody, or a derivative of an antibody, such as a single chain antibody, a chimeric antibody, a humanized antibody (or other species-ized antibody modified for use in another target species), a veneered antibody, etc.
  • the antibody may be glycosylated or aglycosylated, or have a modified glycosylation pattern.
  • Doppel-targeting moieties suitable for use in the ADCs and methods described herein include the doppel-targeting molecules described in U.S. Patent Application 17/350,763 filed June 17, 2021, the entire contents of which are incorporated herein by reference.
  • An immunoglobulin monomer comprises two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly bound via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the isotype of the antibody) and a variable region.
  • the variable region comprises three hypervariable regions (or complementarity determining regions) which are designated CDRH1, CDRH2 and CDRH3 and which are supported within framework regions.
  • Each light chain comprises a constant region and a variable region, with the variable region comprising three hypervariable regions (designated CDRL1, CDRL2 and CDRL3) supported by framework regions in an analogous manner to the variable region of the heavy chain.
  • the hypervariable regions of each pair of heavy and light chains mutually cooperate to provide an antigen binding site that is capable of binding a target antigen.
  • the binding specificity of a pair of heavy and light chains is defined by the sequence of their respective CDRs.
  • the doppel-targeting moiety is an anti-doppel antibody.
  • the doppel-targeting moiety is a human monoclonal antibody selected from human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, human monoclonal antibody 3H9, and doppel-binding fragments of any thereof.
  • the doppel-targeting moiety has an amino acid sequence that is at least 85%, 90%, 95%, or 99% identical to monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, human monoclonal antibody 3H9.
  • the doppel-targeting moiety is an anti-doppel antibody having CDR sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the CDRs of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • the doppel-targeting moiety is an anti-doppel antibody having heavy chain sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the heavy chain sequence of human monoclonal antibody A12 (SEQ ID NO:9), heavy chain sequence of human monoclonal antibody B2 (SEQ ID NO:10), heavy chain sequence of human monoclonal antibody E9 (SEQ ID NO:11), heavy chain sequence of human monoclonal antibody 3D5 (SEQ ID NO:12), heavy chain sequence of human monoclonal antibody 3D1 (SEQ ID NO:13), heavy chain sequence of human monoclonal antibody 4D1 (SEQ ID NO:14), or human monoclonal antibody 3H9 (SEQ ID NO:15).
  • the doppel-targeting moiety is an anti-doppel antibody having heavy chain variable domain sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the heavy chain variable domain sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • the heavy chain variable region comprises a CDRH1 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRH1 sequence of any one of human monoclonal antibody A12 CDRH1 (SEQ ID NO:16), human monoclonal antibody B2 CDRH1 (SEQ ID NO:17), human monoclonal antibody E9 CDRH1 (SEQ ID NO:18), human monoclonal antibody 3D5 CDRH1 (SEQ ID NO:19), human monoclonal antibody 3D1 CDRH1 (SEQ ID NO:20), human monoclonal antibody 4D1 CDRH1 (SEQ ID NO:21), or human monoclonal antibody 3H9 CDRH1 (SEQ ID NO:22).
  • human monoclonal antibody A12 CDRH1 SEQ ID NO:16
  • human monoclonal antibody B2 CDRH1 SEQ ID NO:17
  • human monoclonal antibody E9 CDRH1 SEQ ID NO:18
  • the heavy chain variable region comprises a CDRH2 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRH2 sequence of any one of human monoclonal antibody A12 CDRH2 (SEQ ID NO:23), human monoclonal antibody B2 CDRH2 (SEQ ID NO:24), human monoclonal antibody E9 CDRH2 (SEQ ID NO:25), human monoclonal antibody 3D5 CDRH2 (SEQ ID NO:26), human monoclonal antibody 3D1 CDRH2 (SEQ ID NO:27), human monoclonal antibody 4D1 CDRH2 (SEQ ID NO:28), or human monoclonal antibody 3H9 CDRH2 (SEQ ID NO:29).
  • the heavy chain variable region comprises a CDRH3 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRH3 sequence of any one of human monoclonal antibody A12 CDRH3 (SEQ ID NO:30), human monoclonal antibody B2 CDRH3 (SEQ ID NO:31), human monoclonal antibody E9 CDRH3 (SEQ ID NO:32), human monoclonal antibody 3D5 CDRH3 (SEQ ID NO:33), human monoclonal antibody 3D1 CDRH3 (SEQ ID NO:34), human monoclonal antibody 4D1 CDRH3 (SEQ ID NO:35), or human monoclonal antibody 3H9 CDRH3 (SEQ ID NO:36).
  • human monoclonal antibody A12 CDRH3 SEQ ID NO:30
  • human monoclonal antibody B2 CDRH3 SEQ ID NO:31
  • human monoclonal antibody E9 CDRH3 SEQ ID NO:32
  • the doppel-targeting moiety is an anti-doppel antibody having light chain sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the light chain sequence of human monoclonal antibody A12 (SEQ ID NO:37), the light chain sequence of human monoclonal antibody B2 (SEQ ID NO:38), the light chain sequence of human monoclonal antibody E9 (SEQ ID NO:39), the light chain sequence of human monoclonal antibody 3D5 (SEQ ID NO:40), the light chain sequence of human monoclonal antibody 3D1 (SEQ ID NO:41), the light chain sequence of human monoclonal antibody 4D1 (SEQ ID NO:42), or human monoclonal antibody 3H9 (SEQ ID NO:43).
  • the doppel-targeting moiety is an anti-doppel antibody having light chain variable domain sequences that are at least 85%, 90%, 95%, 99%, or 100% identical to the light chain variable domain sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • the light chain variable region comprises a CDRL1 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRL1 sequence of any one of human monoclonal antibody A12 CDRL1 (SEQ ID NO:44), human monoclonal antibody B2 CDRL1 (SEQ ID NO:45), human monoclonal antibody E9 CDRL1 (SEQ ID NO:46), human monoclonal antibody 3D5 CDRL1 (SEQ ID NO:47), human monoclonal antibody 3D1 CDRL1 (SEQ ID NO:48), human monoclonal antibody 4D1 CDRL1 (SEQ ID NO:49), or human monoclonal antibody 3H9 CDRL1 (SEQ ID NO:50).
  • human monoclonal antibody A12 CDRL1 SEQ ID NO:44
  • human monoclonal antibody B2 CDRL1 SEQ ID NO:45
  • human monoclonal antibody E9 CDRL1 SEQ ID NO:46
  • human monoclonal antibody 3D5 CDRL1 SEQ ID NO:
  • the light chain variable region comprises a CDRL2 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRL2 sequence of any one of human monoclonal antibody A12 CDRL2 (SEQ ID NO:51), human monoclonal antibody B2 CDRL2 (SEQ ID NO:52), human monoclonal antibody E9 CDRL2 (SEQ ID NO:53), human monoclonal antibody 3D5 CDRL2 (SEQ ID NO:54), human monoclonal antibody 3D1 CDRL2 (SEQ ID NO:55), human monoclonal antibody 4D1 CDRL2 (SEQ ID NO:56), or human monoclonal antibody 3H9 CDRL2 (SEQ ID NO:57).
  • human monoclonal antibody A12 CDRL2 SEQ ID NO:51
  • human monoclonal antibody B2 CDRL2 SEQ ID NO:52
  • human monoclonal antibody E9 CDRL2 SEQ ID NO:53
  • human monoclonal antibody 3D5 CDRL2 SEQ ID NO:
  • the light chain variable region comprises a CDRL3 amino acid sequence at least 85%, 90%, 95%, 99%, or 100% identical to the CDRL3 sequence of any one of human monoclonal antibody A12 CDRL3 (SEQ ID NO:58), human monoclonal antibody B2 CDRL3 (SEQ ID NO:59), human monoclonal antibody E9 CDRL3 (SEQ ID NO:60), human monoclonal antibody 3D5 CDRL3 (SEQ ID NO:61), human monoclonal antibody 3D1 CDRL3 (SEQ ID NO:62), human monoclonal antibody 4D1 CDRL3 (SEQ ID NO:63), or human monoclonal antibody 3H9 CDRL3 (SEQ ID NO:64).
  • human monoclonal antibody A12 CDRL3 SEQ ID NO:58
  • human monoclonal antibody B2 CDRL3 SEQ ID NO:59
  • human monoclonal antibody E9 CDRL3 SEQ ID NO:60
  • human monoclonal antibody 3D5 CDRL3 SEQ ID NO:
  • the doppel-targeting moiety is an anti-doppel antibody having framework region sequence that are at least 85%, 90%, 95%, 99%, or 100% identical to the framework sequence of any one of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • the doppel-targeting moiety is an anti-doppel antibody having heavy chain constant domain sequence at least 85%, 90%, 95%, 99%, or 100% identical to the heavy chain constant domain sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • the doppel-targeting moiety is an anti-doppel antibody having light chain constant domain sequence at least 85%, 90%, 95%, 99%, or 100% identical to the light chain constant domain sequence of human monoclonal antibody A12, human monoclonal antibody B2, human monoclonal antibody E9, human monoclonal antibody 3D5, human monoclonal antibody 3D1, human monoclonal antibody 4D1, or human monoclonal antibody 3H9.
  • an anti-doppel antibody suitable for use in the ADCs and methods described herein may be capable of targeting tumor cells, and thus exhibit one or more properties such as being capable of recognizing tumor cells, being capable of binding to tumor cells, being internalized into tumor cells, and exhibiting cytocidal activity against tumor cells, etc.
  • an anti-doppel antibody suitable for use in the ADCs and methods described herein may be capable of targeting tumor endothelial cells (TECs), and thus exhibit one or more properties such as being capable of recognizing TECs, being capable of binding to TECs, being internalized into TECs, and exhibiting cytocidal activity against TECs, etc.
  • the ADC may include a separate chemotherapeutic agent, it is not essential that the doppel-targeting antibody itself exhibits an anti-tumor effect. Nevertheless, in some embodiments the doppel-targeting antibody itself exhibits an anti-tumor effect. As discussed above, the doppel-targeting antibody advantageous is internalized into tumor cells, to promote the cytotoxic effect of the chemotherapeutic compound in a manner that specifically and selectively targets tumor cells.
  • Binding activity of the antibody with tumor cells can be confirmed using flow cytometry. Internalization of the antibody into tumor cells can be confirmed using (1) an assay comprising visualizing antibody incorporated in cells under a fluorescence microscope using a secondary antibody (fluorescently labeled) bound to the doppel-targeting antibody (see, e.g., Adams et al., Cell Death and Differentiation (2008) 15, 751-761), (2) an assay comprising measuring the amount of fluorescence incorporated in cells using a secondary antibody (fluorescently labeled) bound to the doppel-targeting antibody (Austin et al., Mol. Biol.
  • a Mab-ZAP assay using an immunotoxin bound to the doppel-targeting antibody wherein the toxin is released upon incorporation into cells to inhibit cell growth see, e.g., Kohls et al., BioTechniques, 2000, 28, 162-165.
  • a recombinant complex protein of a diphtheria toxin catalytic domain and protein G may be used as the immunotoxin.
  • antibodies and antibody fragments can be screened for doppel-binding activity using conventional techniques. For example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), western blot assay, and/or immunofluorescence may be used to measure doppel-binding activity.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • western blot assay and/or immunofluorescence
  • a known anti-doppel antibody can be immobilized on a plate, doppel applied to the plate, and then a sample containing a test antibody, such as culture supernatant of antibody-producing cells or purified antibodies, can be applied.
  • a secondary antibody that recognizes the primary antibody and is labeled with an enzyme such as alkaline phosphatase
  • an enzyme substrate such as nitrophenyl phosphate
  • the absorbance is measured to evaluate the antigen-binding activity of the sample.
  • C-terminal or N-terminal fragment of Doppel protein may be used as an antigen.
  • surface plasmon resonance analysis may be used to evaluate the activity of the antibody according to the present invention.
  • the anti-doppel antibody binds one or more forms of doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms discussed above. In some embodiments, the anti-doppel antibody binds one or more forms of human Doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms discussed above. In some embodiments, the anti-doppel antibody binds one or more forms of a non-human species of doppel, such as one or more of the monomeric, dimeric, glycosylated, and non-glycosylated forms discussed above.
  • the anti-doppel antibody preferentially binds to one or more of the forms of doppel described above, such as preferentially binding to one or more forms of doppel described above as compared to one or more of the other forms. In some embodiments, the anti-doppel antibody preferentially binds to one or more forms of human doppel. In some embodiments, the anti-doppel antibody preferentially binds to one or more forms of a non-human species of doppel.
  • the anti-doppel antibody preferentially binds to one of the forms of doppel described above, such as preferentially binding to one of the forms of doppel described above as compared to the other forms. In some embodiments, the anti-doppel antibody preferentially binds to one form of human doppel. In some embodiments, the anti-doppel antibody preferentially binds to one form of a non-human species of doppel.
  • Antitumor activity of the antibody can be confirmed in vitro by determining inhibitory activity against cell growth.
  • a cancer cell line overexpressing a target protein for the antibody can be cultured, and the antibody added at varying concentrations into the culture system to determine inhibitory activity against focus formation, colony formation, and spheroid growth.
  • Antitumor activity can be confirmed in vivo , for example, by administering the antibody to a nude mouse with a transplanted tumor cell line highly expressing the target protein and determining change in cancer (tumor) cells.
  • Doppel-targeting moieties suitable for use in the ADCs and methods described herein can be prepared using methodology known in the art.
  • An anti-doppel antibody suitable for use in the ADCs and methods described herein can be derived from (e.g., raised in) any species. Examples of typical species include human, rat, mouse, and rabbit.
  • An anti-doppel antibody derived from a non-human species may be chimeric or humanized.
  • An anti-doppel antibody suitable for use in the ADCs and methods described herein can be a polyclonal antibody or a monoclonal antibody.
  • antibodies can be raised in a host (such as a mammalian host) using an antigen comprising the doppel protein or a fragment thereof, such as an N-terminal or globular domain thereof, and screened for their ability to bind to doppel and, optionally, inhibit its interaction with a tyrosine kinase receptor (e.g. , VEGFR2, etc.).
  • a host such as a mammalian host
  • an antigen comprising the doppel protein or a fragment thereof, such as an N-terminal or globular domain thereof
  • a tyrosine kinase receptor e.g. , VEGFR2, etc.
  • polyclonal antibodies against doppel may be prepared by collecting blood from a mammal immunized with doppel and having desired antibodies in the serum, and by separating the serum from the blood by methods known in the art. Serum containing polyclonal antibodies and/or fraction containing polyclonal antibodies may be isolated and purified.
  • Monoclonal antibodies for use in the ADCs and methods described herein can be produced by methods known to those skilled in the art.
  • immune cells may be collected from an antigen-immunized non-human mammal having desired antibodies in its serum and subjected to cell fusion.
  • the immune cells used for cell fusion are typically obtained from the spleen.
  • suitable parental cells that can be fused with the immunocyte include, for example, mammalian myeloma cells, such as mammalian myeloma cells having an acquired property for selection of fused cells by drugs.
  • the immunocyte and myeloma cells can be fused according to known methods.
  • Hybridomas obtained by the cell fusion may be selected by cultivating them in a standard selection medium, such as HAT medium (hypoxanthine, aminopterin, and thymidine-containing medium).
  • HAT medium hyperxanthine, aminopterin, and thymidine-containing medium.
  • the cell culture is typically grown in HAT medium for several days to several weeks, e.g., a time sufficient to allow all other cells except for the desired hybridoma (non-fused cells) to die. Then, a standard limiting dilution can be performed to screen and clone a hybridoma cell producing the desired antibody.
  • Humanized forms of non-human (e.g., murine) antibodies can be obtained as chimeric antibodies, which contain minimal sequences derived from non-human immunoglobulin.
  • a humanized antibody comprises at least one or two variable domains in which variable regions are derived from non-human immunoglobulin and framework regions (FR) correspond to a human immunoglobulin sequence.
  • the anti-doppel antibody used herein comprises a human antibody framework region.
  • Such antibodies can be prepared by know techniques.
  • a humanized antibody optionally may contain at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • transgenic animals with human antibody genes may be immunized with the doppel protein, doppel protein-expressing cells, or their lysates.
  • the resulting antibody-producing cells can be collected and fused with myeloma cells to obtain a hybridoma, from which human antibodies against doppel can be prepared generally as outlined above.
  • an immune cell such as an immunized lymphocyte, producing antibodies may be immortalized by an oncogene and used for preparing monoclonal antibodies.
  • Monoclonal antibodies against doppel useful in the ADCs and methods described herein can be also prepared using recombinant genetic engineering techniques. See, e.g., Borrebaeck et al., Therapeutic Monoclonal Antibodies (MacMillan Publishers Ltd. (1990).
  • a DNA encoding an antibody against doppel may be cloned from an immune cell, such as a hybridoma or an immunized lymphocyte producing the antibody, inserted into an appropriate vector, and introduced into host cells to prepare a recombinant anti-doppel antibody.
  • the doppel-targeting moiety of the ADCs described herein may be an antibody fragment that binds to doppel.
  • antibody fragment that binds to doppel includes any doppel-binding fragment of an antibody or antibody-like molecule, including, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, and smaller fragments, diabodies, etc.
  • An antibody "fragment” may be prepared from a full-length antibody or may be synthesized as a "fragment” for example, using recombinant techniques.
  • a doppel-binding antibody fragment may be generated by treating a doppel-binding antibody with an enzyme, such as papain or pepsin.
  • an enzyme such as papain or pepsin.
  • a gene encoding a doppel antibody fragment may be constructed, inserted into an expression vector, and expressed in an appropriate host cell. See, e.g., Co et al., J. Immunol. 152: 2968-2976 (1994); Better and Horwitz, Methods Enzymol. 178:476-496 (1989); Pluckthun and Skerra, Methods Enzymol. 178:497-515 (1989); Lamoyi, Methods Enzymol. 121:652-663 (1986); Rousseaux et al., Methods Enzymol. 121:663-669 (1986); Bird and Walker, Trends Biotechnol. 9:132-137 (1991).
  • the anti-doppel ADCs described herein include a cleavable linker.
  • linkage of the anti-doppel moiety to the therapeutic agent drug via a cleavable linker may serve a dual purpose of linking the therapeutic agent to the anti-doppel moiety in an inactive form until it reaches the target site (therefore preventing systemic toxicities that may be associated with a chemotherapeutic agent, for example), and releasing the therapeutic agent at the target site (thereby maximizing efficacy at the desired site).
  • Suitable cleavable linkers for ADCs are known in the field, and include peptide linkers and non-peptide linkers.
  • the cleavable linker of an anti-doppel ADC conjugate as described herein is cleavable under one or both of intracellular conditions and extracellular conditions.
  • the linker is cleavable by an intracellular protease, such as lysosomal proteases or endosomal proteases.
  • the linker is hydrolysable at a pH of less than 5.5.
  • the linker is cleavable by a caspase, as discussed in more detail below.
  • the linker comprises or consists of a dipeptide linker.
  • the dipeptide linker is a valine-citrulline (Val-Cit) linker, a valine-alanine (Val-Ala) linker, or a phenylalanine-lysine (Phe-Lys) linker, which are cleavable by cathepsin B.
  • the linker is or comprises a disulfide linker that is cleaved in a reduced environment.
  • the linker is or comprises a hydrazone linker that is hydrolyzable at a pH of less than 5.5.
  • the linker is a caspase-cleavable peptide linker.
  • suitable caspase-cleavable peptide linkers are described in U.S. Patent No.10,357,572, the entire contents of which are incorporated herein by reference.
  • caspase refers to cysteine-aspartic proteases and cysteine-dependent aspartate-directed proteases that are activated (e.g., expressed) by cells undergoing apoptosis.
  • the caspase is caspase-3, caspase-7, and/or caspase-9.
  • Caspases are not specific to tumor cells per se , but are expressed as a result of cell apoptosis. Therefore, caspase has to be activated by another agent. This gives drugs which have caspase-cleavable peptide linkers (such as linkers comprising or consisting of the DEVD peptide sequence (SEQ ID NO: 4)) several unique advantages.
  • caspase-cleavable peptide linkers are cleaved independently of tumor enzyme expression, which can be quite variable.
  • caspase-cleavable peptide linkers only are cleaved in the presence of caspase, e.g., in the presence of cells undergoing apoptosis.
  • chemotherapeutic agent payload itself is non-selective
  • cleavage of the linker will release chemotherapeutic agent that can kill nearby tumor cells regardless of heterogeneity (this is known as the bystander killing effect).
  • cleavage of a caspase-cleavable peptide linker can propagate cleavage of additional linkers, exerting an amplification effect.
  • cells that are killed by the chemotherapeutic agent will expresses caspase, which will in turn cleave caspase-cleavable peptide linkers of additional ADCs, which will release their chemotherapeutic agents, which will kill more tumor cells, and so on, effectively amplifying both the strength and duration of the response.
  • caspase which will in turn cleave caspase-cleavable peptide linkers of additional ADCs, which will release their chemotherapeutic agents, which will kill more tumor cells, and so on, effectively amplifying both the strength and duration of the response.
  • caspase-cleavable peptide linker refers to a peptide sequence of two or more amino acid residues that is capable of being cleaved by caspase.
  • the caspase cleavable peptide linker is cleavable by caspase-3 or caspase-7, such as peptides comprising the sequence Asp-Xaa-Xaa-Asp (where "Xaa” represents any amino acid, in L- or D- isomer form).
  • the caspase-cleavable peptide linker is cleavable by caspase-9, such as peptides comprising the amino acid sequence Leu -Xaa-Xaa-Asp or Val-Xaa-Xaa-Asp (where "Xaa” represents any amino acid, in L- or D- isomer form).
  • the caspase-cleavable peptide linker comprises or consists of one of the following sequences:
  • the caspase-cleavable peptide linker comprises or consists of the sequence Lys-Gly-Asp-Glu-Val-Asp (SEQ ID NO:8), also denoted as KGDEVD (SEQ ID NO:8).
  • the anti-doppel ADCs described herein are inactive until the linker is cleaved.
  • the anti-doppel ADCs described herein comprising a chemotherapeutic agent exert minimal damage to healthy cells.
  • anti-doppel ADCs described herein that have a caspase-cleavable peptide linker only are activated in the presence of caspase, e.g., in the presence of cells undergoing apoptosis, such as tumor cells undergoing apoptosis.
  • anti-doppel ADCs described herein that have a caspase-cleavable peptide linker are specific for their target via two modalities: (i) target specificity provided by the doppel-targeting moiety and (ii) target specificity provided by the caspase-cleavable peptide linker.
  • anti-doppel ADCs described herein that have a caspase-cleavable peptide linker have an additional advantage due to the amplification effect.
  • anti-doppel ADCs described herein that have a caspase-cleavable peptide linker wherein the four C-terminal amino acid residues of the caspase-cleavable peptide linker are selected from Asp-Xaa-Xaa-Asp, Leu-Xaa-Xaa-Asp, and Val-Xaa-Xaa-Asp, where Xaa represents any amino acid residue, have an additional advantage in that the linker is cleaved at its point of conjugation to the therapeutic agent, releasing the therapeutic agent in an unmodified form that will exhibit its intrinsic therapeutic effect (e.g., an antitumor or cyctotoxic effect).
  • the linker is cleaved at its point of conjugation to the therapeutic agent, releasing the therapeutic agent in an unmodified form that will exhibit its intrinsic therapeutic effect (e.g., an antitumor or cyctotoxic effect).
  • Xaa represents any amino acid residue
  • Xaa represents any amino acid residue
  • Xaa represents any amino acid residue
  • Val-Xaa-Xaa-Asp SEQ ID NO:4 Asp-Glu-Val-Asp SEQ ID NO:5 Asp-Leu-Val-Asp SEQ ID NO:6 Asp-Glu-Ile-Asp SEQ ID NO:7 Leu-Glu-His-Asp SEQ ID NO:8 Lys-Gly-Asp-Glu-Val-Asp
  • the anti-doppel ADCs described herein include a therapeutic agent.
  • the therapeutic agent is or comprises a chemotherapeutic agent.
  • chemotherapeutic agent and “cytotoxic agent” generally are used interchangeably herein.
  • the term “chemotherapeutic agent” refers to a moiety useful to treat cancer, such as a small molecule chemical compound used to treat cancer.
  • the term “cytotoxic agent” refers to a moiety useful to induce cell death (apoptosis), such as a small molecule chemical compound used to induce cell death in target cells.
  • the chemotherapeutic agent induces apoptosis in target cells, e.g., in tumor cells and tumor tissue.
  • chemotherapeutic agent known in the art can be used as a chemotherapeutic agent in the anti-doppel ADCs described herein.
  • a chemotherapeutic agent is used that has, or can be prepared to have, a substituent, structure, or moiety that can be conjugated to another moiety of the ADC (directly or through a linkage).
  • Suitable chemotherapeutic agents include the cytotoxic agents described in U.S. Patent No. 10,357,572, the entire contents of which are incorporated herein by reference.
  • Suitable cytotoxic agents include, for example, an auristatin, a DNA minor groove binding agent, a DNA minor groove alkylating agent, an enediyne, a lexitropsin, a duocarmycin, a taxane, a puromycin, a dolastatin, a maytansinoid, and a vinca alkaloid.
  • the cytotoxic agent is selected from auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, calicheamicin, maytansine, DM1, netropsin, and derivatives thereof.
  • AFP auristatin phenylalanine phenylenediamine
  • MMAF monomethyl auristatin F
  • MMAE monomethyl auristatin E
  • auristatin E paclitaxel
  • docetaxel CC-1065, SN-38
  • topotecan topotecan
  • cytotoxic agents include anti-tubulin agents, such as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, and a dolastatin.
  • anti-tubulin agents such as an auristatin, a vinca alkaloid, a podophyllotoxin, a taxane, a baccatin derivative, a cryptophysin, a maytansinoid, a combretastatin, and a dolastatin.
  • the anti-tubulin agent is auristatin phenylalanine phenylenediamine (AFP), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), auristatin E, Vincristine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM1, or eleutherobin.
  • the anti-tubulin is monomethyl auristatin E (MMAE).
  • the chemotherapeutic agent is an anthracycline, such as doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, or a derivative thereof; an antibiotic, such as actinomycin-D, bleomycin, mitomycin-C, calicheamicin, or a derivative thereof; an alkylating agent, such as cyclophosphamide, mechlorethamine, uramustine, melphalan, chlorambucil, ifosfamide, bendamustine, carmustine, lomustine, streptozocin, busulfan, dacarbazine, temozolomide, thiotepa, altretamine, duocarmycin, or a derivative thereof; a platinum-based agent, such as cisplatin, carboplatin, nedaplatin, oxaliplatin, satraplatin, triplatin tetranitrate, or
  • the chemotherapeutic agent is selected from 5-FU, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, exatecan, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celebrex, chlorambucil, cisplatin, COX-2 inhibitors, irinotecan, SN-38, cladribine, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, epidophyllotoxin, erlotin
  • a chemotherapeutic agent useful in the anti-doppel ADCs described herein include monomethyl auristatin E (MMAE), which has the chemical name:
  • chemotherapeutic agent useful in the anti-doppel ADCs described herein includes exatecan which has the chemical name:
  • chemotherapeutic agents include doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, platinum-based antitumor agent (cisplatin or derivatives thereof), taxol and derivatives thereof, and camptothecin and derivatives thereof, including exatecan.
  • the therapeutic agent is or comprises an immunomodulatory agent.
  • immunomodulatory agent refers to agents that can help the body defend against pathogens, e.g., tumor cells, by adjusting the immune response.
  • the immunomodulator is selected from cytokines, lymphokines, monokines, stem cell growth factors, lymphotoxins, hematopoietic factors, colony stimulating factors (CSF), interrerons (IFN), parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, transforming growth factor (TGF), TGF-alpha, TGF-beta, insulin-like growth factor (IGF), erythropoietin, thrombopoietin
  • the therapeutic agent is or comprises a toxin.
  • toxin refers to naturally occurring organic elements produced by metabolic activities of living cells or organisms.
  • toxin is selected from ricin, abrin, ribonuclease (RNase), DNase 1, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
  • the therapeutic agent is or comprises a radionuclide.
  • radionuclide refers to a nuclide that has excess nuclear energy making it unstable. Radionuclides occur naturally or are artificially produced. Expose to radionuclides can have harmful effects on living organisms.
  • the radionuclide is selected from 11C, 13N, 15O, 32P, 33P, 47Sc, 51Cr, 57Co, 58Co, 59Fe, 62Cu, 67Cu, 67Ga, 75Br, 75Se, 76Br, 77As, 77Br, 80mBr, 89Sr, 90Y, 95Ru, 97Ru, 99Mo, 99mTc, 103mRh, 103Ru, 105Rh, 105Ru, 107Hg, 109Pd, 109Pt, 111Ag, 111In, 113mIn, 119Sb, 121mTe, 122mTe, 125I, 125mTe, 126I, 131I, 133I, 142Pr, 143Pr, 149Pm, 152Dy, 153Sm, 161Ho, 161Tb, 165Tm, 166Dy, 166Ho, 167Tm,
  • the therapeutic agent is or comprises a DNA cross-linking agent.
  • the DNA cross-linking agent is selected from indolionobenzodiazepine dimer (IGN), Pyrrolobenzodiazepine (PBD) and derivatives thereof.
  • the doppel-targeting moiety is joined directly or through a linker to the cleavable linker, and the cleavable linker may be joined directly or through a linker to the therapeutic agent.
  • the doppel-targeting moiety is conjugated directly to, e.g., the caspase-cleavable peptide linker, such as by a covalent bond between a moiety on the doppel-targeting moiety and a moiety at the N-terminus of the peptide linker or on a side chain of the peptide linker.
  • the cleavable linker (such as the caspase-cleavable peptide linker) is conjugated directly to the therapeutic agent, such as by a covalent bond between a moiety at the C-terminus of the peptide linker or on a side chain of the peptide linker and a moiety on the therapeutic agent.
  • linkages may be through a linker.
  • Any linker suitable for use in pharmaceutical compounds may be used for this purpose. Suitable linkers include p-aminobezylcarbamate (PABC).
  • the doppel-targeting moiety is linked to the N-terminus of the caspase-cleavable peptide linker, while the therapeutic agent is linked to the C-terminus of the caspase-cleavable peptide linker
  • ADCs wherein the therapeutic agent is linked to the N-terminus of the caspase-cleavable peptide linker, while the doppel-targeting moiety is linked to the C-terminus of the caspase-cleavable peptide linker.
  • an anti-doppel ADC as described herein includes a caspase-cleavable peptide joined directly or through a linker to a therapeutic agent, which is joined directly or through a linker to a doppel-targeting moiety.
  • daunorubicin exhibits its chemotherapeutic effect when it is conjugated at its 14-CH 3 position to another moiety.
  • caspase-induced cleavage need not release free daunorubicin in order to provide a chemotherapeutic effect.
  • an ADC comprises a caspase-cleavable peptide joined directly or through a linker to daunorubicin, which is joined at its 14-CH 3 position directly or through a linker to a doppel-targeting moiety.
  • anti-doppel ADCs described herein can be made by methods known in the art for preparing conjugates.
  • the anti-doppel ADCs described herein can be made in a step wise approach, such as by first making a cleavable linker-therapeutic agent conjugate, and then conjugating one or more cleavable linker-therapeutic agent conjugates to a doppel-targeting moiety.
  • a caspase cleavable peptide linker-therapeutic agent conjugate can be prepared as described in U.S. Patent No.10,357,572 (the entire contents of which are incorporated herein by reference), and then one or more thereof can be conjugated to a doppel-targeting moiety as described herein.
  • Other suitable methodologies for preparing ADCs as described herein are known in the field and illustrated in the examples below.
  • the number of therapeutic agent drug molecules per doppel-targeting moiety may impact the efficacy and safety of the ADC.
  • the ADC may be prepared under reaction conditions that control the number of therapeutic agent drug molecules per doppel-targeting moiety, such as the relative amounts of reactants used, other reagents used, and other reaction conditions. Still, a mixture of ADCs having different numbers of conjugated therapeutic agent drug molecules may be obtained.
  • the number of therapeutic agent drug molecules conjugated to an ADC may be specified by an average value representing the average number of conjugated therapeutic agent drug molecules per doppel-targeting moiety.
  • the number of conjugated therapeutic agent drug molecules per doppel-targeting moiety refers to an average value.
  • the anti-doppel ADC is provided in a pharmaceutical composition, such as a composition comprising the anti-doppel ADC and a pharmaceutically acceptable carrier, excipient, and/or diluent.
  • suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, minerals, and the like.
  • the pharmaceutical composition may be prepared for any route of administration, including any parenteral or local route of administration.
  • the pharmaceutical composition is suitable for injection or infusion, such as for intravenous injection or infusion, such as being prepared as a sterile composition for injection or infusion.
  • Appropriate components and excipients for such compositions are known in the art.
  • Non-aqueous solutions and suspensions may include, for example, propylene glycol, polyethylene glycol, a plant oil such as olive oil, or injectable ester such as ethyloleate.
  • a base for a suppository formulation may include, for example, witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin or the like.
  • the conjugate is dissolved in water or another pharmaceutically acceptable aqueous carrier in which the conjugate exhibits good solubility, optionally with or without other pharmaceutical acceptable excipients, preservatives, and the like.
  • kits may comprise one or more of the ADCs as disclosed herein, contained in a suitable container, optionally together with instructions for use in a method as disclosed herein.
  • the anti-doppel ADCs described herein are useful in methods of treating doppel-associated diseases and conditions, including cancer.
  • the anti-doppel ADCs may be administered by any route of administration.
  • the ADCs are administered intravenously.
  • the dose of ADC administered will vary depending on the subject and the condition for which it is administered, and can be determined by someone of skill in the art.
  • the dose administered to a subject may be between about 1 mg/kg and about 100 mg/kg, including from about 5 mg/kg to about 75 mg/kg, such as from about 10 mg/kg to about 50 mg/kg, or greater.
  • ADC's comprising chemotherapeutic agents typically exhibits lower toxicity than the chemotherapeutic agent alone, such that the dose of ADC administered may be higher than that which would be non-toxic for the chemotherapeutic agent alone.
  • Apoptosis is induced by an apoptosis inducing treatment, as disclosed above, resulting in expression of caspase.
  • the ADC is administered, and the caspase-cleavable peptide linker is cleaved by the caspase, releasing the chemotherapeutic agent.
  • the chemotherapeutic agent induces apoptosis of additional cells, resulting in additional expression of caspase, resulting in the caspase-induced cleavage/activation of additional ADC, resulting in amplified apoptosis.
  • This amplification yields methods with high efficiency and specificity in killing target cells, such as target tumor cells. Moreover, this amplification effect can prolong the time interval between apoptosis inducing treatments and/or between administrations of doses of the ADC. Thus, in some embodiments, this amplification effect may reduce the amount of chemotherapeutic agent needed to treat a certain number of cancer cells.
  • the ADC is inactive prior to cleavage of the caspase-cleavable peptide linker.
  • the ADC is not toxic (or apoptotic) to healthy cells.
  • the methods described herein reduce damage to normal cells by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or more, as compared with administration of the same chemotherapeutic agent in non-conjugated form.
  • the apoptotic effect of the ADC is selective to cells expressing caspase, e.g., cells undergoing apoptosis.
  • caspase e.g., cells undergoing apoptosis.
  • MMAE cytotoxic drug monomethyl auristatin E
  • mAb 3H9 is partially reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced Cys residues with the maleimide-KGDEVD-MMAE (SEQ ID NO:77) conjugate (e.g., maleimide-terminated linker-payload).
  • TCEP tris(2-carboxyethyl)phosphine
  • the reduced mAb is chilled on ice.
  • the maleimide-terminated linker-payload is added to the reaction mixture at a linker-payload/mAb-thiol molar ratio of 2:1, and reacted for an additional 1 hour at 4°C.
  • the reaction mixture is concentrated by centrifugal ultrafiltration and purified by elution through de-salting G25 in PBS at 4°C.
  • the ADC is analyzed for (1) concentration, by BCA protein assay; (2) aggregation, by size exclusion chromatography; (3) residual free drug, by reverse phase HPLC; and (4) drug-antibody ratio (DAR), by hydrophobic interaction chromatography.
  • An ADC was prepared comprising MMAE as the cytotoxic drug, mAb 3H9 as the doppel-targeting moiety, and a cathepsin B-cleavable peptide linker consisting of Val-Cit to give the ADC named 3H9-vc-MMAE:
  • mAb 3H9 is partially reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced Cys residues with the maleimide-terminated linker-payload (maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl-MMAE, also known as mc-Val-Cit-PABA-MMAE, which can be purchased from MedChemExpress).
  • TCEP tris(2-carboxyethyl)phosphine
  • 3H9 mAb is partially reduced via addition of 4.2 molar excess of tris(2-carboxyethyl)phosphine (TCEP) in 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), pH 7.0, and 1mM diethylenetriamine pentaacetic acid (DTPA) for 1 hour at 37°C.
  • TCEP tris(2-carboxyethyl)phosphine
  • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer
  • DTPA diethylenetriamine pentaacetic acid
  • the reduced mAb is chilled on ice.
  • the maleimide-terminated linker-payload (mc-Val-Cit-PABA-MMAE, MedChemExpress) is added to the reaction mixture at a linker-payload/mAb-thiol molar ratio of 2:1, and reacted for an additional 1 hour at 4°C.
  • the reaction mixture is concentrated by centrifugal ultrafiltration and purified by elution through de-salting G25 in PBS at 4°C.
  • 3H9-vc-MMAE is then filtered through a 0.2 micron filter under sterile conditions and immediately frozen at -80°C.
  • a maleimide-KGDEVD-Exatecan (SEQ ID NO:2) conjugate is prepared as described in U.S. Patent No.10,357,572 (the entire contents of which are incorporated herein by reference).
  • ⁇ -maleimidocaproylate and exatecan are bound to the amino group of the Lys side chain and the C-terminus of an AcKGDEVD peptide (SEQ ID NO:1), respectively.
  • mAb 3H9 is partially reduced with tris(2-carboxyethyl)phosphine (TCEP) followed by reaction of reduced Cys residues with the maleimide-KGDEVD-Exatecan (SEQ ID NO:2) conjugate (e.g., maleimide-terminated linker-payload).
  • TCEP tris(2-carboxyethyl)phosphine
  • 3H9 mAb is partially reduced via addition of 4.2 molar excess of tris(2-carboxyethyl)phosphine (TCEP) in 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer), pH 7.0, and 1mM diethylenetriamine pentaacetic acid (DTPA) for 1 hour at 37°C.
  • TCEP tris(2-carboxyethyl)phosphine
  • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer
  • DTPA diethylenetriamine pentaacetic acid
  • the reduced mAb is chilled on ice.
  • the maleimide-terminated linker-payload is added to the reaction mixture at a linker-payload/mAb-thiol molar ratio of 2:1, and reacted for an additional 1 hour at 4°C.
  • the reaction mixture is concentrated by centrifugal ultrafiltration and purified by elution through de-salting G25 in PBS at 4°C.
  • the ADC is analyzed for (1) concentration, by BCA protein assay; (2) aggregation, by size exclusion chromatography; (3) residual free drug, by reverse phase HPLC; and (4) drug-antibody ratio (DAR), by hydrophobic interaction chromatography.
  • anti-doppel ADCs as described herein to deliver a potent cytotoxic drug and eliminate doppel-expressing cells was assessed.
  • a cell line expressing doppel, HCT116 was selected and cultured with increasing concentrations of ADC. After 72 hours, viability of each culture was assessed. IC 50 values were calculated by logistic non-linear regression and reported as nM.
  • the ADCs 3H9-KGDEVD-MMAE (name SEQ ID NO:65; structure - SEQ ID NO:66) and 3H9-vc-MMAE prepared as described above inhibited growth of the doppel expressing cell line (HCT116), while 3H9 mAb alone did not. (FIG. 1)
  • ADC IC 50 values of anti-doppel ADCs on HCT116 cells.
  • HCTECs human colorectal tumor endothelial cells
  • a total of 1 x 10 4 HCTEC cells cultured in coverslips were treated with 0.1 mg/ml of 3H9 mAb conjugated with FITC. After incubating for 3 hours, the cells were washed with PBS and fixed in 10% neutral buffered formalin solution. Then, the cells were stained with 5 ug/ml WGA-Texas red solution overnight at 4°C. Subsequently, cells were washed with PBS, in situ mounting media with DAPI was added, and the cells were observed in a microscope. The results show that anti-doppel mAb 3H9 was internalized into doppel-expressing HCTEC cells. (FIG. 2)
  • HCTECs human colorectal tumor endothelial cells
  • a total of 1 x 10 4 HCTEC cells cultured in coverslips were treated with 0.1 mg/ml of 3H9 mAb conjugated with Cy5.5. After incubating for 3 hours, the cells were washed with PBS and treated with lysosensor for 2 hours at 37°C. Then, the cells were washed again with PBS and fixed in 10% neutral buffered formalin solution. The cells were washed with PBS, in situ mounting media with DAPI was added, and the cells were observed in a microscope.
  • the results show that anti-doppel mAb 3H9 was colocalized with lysosomal marker in doppel-expressing HCTEC cells. (FIG. 3)
  • HCT116 xenograft tumor model was used. HCT116 cells (1 x 10 ⁇ 7 cells/mouse) were inoculated into the left flank of balb-c/nu mice. When the average tumor volume reach 70-100 mm 3 , ADC was administered intravenously every 4 days for a total of 4 times. Tumor volume was measured every 4 days until the end of the experiments.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Pulmonology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Vascular Medicine (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

Sont décrits des conjugués anticorps-médicament anti-Doppel, des compositions les comprenant, et des méthodes associées de traitement de maladies et d'états associés à Doppel, y compris le cancer. L'invention propose des conjugués anticorps-médicament anti-Doppel (ADC) comprenant : (i) une fraction de ciblage de Doppel, (ii) un lieur clivable et (iii) un agent thérapeutique. L'invention propose également des méthodes de traitement de maladies et d'états associés à Doppel, y compris des cancers associés à Doppel, à l'aide des ADC décrits ici et des kits comprenant les ADC décrits dans la description.
PCT/KR2023/012720 2023-08-28 2023-08-28 Conjugués anticorps-médicament anti-doppel Pending WO2025047993A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/KR2023/012720 WO2025047993A1 (fr) 2023-08-28 2023-08-28 Conjugués anticorps-médicament anti-doppel
KR1020247029994A KR20250034273A (ko) 2023-08-28 2023-08-28 항-도펠 항체-약물 접합체

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2023/012720 WO2025047993A1 (fr) 2023-08-28 2023-08-28 Conjugués anticorps-médicament anti-doppel

Publications (1)

Publication Number Publication Date
WO2025047993A1 true WO2025047993A1 (fr) 2025-03-06

Family

ID=94819889

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2023/012720 Pending WO2025047993A1 (fr) 2023-08-28 2023-08-28 Conjugués anticorps-médicament anti-doppel

Country Status (2)

Country Link
KR (1) KR20250034273A (fr)
WO (1) WO2025047993A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180185498A1 (en) * 2014-11-20 2018-07-05 Pharosgen Co., Ltd. Prodrugs activated by caspase
US10501549B2 (en) * 2015-11-30 2019-12-10 Seoul National University R&Db Foundation Methods of inhibiting pathological angiogenesis with doppel-targeting molecules
EP3292150B1 (fr) * 2015-05-04 2020-02-05 Cytomx Therapeutics Inc. Anticorps anti-cd166 activables, et leurs procédés d'utilisation
US20240091372A1 (en) * 2022-07-19 2024-03-21 Seoul National University R&Db Foundation Anti-doppel antibody drug conjugates

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180185498A1 (en) * 2014-11-20 2018-07-05 Pharosgen Co., Ltd. Prodrugs activated by caspase
EP3292150B1 (fr) * 2015-05-04 2020-02-05 Cytomx Therapeutics Inc. Anticorps anti-cd166 activables, et leurs procédés d'utilisation
US10501549B2 (en) * 2015-11-30 2019-12-10 Seoul National University R&Db Foundation Methods of inhibiting pathological angiogenesis with doppel-targeting molecules
US20240091372A1 (en) * 2022-07-19 2024-03-21 Seoul National University R&Db Foundation Anti-doppel antibody drug conjugates

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHUNG SEUNG WOO, CHOI JEONG UK, CHO YOUNG SEOK, KIM HA RIN, WON TAE HYUNG, DIMITRION PETER, JEON OK‐CHEOL, KIM SEONG WHO, KIM IN‐S: "Self‐Triggered Apoptosis Enzyme Prodrug Therapy (STAEPT): Enhancing Targeted Therapies via Recurrent Bystander Killing Effect by Exploiting Caspase‐Cleavable Linker", ADVANCED SCIENCE, JOHN WILEY & SONS, INC, GERMANY, vol. 5, no. 7, 1 July 2018 (2018-07-01), Germany, XP093285391, ISSN: 2198-3844, DOI: 10.1002/advs.201800368 *
LEE HA, KIM BYOUNGMO, KWEON SEHO, CHOI JEONG, KIM SANG, BYUN YOUNGRO: "Abstract 2710: Developing a novel doppel-targeting antibody-drug conjugate (ADC) for solid cancer", CANCER RESEARCH, vol. 83, no. 7 Suppl., 4 April 2023 (2023-04-04), pages 2710 - 7445, XP009561871, ISSN: 0008-5472, DOI: 10.1158/1538-7445.AM2023-2710 *

Also Published As

Publication number Publication date
KR20250034273A (ko) 2025-03-11

Similar Documents

Publication Publication Date Title
JP7696635B2 (ja) 抗－ｉｌ１ｒａｐ抗体および抗体薬物コンジュゲート
KR102498405B1 (ko) 글리신아미드 화합물의 제조 방법
JP2022046678A (ja) チェックポイント阻害薬に再発／耐性を示す腫瘍を治療するための抗Ｔｒｏｐ－２－ＳＮ－３８抗体薬物複合体の効果
KR102342934B1 (ko) 항체-약물 접합체 및 면역독소
JP2022512778A (ja) 抗－ｃｄ４５抗体及びそのコンジュゲート
CN110121507B (zh) 抗sez6l2抗体和抗体药物缀合物
JP2015110667A (ja) Ｄｒ５リガンド薬物結合体
WO2018217058A1 (fr) Anticorps interleukine-2 anti-humains et leurs utilisations
CA2947238A1 (fr) Medicaments lieurs conjugues en un site specifique a des anticorps et cam resultants
TW201638108A (zh) 抗ｃ－Ｍｅｔ抗體和抗ｃ－Ｍｅｔ抗體－細胞毒性藥物偶聯物及其醫藥用途
RS64951B1 (sr) Konjugati leka koji sadrže antitela protiv klaudina 18.2
KR20150003836A (ko) Dr5 리간드 약물 접합체
CN110575547A (zh) 靶向于tf的抗体-药物偶联物及其制法和用途
JP2025011139A (ja) 同種異系の細胞療法における抗－ｃｄ５抗体薬物コンジュゲート（ａｄｃ）の使用
KR20240159839A (ko) FRα에 대한 결합 분자
CN110575548A (zh) 靶向于cd73的抗体-药物偶联物及其制法和用途
JP2021526525A (ja) 抗体薬物コンジュゲート（ａｄｃｓ）を用いた治療方法
JP2021530545A (ja) 同種異系の細胞療法における抗−ｃｄ２抗体薬物コンジュゲート（ａｄｃ）の使用
CN119604314A (zh) 抗gd2抗体、免疫偶联物及其治疗用途
JP2023509760A (ja) Ａｌｋ５阻害剤複合体およびその使用
CN119487076A (zh) 抗ceacam5抗体及其缀合物和用途
US20240091372A1 (en) Anti-doppel antibody drug conjugates
WO2025047993A1 (fr) Conjugués anticorps-médicament anti-doppel
JP2021530546A (ja) 同種異系の細胞療法における抗−ｃｄ１３７抗体薬物コンジュゲート（ａｄｃ）の使用
KR20250084935A (ko) 링커-페이로드 화합물, 접합체 및 이의 응용 분야

Legal Events

Date Code Title Description
WWP Wipo information: published in national office

Ref document number: 1020247029994

Country of ref document: KR

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23950868

Country of ref document: EP

Kind code of ref document: A1