WO2023212646A2 - Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease - Google Patents

Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease Download PDF

Info

Publication number
WO2023212646A2
WO2023212646A2 PCT/US2023/066299 US2023066299W WO2023212646A2 WO 2023212646 A2 WO2023212646 A2 WO 2023212646A2 US 2023066299 W US2023066299 W US 2023066299W WO 2023212646 A2 WO2023212646 A2 WO 2023212646A2
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
cancer
npm1
seq
conjugate
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.)
Ceased
Application number
PCT/US2023/066299
Other languages
English (en)
French (fr)
Other versions
WO2023212646A3 (en
Inventor
Ryan A. FLYNN
Benson M. GEORGE
Konstantinos TZELEPIS
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.)
Cambridge Enterprise Ltd
Boston Childrens Hospital
Original Assignee
Cambridge Enterprise Ltd
Boston Childrens Hospital
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 Cambridge Enterprise Ltd, Boston Childrens Hospital filed Critical Cambridge Enterprise Ltd
Priority to US18/860,335 priority Critical patent/US20250313647A1/en
Priority to JP2024563536A priority patent/JP2025514288A/ja
Priority to CN202380050132.2A priority patent/CN119487077A/zh
Priority to EP23797543.8A priority patent/EP4514858A2/en
Publication of WO2023212646A2 publication Critical patent/WO2023212646A2/en
Publication of WO2023212646A3 publication Critical patent/WO2023212646A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • A61K47/6825—Ribosomal inhibitory proteins, i.e. RIP-I or RIP-II, e.g. Pap, gelonin or dianthin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6815—Enzymes
    • 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
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/02—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2) hydrolysing N-glycosyl compounds (3.2.2)
    • C12Y302/02022—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2) hydrolysing N-glycosyl compounds (3.2.2) rRNA N-glycosylase (3.2.2.22)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57505—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the blood, e.g. leukaemia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/5758—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites
    • G01N33/5759—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumours, cancers or neoplasias, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides or metabolites involving compounds localised on the membrane of tumour or cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants

Definitions

  • Nucleophosmin 1 is a protein that typically resides in the nucleus and cytosol of cells.
  • NPM1 can be mislocalized to the cell surface.
  • Some cancers are also characterized by a mutation in NPM1, which leads to the translation of a mutant NPM1, referred to as NPMlc, which is mislocalized to the cell surface in even greater quantities than wild-type NPM1.
  • the NPMlc mutation is present in a considerable subpopulation of patients with AML.
  • the present disclosure is based on the identification of antibodies that bind specifically to nucleophosmin 1 (NPM1). These antibodies are capable of targeting wild-type (WT) and/or mutant NPM1 located on the surface of cells, including cancer cells, and are useful for targeting cytotoxic payloads to cells with cell surface expression of WT and/or mutant NPM1.
  • WT wild-type
  • NPM1 nucleophosmin 1
  • some aspects of the present disclosure relate to an antibody that binds to NPM1.
  • the antibody binds to WT NPM1.
  • the antibody comprises a heavy chain variable region comprising a heavy chain (HC) complementarity determining region (CDR) 1 comprising the amino acid sequence NIFVH (SEQ ID NO: 1), a HC CDR2 comprising the amino acid sequence KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and a HC CDR3 comprising the amino acid sequence DSSGYDAVDY (SEQ ID NO: 3), and a light chain variable region comprising a light chain (LC) CDR1 comprising the amino acid sequence RASESVYTYLA (SEQ ID NO: 9), a LC CDR2 comprising the amino acid sequence NAKTLTE (SEQ ID NO: 10), and a LC CDR3 comprising the amino acid sequence QHHYGTPYT (SEQ ID NO: 11).
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 28 and/or the light chain variable region comprises
  • the antibody binds to mutant NPM1.
  • the antibody comprises a heavy chain variable region comprising a heavy chain (HC) complementarity determining region (CDR) 1 comprising the amino acid sequence SYAMS (SEQ ID NO: 15), a HC CDR2 comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 16), and a HC CDR3 comprising the amino acid sequence WRNNAFDY (SEQ ID NO: 17), and a light chain variable region comprising a light chain (LC) CDR1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 22), a LC CDR2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 23), and a LC CDR3 comprising the amino acid sequence NSSPRLKHRVV (SEQ ID NO: 24).
  • the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 30, and/or in the light chain variable region comprises the amino acid
  • the antibody is a full-length antibody or an antigen-binding fragment thereof.
  • the antibody is a full-length antibody selected from an immunoglobulin G (IgG), an immunoglobulin A (IgA), an immunoglobulin D (IgD), an immunoglobulin E (IgE), and an immunoglobulin M (IgM). In some embodiments, the antibody is an IgG.
  • the antibody is an antigen-binding fragment selected from a Fab fragment, a F(ab’)2 fragment, an Ig monomer, a Fd fragment, a scFv, a scAb, a dAb, a Fv, an affibody, a diabody, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the antibody is a human antibody or a humanized antibody.
  • the antibody further comprises a heavy chain constant region.
  • the heavy chain constant region comprises the amino acid sequence set for in SEQ ID NO: 32 or SEQ ID NO: 46.
  • the antibody further comprises a light chain constant region.
  • the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 47.
  • the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44 and/or a light chain comprising the amino acid sequence of SEQ ID NO: 45.
  • the antibody preferentially binds to wild-type NPM1. In some embodiments, the antibody preferentially binds to mutant NPM1. In some embodiments, the antibody binds to both wild-type NPM1 and mutant NPM1.
  • compositions comprising an antibody that binds NPM1 described herein.
  • the composition further comprises a pharmacologically acceptable excipient.
  • nucleic acid or nucleic acid set that encodes an antibody that binds NPM1 described herein.
  • the nucleic acid or nucleic acid set comprises a vector or vector set.
  • the vector or vector set is an expression vector or vector set.
  • nucleic acid or nucleic acid set that encodes an antibody that binds NPM1 described herein.
  • aspects of the present disclosure relate to a method of producing an antibody that binds NPM1 described herein, the method comprising culturing a cell that comprises a nucleic acid or nucleic acid set encoding an antibody that binds NPM1 described herein, collecting the cultured cells and/or culture media, and isolating the antibody from the cultured cells and/or culture media.
  • conjugate comprising an antibody that binds NPM1 described herein conjugated to an agent.
  • the agent is a drug.
  • the drug is selected from the group consisting of: auri statin E, auri statin F, monomethyl auri statin D (MMAD), monomethyl auri statin F (MMAF), monomethyl auri statin E (MMAE), actinomycin, actinomycin X2, a-amanitin, P-amanitin, y-amanitin, s-amanitin, aeroplysinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacitidine, bafilomycin Al, bafilomycin Bl, bafilomycin B2, bafilomycin Cl, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, campathecin, chaetocin, chaetoglobosin,
  • the antibody and the drug are conjugated via a linker.
  • the linker is a cleavable linker.
  • the linker is a pH-sensitive linker, a glutathione-sensitive linker, or a protease-cleavable linker.
  • the cleavable linker is selected from the group consisting of N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), Sulfo-SPDB, valine-citrulline (Val-cit), acetyl butyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal.
  • the linker is a non-cleavable linker.
  • the non-cleavable linker is selected from the group consisting of N-succinimidyl 4-(N- mal eimidomethyl)cy cl ohexane-1 -carboxylate (SMCC) and maleimidomethyl cyclohexane- 1 -carboxylate (MCC), MC-VC-PAB.
  • the ratio of the antibody to the drug is between 1 : 1 and 1 : 10. In some embodiments, the ratio of the antibody to the agent is 1 :4. In some embodiments, the agent is a radioisotope. In some embodiments, the radioisotope is selected from the group consisting of: Iodine-131, Rhenium-188, Yttrium-90, Bismuth-213, and Actinium-225.
  • the agent is an imaging agent.
  • the imaging agent is a luminescent or fluorescent imaging agent.
  • the imaging agent is an agent that is detectable by magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the imaging agent is gadolinium-diethylenetriamine (Gd-DTPA).
  • nucleophosmin 1 (NPMl)-expressing cancer
  • the method comprising administering to a subject in need thereof an effective amount of an antibody or a conjugate described herein.
  • the NPM1 -expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells.
  • the cancer is a solid or liquid cancer selected from the group consisting of: a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer.
  • the cancer is selected from the group consisting of: acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • the cancer is a metastatic cancer.
  • the cancer is a therapy -related cancer or a secondary malignancy.
  • the cancer is therapy-related AML (t-AML) or a secondary malignancy of nonHodgkin’s lymphoma.
  • the antibody or conjugate is administered systemically or locally. In some embodiments, the antibody or conjugate is administered via injection. In some embodiments, the injection is intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection. In some embodiments, the antibody or conjugate is administered orally.
  • the administration occurs more than once. In some embodiments, the administration occurs between once per day and once per six months.
  • the subject is a mammal. In some embodiments, the subject is a human.
  • NPM1 cell surface nucleophosmin 1
  • nucleophosmin 1 nucleophosmin 1 (NPMl)-expressing cancer
  • the method comprising administering to a subject in need thereof an effective amount of a conjugate described herein, imaging the conjugate in the subject, and determining the presence of a NPM1 -expressing cancer in the subject, based on the level and position of the antibody conjugate imaged in the subject.
  • the NPM1 -expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells.
  • the cancer is selected from the group consisting of: acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • non-Hodgkin’s lymphoma and myelodysplastic syndrome (MDS).
  • MDS myelodysplastic syndrome
  • the cancer is a metastatic cancer.
  • the cancer is a therapy-related cancer or a secondary malignancy.
  • the cancer is therapy -related AML (t-AML) or a secondary malignancy of non-Hodgkin’s lymphoma.
  • the imaging is luminescent or fluorescent imaging. In some embodiments, the imaging is magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the method further comprises administering to the subject an effective amount of an antibody or a conjugate described herein, if a NPM1 -expressing cancer is determined to be present in the subject.
  • nucleophosmin 1 nucleophosmin 1 (NPMl)-expressing cancer
  • the method comprising collecting a biological sample from a subject in need thereof, contacting the biological sample with a conjugate described herein, analyzing binding between the conjugate and NPM1 -expressing cancer cells in the biological sample, and determining the presence of a NPM1 -expressing cancer in the subject, based on the level of binding between the conjugate and NPM1 -expressing cancer cells analyzed in the sample.
  • the NPM1 -expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells.
  • the cancer is selected from the group consisting of: acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • the cancer is a metastatic cancer.
  • the cancer is a therapy-related cancer or a secondary malignancy.
  • the cancer is therapy -related AML (t-AML) or a secondary malignancy of non-Hodgkin’s lymphoma.
  • the biological sample collected from the subject is a blood sample, a serum sample, or a plasma sample.
  • the analysis is luminescent or fluorescent analysis. In some embodiments, the analysis is performed via flow cytometry.
  • the method further comprises administering to the subject an effective amount of an antibody or a conjugate described herein, if a NPM1 -expressing cancer is determined to be present in the subject.
  • FIGs. 1A-1E show that NPMl is localized on the surface of cancerous cell types.
  • FIG. 1A shows immunoblotting of WT and mutant NPM1 in cytosolic and membrane fractions collected from various human leukemia cell lines.
  • FIG. IB shows FACS analysis of NPM1 surface expression in human leukemia cell lines.
  • FIGs. 1C and ID show relatively low levels of NPM1 expressed on the surface of murine bone marrow (BM) or peripheral blood (PB) cells.
  • FIG. IE shows FACS analysis that NPM1 is not robustly expressed on healthy human BM cells. FACS analysis was conducted with isolated anti-NPMl antibodies, shown in FIG. 2A and FIG. 2B, as well as commercial anti-NPMl antibody (Santa Cruz Biotechnology anti-WT NPM1 # sc-32256).
  • FIGs. 2A and 2B show preparation of anti-NPMl antibodies.
  • FIG. 2A shows isolation of an antibody that binds to WT NPM1.
  • FIG. 2B shows isolation of an antibody that binds to mutant NPM1.
  • FIGs. 3A and 3B shows that an anti-NPMl-ADC is toxic toward human cancer cells.
  • FIG. 3A shows cytotoxicity of an ADC comprising an antibody that binds WT NPM1 toward 0CI-AML3 cells. Streptavidin-saporin was bound to either biotinylated anti-WT NPM1 or antimouse IgG, after which complexes were added to 0CI-AML3 cells and incubated for 24 hours. After incubation, cells were washed and cell killing was analyzed by FACS.
  • FIG. 3B shows the same as in FIG. 3A, with incubation for 48 hours.
  • FIGs. 4A and 4B show that NPM1 is highly conserved between mammalian species.
  • FIG. 4A shows a pairwise alignment depicting conservation between human and murine NPM1 amino acid sequences (SEQ ID NOs: 39-40).
  • FIG. 4B shows a pairwise alignment depicting conservation between human and Rhesus macaque NPM1 amino acid sequences (SEQ ID NOs: 39 and 41).
  • FIGs. 5A-5C shows that NPM1 is present on the surface of human AML cell lines and primary murine AML cells.
  • FIG. 5A shows binding of an anti-NPMl antibody (Merck/Sigma anti-B23 # B0556) to NPM1 localized on the surface of human 0CI-AML3 cells and M0LM13 cells. Binding to NPM1 was assayed in both live cells and in fixed cells. 0CI-AML3 cells express mutant NPM1, while M0LM13 cells express relatively lower amounts of WT NPM1.
  • FIG. 5A shows binding of an anti-NPMl antibody (Merck/Sigma anti-B23 # B0556) to NPM1 localized on the surface of human 0CI-AML3 cells and M0LM13 cells. Binding to NPM1 was assayed in both live cells and in fixed cells. 0CI-AML3 cells express mutant NPM1, while M0LM13 cells express relatively lower amounts of WT NPM1.
  • FIG, 5C show binding of an anti-NPMl antibody (Merck/Sigma anti-B23 # B0556) to NPM1 localized on the surface of fixed primary murine MLL-AF4/FLT3 ltd/+ , MLL- AF9/FLT3 ltd/+ , MLL-ENL/FLT3 ltd/+ , and Npmlc/FLT3 ltd/+ AML cells.
  • NPMl antibody Merck/Sigma anti-B23 # B0556
  • FIGs. 6A-6F show that NPMlc (mutant NPM1) is present on the surface of a human myelogenous leukemia cell line.
  • FIG. 6A shows that an anti-TY 1 tag antibody (Diagenode TY 1 # Cl 5200054) binds the surface of human K562 myelogenous leukemia cells expressing TY1- tagged WT NPM1 or TYl-tagged NPMlc.
  • FIG. 6B shows the data in FIG. 6A as separate panels with quantification of the percentage of cells positively bound by the TY1 antibody and anti-NPMl antibody (Merck/Sigma anti-B23 # B0556).
  • FIG. 6A shows that an anti-TY 1 tag antibody (Diagenode TY 1 # Cl 5200054) binds the surface of human K562 myelogenous leukemia cells expressing TY1- tagged WT NPM1 or TYl-tagged NPMlc.
  • FIG. 6B shows the data in FIG. 6A as separate
  • FIG. 6C shows binding of anti-NPMl antibodies (Diagenode TY1 # C15200054; Merck/Sigma anti-B23 # B0556) to intracellular and cell surface WT and mutant NPM1.
  • the relevant cells expressed an empty TY1 lentiviral vector, a TYl-tagged NPM1 -wild-type lentiviral vector, or a TYl-tagged NPMlc lentiviral vector.
  • FIG. 6D shows binding between anti-NPMl antibodies (Diagenode TY1 # Cl 5200054; Merck/Sigma anti-B23 # B0556) and intracellular WT and mutant NPM1.
  • the relevant cells expressed an empty TY1 lentiviral vector, a TYl-tagged NPM1 -wild-type lentiviral vector, or a TYl-tagged NPMlc lentiviral vector.
  • FIG. 6E shows binding between anti-NPMl antibodies (Diagenode TY1 # C15200054; Merck/Sigma anti-B23 # B0556) and WT and mutant NPM1 on the surface of K562 cells or M0LM13 cells (negative control).
  • the relevant cells expressed an empty TY1 lentiviral vector, a TYl-tagged NPM1 -wild-type lentiviral vector, or a TYl-tagged NPMlc lentiviral vector.
  • 6F shows immunofluorescence depicting binding between anti- NPMl antibodies (Diagenode TY1 # C15200054; Merck/Sigma anti-B23 # B0556) and WT and mutant NPM1 on the surface of K562 cells or M0LM13 cells (negative control).
  • the relevant cells expressed an empty TY1 lentiviral vector, a TYl-tagged NPM1 -wild-type lentiviral vector, or a TYl-tagged NPMlc lentiviral vector.
  • FIGs. 7A and 7B show that anti-NPMl antibodies bind to NPM1 on the surface of primary murine AML cells.
  • FIG 7A shows binding between isolated WT and mutant anti- NPMl antibodies and NPM1 localized on the surface of primary murine MLL-rearranged (MLL-r) AML cells.
  • FIG. 7B shows binding between isolated WT and mutant anti-NPMl antibodies and NPM1 localized on the surface of primary murine NPMlc AML cells.
  • FIGs. 8A-8D show that anti-NPMl antibodies bind to NPM1 on the surface of human AML patient-derived xenografts (PDX) implanted in a murine model.
  • FIG. 8A shows binding between isolated WT and mutant anti-NPMl antibodies and NPM1 localized on the surface of PDX-1 cells (MLL-r). In contrast to PDX-1 cells, host (mouse) bone marrow cells (bottom panels) display relatively low binding.
  • FIG. 8B shows binding between isolated WT and mutant anti-NPMl antibodies and NPM1 localized on the surface of PDX-2 (NPMlc) cells.
  • FIG. 8C shows binding between isolated WT and mutant anti-NPMl antibodies and NPM1 localized on the surface of PDX-3 (MLL-r, BCOR) cells.
  • FIG. 8D shows binding between isolated WT and mutant anti-NPMl antibodies and NPM1 localized on the surface of PDX-4 (DNMT3A, N/KRAS) cells.
  • FIGs. 10A-10B show the results from live cell staining of cultured human suspension cell lines (FIG. 10 A) and primary murine cell lines (FIG. 10B) with anti-NPMl antibody (AF647 signal).
  • FIGs. 11A-11B show western blots of cells after cell surface biotinylation with a cell- impermeable biotinylation reagent.
  • a western blot detects NPM1 in the membrane fraction of these cells and NPM1 IP is able to enrich NPM1 more robustly from the membrane lysate (FIG. 11 A).
  • Examination of the biotin signal from these fractions demonstrates the isolation of a single band in the NPM1 IP (FIG. 1 IB), showing that full length NPM1 is exposed to the surface of live cells.
  • FIGs. 12A-12B are diffraction limited (DL) and super resolution reconstructions (SR) of anti-NPMl staining the cell surface of both HL-60 and OCI-AML3 (FIG. 12 A) and the adherent cell line PANCI (FIG. 12B). Cell surface NPM1 appears as distinct clusters.
  • DL diffraction limited
  • SR super resolution reconstructions
  • FIG. 13 are fluorescent images of live (top) or fixed and permeabilized (bottom) OCI- AML3 cells stained with either Ab2.2 or commercially available NPM1 antibodies.
  • FIGs. 14A-14B shows three healthy donor bone marrow samples being stained with Ab2.2 and sorted for various markers of the hematopoietic system. Ab2.2 partially binds to CD33+ cells however there is no observable binding to CD34+ (HSC) cells.
  • FIG. 14B shows the binding of a commercial anti-NPMl antibody as well as Ab2.2 binding to protein lysate samples by western blotting. The banding patterns are identical.
  • FIG. 15 is a table describing 12 AML patients.
  • FIGs. 16A-16B show the flow cytometry analysis of 12 AML.
  • FIG. 17 is a table describing samples collected from 15 AML patients.
  • FIGs. 18A-18E show the flow cytometry analysis of the 15 AML patients.
  • FIGs. 19A-19B show how the toxicity of Ab2.2 was assessed in vivo.
  • FIG. 19A is a schematic of the experimental protocol.
  • FIG. 19B are the weight results, and results from blood sample analysis of WBC, PLT and HGB.
  • FIGs. 20A-20G show how the efficacy of the Ab2.2 treatment was assessed in vivo.
  • FIG. 20A is a schematic of the experimental design.
  • FIG. 20B shows antibody binding to this AML model.
  • FIG. 20C shows WBC count.
  • FIG. 20D are results from qPCR analysis of the AML-allele.
  • FIG. 20E shows spleens of IgG and Ab2.2 treated mice and the weight of the spleens.
  • FIG. 20F are results of HGB and HCT analysis of blood samples.
  • FIG. 20G show the results from a survival assay.
  • FIGs. 21A-21C shows how mice were subjected to sub-lethal irradiation and transplantation of primary murine AML cells to assess the efficacy (efficacy model 2).
  • FIG. 21A is a schematic of the experimental design.
  • FIG. 2 IB shows antibody binding.
  • FIG. 21C show the results from a survival assay.
  • FIGs. 22A-22I show treatment with Ab2.2 reduces tumor burden in transplantation model.
  • FIG. 22A is a schematic of the experimental design to test the effect of secondary transplant after Ab2.2 treatment.
  • FIG. 22B are weights of the spleen, lung and liver in mice who received a transplant from IgG or Ab2.2 treated mice.
  • FIG. 22C show the results from a survival assay.
  • FIGs. 22D-22E shows flow cytometry results.
  • FIG. 22F are the WBC count and PLT levels from blood samples.
  • FIG. 22G shows the WBC over time.
  • FIG. 22H shows images of the spleen and graphs of weight of spleens in isotype or Ab2.2 treated mice.
  • FIG. 221 shows the AML% in BM and PB.
  • FIGs. 23A-23B shows immune dependency for tumor killing activity of Ab2.2; it on an intact immune system.
  • FIG. 23 A shows the experimental design.
  • FIG. 23B shows the survival curve of either Ab2.2 or IgG treated mice.
  • FIGs. 24A-24C shows the solid tumor activity of Ab2.2.
  • FIG. 24A shows the experimental design.
  • FIG. 24B shows the tumor volume on day 10 and day 13.
  • FIG. 24C shows the tumor volume over time from day 7 to day 13.
  • FIGs. 25A-25B shows wild type, healthy mice that were treated with sub-lethal irradiation and Ab2.2 did not exhibit an observable effect of Ab2.2 treatment.
  • FIG. 25A shows the experimental design.
  • FIG. 25B shows the weight and WBC count, HGB and PLT analysis from blood samples.
  • FIGs. 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or murine tumors.
  • FIG. 27 shows high surface detection of Npmlc on single Dmnt3a R882H mutant cells (pre-leukemia cells).
  • FIG. 28 shows how NPMl can serve as an ADC target in some models.
  • 0CI-AML3 cells were treated with negative control, isotype Saporin conjugate or Ab2.2-Saporin conjugate to assess the in vitro activity of Ab2.2 with an ADC.
  • FIGs. 29A-29C show Ab2.2 extends lifespan in a transplantation model of human AML cell line.
  • FIG. 29A is a schematic of the experimental design.
  • FIG. 29B shows antibody binding using flow cytometry.
  • FIG. 29C is a graph of the results of a survival assay.
  • FIGs. 30A-30C show Ab2.2 extends lifespan in a transplantation model of human AML- PDX cell line.
  • FIG. 30A is a schematic of the experimental design.
  • FIG. 30B shows antibody binding using flow cytometry.
  • FIG. 30C is a graph of the results of a survival assay.
  • nucleophosmin 1 also known as nucleolar phosphoprotein B23 or numatrin
  • target agents e.g., chemotherapeutic drugs, radioactive isotopes
  • cell types e.g., cancer cells
  • NPM1 is a multifunctional protein that typically binds to single-stranded and double-stranded nucleic acids and is present in the nucleus and cytosol of cells.
  • quantities of NPM1 may instead be mislocalized to the cell surface where they are exposed to the extracellular environment.
  • NPM1 cell membrane localized NPM1
  • leukemia e.g., acute myeloid leukemia (AML)
  • AML acute myeloid leukemia
  • NPMlc mutant variant of NPM1
  • NPMlc is also only known to occur in cancer cells, as it drives malignant transformation in AML.
  • an antibody or other agent that is specific for wild-type NPM1 or mutant NPM1 could be conjugated to a cytotoxic payload and used to target cancer cells.
  • an NPM1 -specific antibody may itself be sufficient to stimulate antibody dependent cellular cytotoxicity (ADCC) or antibody dependent cell phagocytosis (ADCP) upon binding to cancer cells, without conjugation to a cytotoxic payload.
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody dependent cell phagocytosis
  • antibodies specific for NPM1 are commercially available, antibodies that can effectively bind to wild-type and/or mutant NPM1 on the surface of live cells are lacking. Additionally, most commercially available anti-NPMl antibodies are polyclonal and there are currently no available monoclonal antibodies that bind to NPMlc.
  • new antibodies have been developed that are capable of effectively and specifically binding to wildtype NPM1 and mutant NPM1 on the surface of cells, including cancer cells.
  • These antibodies and molecular conjugates thereof e.g., antibody-drug conjugates
  • the present disclosure provides antibodies that bind to NPM1, for example, wild-type (WT) NPM1 or mutant NPM1.
  • WT wild-type
  • Such antibodies may have higher affinity for WT NPM1 than for mutant NPM1, or may have higher affinity for mutant NPM1 than for WT NPM1.
  • antibody refers to an immunoglobulin molecule capable of specific binding to a target, referred to as an “antigen,” such as but not limited to a protein or peptide, through at least one recognition site on the antigen.
  • antibody encompasses both full-length immunoglobulin molecules (immunoglobulins having two heavy chains and two light chains, e.g., a monoclonal or polyclonal full-length antibody) and antigenbinding fragments thereof, such as, but not limited to chimeric antibodies, diabodies, linear antibodies, nanobodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), or another antigen-binding fragment that retains specific binding for the antigen.
  • An “immunoglobulin (Ig)” is a large, Y-shaped protein produced mainly by plasma cells that is used by the immune system to neutralize an exogenous substance (e.g., a pathogen such as bacteria and viruses).
  • Antibody fragments include any antigen binding fragment (i.e., “antigen-binding portion”) or single chain thereof.
  • an “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CH 1, CH 2 and CH 3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • an antibody is an immunoglobulin (Ig) monomer.
  • An antibody may be a polyclonal antibody or a monoclonal antibody.
  • an antibody is a heterotetrameric glycoprotein composed of two identical L chains and two H chains
  • an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain).
  • the 4-chain unit is generally about 150,000 daltons.
  • Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype.
  • Each H and L chain also has regularly spaced intrachain disulfide bridges.
  • Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and a isotypes.
  • Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end.
  • the VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain.
  • Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site.
  • an antibody is an IgG.
  • immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated a, 8, a, y and p, respectively.
  • the y and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.
  • variable domain mediates antigen binding and define specificity of a particular antibody for its particular antigen.
  • variability is not evenly distributed across the 110-amino acid span of the variable domains.
  • the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long.
  • FRs framework regions
  • hypervariable regions regions of extreme variability
  • the hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference).
  • the constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC) and/or antibody dependent cell phagocytosis (ADCP).
  • the antibody is a monoclonal antibody.
  • a “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567).
  • Monoclonal antibodies may also be isolated from phage antibody libraries, e.g., using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol.
  • the monoclonal antibodies described herein encompass “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Set. USA, 81 :6851-6855 (1984)).
  • Chimeric antibodies of interest herein include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.), and human constant region sequences.
  • the antibody is a polyclonal antibody.
  • a “polyclonal antibody” is a mixture of different antibody molecules which react with more than one immunogenic determinant of an antigen. Polyclonal antibodies may be isolated or purified from mammalian blood, secretions, or other fluids, or from eggs. Polyclonal antibodies may also be recombinant. A recombinant polyclonal antibody is a polyclonal antibody generated by the use of recombinant technologies.
  • Recombinantly generated polyclonal antibodies usually contain a high concentration of different antibody molecules, all or a majority of (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or more) which are displaying a desired binding activity towards an antigen composed of more than one epitope.
  • the antibodies are “humanized” for use in human (e.g., as therapeutics).
  • “Humanized” forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • donor antibody such as mouse, rat, rabbit or non-human primate having the desired antibody specificity, affinity, and capability.
  • framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Some aspects of the present disclosure relate to an antibody that binds to NPM1. Such an antibody may preferentially bind to WT NPM1 or mutant NPM1.
  • the term “preferentially binds” refers to binding that occurs more frequently, at a higher rate, over a greater duration, and/or with greater affinity with a particular antigen than with other antigens.
  • the terms “preferentially binds” and “specifically binds” may be used interchangeably.
  • the terms “preferentially binds” and “specifically binds” do not necessarily denote exclusive binding, i.e., an antibody that preferentially bind to an antigen may or may not bind to one or more additional antigens.
  • an antibody described herein may or may not bind to other forms (variants) of a specific antigen.
  • an antibody that binds to WT NPM1 may or may not bind to a variant of NPM1 comprising one or more amino acid insertions, deletions, or substitutions (i.e., a mutant NPM1), and a variant ofNPMl comprising one or more amino acid insertions, deletions, or substitutions (i.e., a mutant NPM1) may or may not bind to WT NPM1.
  • wild-type NPM1 or “WT NPM1” refers to a NPM1 protein comprising an amino acid sequence that is identical to that of a generally accepted reference sequence for intact, fully functional NPM1.
  • a WT NPM1 may be an NPM1 isoform (e.g., NCBI Reference Sequence: NP_001341935.1) that is expressed from a gene encoding WT NPM1 (NCBI Reference Sequence: NG_016018.1).
  • a “mutant NPM1” refers to a NPM1 protein comprising an amino acid sequence that comprises one or more amino acid insertions, deletions, or substitutions, relative to a WT NPM1 sequence.
  • a mutant NPM1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid insertions, deletions, or substitutions, relative to a WT NPM1 sequence.
  • a mutant NPM1 may have reduced activity and/or altered subcellular localization compared to WT NPM1.
  • a WT NPM1 or mutant NPM1 described herein may be a mammalian WT NPM1 or mammalian mutant NPM1.
  • a WT NPM1 or mutant NPM1 described herein is a human WT NPM1 or human mutant NPM1.
  • an antibody that binds to NPM1 is a full-length antibody.
  • a full- length antibody described herein may be of any antibody class based on the amino acid sequence of its heavy chain constant region.
  • a full-length antibody that binds to NPM1 may be an immunoglobulin G (IgG), an immunoglobulin A (IgA), an immunoglobulin D (IgD), an immunoglobulin E (IgE), and an immunoglobulin M (IgM), or a subclass thereof (e.g., IgGl, IgG2, IgG3, IgG4).
  • an antibody described herein is an antigen-binding fragment.
  • An antigen-binding fragment may be in the format of a Fab fragment, a F(ab’)2 fragment, an Ig monomer, a Fd fragment, a scFv, a scAb, a dAb, a Fv, an affibody, a diabody, a single domain heavy chain antibody, and a single domain light chain antibody.
  • Antibodies described herein may be of murine, rat, human, or any other origin. In some embodiments, an antibody described herein is a human antibody or a humanized antibody.
  • an antibody that binds to NPM1 comprises a heavy chain variable region and a light chain variable region.
  • the heavy chain variable region and light chain variable region each comprise a set of complementarity determining region (CDR) sequences that determine substrate specificity.
  • CDR sequences may be determined using any numbering scheme that is generally known in the art (e.g., Kabat, Chothia, Contact, IGMT).
  • an antibody that binds to NPM1 comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence NIFVH (SEQ ID NO: 1), a HC CDR2 comprising the amino acid sequence KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and a HC CDR3 comprising the amino acid sequence DSSGYDAVDY (SEQ ID NO: 3).
  • an antibody that binds to NPM1 comprises a light chain comprising a light chain CDR1 comprising the amino acid sequence RASESVYTYLA (SEQ ID NO: 9), a LC CDR2 comprising the amino acid sequence NAKTLTE (SEQ ID NO: 10), and a LC CDR3 comprising the amino acid sequence QHHYGTPYT (SEQ ID NO: 11).
  • an antibody that binds to NPM1 comprises a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence SYAMS (SEQ ID NO: 15), a HC CDR2 comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 16), and a HC CDR3 comprising the amino acid sequence WRNNAFDY (SEQ ID NO: 17).
  • an antibody that binds to NPM1 comprises a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence QGDSLRSYYAS (SEQ ID NO: 22), a LC CDR2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 23), and a LC CDR3 comprising the amino acid sequence NSSPRLKHRVV (SEQ ID NO: 24).
  • Table 1 provides the amino acid sequences for heavy chain and light chain CDRs for exemplary antibodies (“Abl” and “Ab2”) that are specific for NPM1 (WT and/or mutant NPM1).
  • an antibody that binds to NPM1 comprises a heavy chain variable region (VH) sequence and/or light chain variable region (VL) sequence provided in Table 2. In some embodiments, an antibody that binds to NPM1 further comprises a heavy chain constant region (CH) sequence and/or light chain variable region (CL) sequence provided in Table 2. In some embodiments, an antibody that binds to NPM1 comprises a heavy chain sequence and/or light chain sequence provided in Table 2.
  • an antibody that binds to NPM1 may comprise one or more mutations (e.g., amino acid insertions, deletions, or substitutions) relative to a CDR, VH, VL, CH, CL, heavy chain, or light chain sequence provided in Table 1 or Table 2.
  • an antibody that binds to NPM1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., amino acid insertions, deletions, or substitutions) relative to a CDR, VH, VL, CH, CL, heavy chain, or light chain sequence provided in Table 1 or Table 2.
  • an antibody that binds to NPM1 may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to a CDR, VH, VL, CH, CL, heavy chain, or light chain sequence provided in Table 1 or Table 2.
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the Kabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the Kabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 10 (according to the Kabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 11 (according to the Kabat definition system).
  • CDR-H1 heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 CDR
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 15 (according to the Kabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 16 (according to the Kabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Kabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Kabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Kabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Kabat definition system).
  • CDR-H1 heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complement
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 4 (according to the IMGT definition system), a heavy chain complementarity determining region 5(CDR-H2) of SEQ ID NO: 2 (according to the Chothia definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the Chothia definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 9 (according to the Chothia definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 10 (according to the Chothia definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 11 (according to the Chothia definition system).
  • CDR-H1 of SEQ ID NO: 4
  • CDR-H2 heavy chain complementarity determining region 5(CDR-H2)
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 18 (according to the Chothia definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 19 (according to the Chothia definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Chothia definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Chothia definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Chothia definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Chothia definition system).
  • CDR-H1 heavy chain complementarity determining region 1
  • CDR-H2 heavy chain complementarity determining region 2
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 6 (according to the IMGT definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 7 (according to the Contact definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 8 (according to the Contact definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 12 (according to the Contact definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 13 (according to the Contact definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 14 (according to the Contact definition system).
  • CDR-H1 of SEQ ID NO: 6
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complementarity determining region 3
  • the antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 20 (according to the Contact definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 48 (according to the Contact definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 21 (according to the Contact definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 25 (according to the Contact definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 26 (according to the Contact definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 27 (according to the Contact definition system).
  • CDR-H1 of SEQ ID NO: 20
  • CDR-H2 heavy chain complementarity determining region 2
  • CDR-H3 heavy chain complementarity determining region 3
  • the antibody of the present disclosure comprises a heavy chain variable region (VH) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH comprising the amino acid sequence of SEQ ID NO: 28.
  • VH heavy chain variable region
  • the antibody of the present disclosure comprises a light chain variable region (VL) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL comprising the amino acid sequence of SEQ ID NO: 29.
  • VL light chain variable region
  • the antibody of the present disclosure comprises a heavy chain variable region (VH) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VH comprising the amino acid sequence of SEQ ID NO: 30.
  • VH heavy chain variable region
  • the antibody of the present disclosure comprises a light chain variable region (VL) containing no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) in the framework regions as compared with the VL comprising the amino acid sequence of SEQ ID NO: 31.
  • VL light chain variable region
  • the antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH comprising the amino acid sequence of SEQ ID NO: 28.
  • the antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the antibody of the present disclosure comprises a VH comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VH comprising the amino acid sequence of SEQ ID NO: 30.
  • the antibody of the present disclosure comprises a VL comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical in the framework regions to the VL comprising the amino acid sequence of SEQ ID NO: 31.
  • the antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 28.
  • the antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 29.
  • the antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 30.
  • the antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 31.
  • the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 35.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 37.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 37.
  • the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 36.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 38.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 38.
  • the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 44.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 45.
  • the antibody of the present disclosure comprises a light chain comprising an amino acid sequence least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 45.
  • the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37.
  • the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.
  • the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45. Alternatively or in addition (e.g., in addition), the antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45.
  • Antibodies capable of binding NPM1 as described herein can be made by any method known in the art. See, for example, Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some examples, an anti-NPMl antibody is prepared by recombinant technology.
  • hybridoma cell lines can be prepared from lymphocytes and immortalized myeloma cells expressing an antibody described herein using techniques that are broadly known in the art (see, e.g., Kohler and Milstein, (1975) Nature 256:495-497; or Buck, et al., (1982) In Vitro, 18:377-381).
  • the generation of a hybridoma involves fusing a myeloma cell with a lymphoid cell using a fusogen (e.g., polyethylene glycol or an electrical current) to produce an immortalized cell line capable of producing and secreting the desired antibody.
  • Hybridomas may be cultured in culture media under conditions that are known in the art.
  • nucleic acids encoding the heavy and light chain of an anti-NPMl antibody as described herein can be cloned using recombinant technology known in the art into one or more vectors, wherein each nucleotide sequence is operably linked to a suitable promoter (e.g., a constitutive or inducible promotor, as known in the art).
  • the vectors may be expression vectors, from which polypeptide sequences for an anti-NPMl antibody can be expressed in a particular cell type.
  • the one or more expression vectors comprise one or more promoters for driving the expression of an operably linked gene (typically 3’ to the promotor sequence).
  • Each of nucleotide sequence encoding the heavy chain and light chain may be operably linked to different promoters.
  • the nucleotide sequences encoding the heavy chain and the light chain may be operably linked to the same promoters (e.g., within the same expression vector).
  • the selection of expression vector(s) and/or promoter(s) depends on the type of host cells for use in producing the antibodies.
  • an internal ribosomal entry site (IRES) can be inserted between the heavy chain and light chain encoding sequences to enhance recombinant expression.
  • Expression vectors encoding an anti-NPMl antibody may be inserted into a cell for expression using any technique known in the art (e.g., chemical transfection, viral transfection).
  • the cell may be a cell which can be cultured using techniques known in the art.
  • the cell may be any cell type capable of expressing heterologous antibody sequences, such as, for example, a human cell, a murine cell, a rat cell, a non-human primate cell, or an insect cell that can be cultured in vitro.
  • the anti-NPMl antibody may be isolated from either the culture media (if the antibody is secreted) or from the cells following lysis. Techniques for isolating antibodies from cells and/or culture media are generally known in the art, but typically include affinity chromatography.
  • Anti-NPMl antibody conjugates are generally known in the art, but typically include affinity chromatography.
  • the present disclosure further provides molecular conjugates that comprise an anti- NPM1 antibody described herein (e.g., an antibody that binds to WT NPM1 or mutant NPM1) which is linked through a chemical linker to an agent.
  • an anti- NPM1 antibody described herein e.g., an antibody that binds to WT NPM1 or mutant NPM1 which is linked through a chemical linker to an agent.
  • a conjugate described herein comprises an anti-NPMl antibody that is linked to a drug.
  • a conjugate comprising an anti-NPMl antibody that is conjugated to a drug may be referred to as an “ADC.”
  • the drug is a small molecule.
  • small molecule refers to molecules, whether naturally occurring or artificially created (e.g., via chemical synthesis) that have a relatively low molecular weight.
  • a small molecule is an organic compound (e.g., it contains carbon).
  • the small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyl, carbonyls, and heterocyclic rings, etc.).
  • the molecular weight of a small molecule is not more than about 1,000 g/mol, not more than about 900 g/mol, not more than about 800 g/mol, not more than about 700 g/mol, not more than about 600 g/mol, not more than about 500 g/mol, not more than about 400 g/mol, not more than about 300 g/mol, not more than about 200 g/mol, or not more than about 100 g/mol.
  • the molecular weight of a small molecule is at least about 100 g/mol, at least about 200 g/mol, at least about 300 g/mol, at least about 400 g/mol, at least about 500 g/mol, at least about 600 g/mol, at least about 700 g/mol, at least about 800 g/mol, or at least about 900 g/mol, or at least about 1,000 g/mol. Combinations of the above ranges (e.g., at least about 200 g/mol and not more than about 500 g/mol) are also possible.
  • the drug is a cytotoxic drug (e.g., a cytotoxic small molecule).
  • a drug is a cytostatic drug (e.g., a cytostatic small molecule).
  • the drug is a chemotherapeutic drug.
  • Non-limiting examples of drugs suitable for use in the ADCs described herein include auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), actinomycin, actinomycin X2, a-amanitin, P-amanitin, y-amanitin, s-amanitin, aeroplysinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacitidine, bafilomycin Al, bafilomycin Bl, bafilomycin B2, bafilomycin Cl, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, campathecin, chaetocin, chaetoglobosin, chlamydocin, cinerub
  • a conjugate described herein comprises an anti-NPMl antibody that is linked to an imaging agent.
  • An imaging agent may be a small molecule.
  • An imaging agent has one or more properties by which presence of the agent may be detected.
  • an imaging agent described herein may be a luminescent imaging agent (e.g., luciferase) or a fluorescent imaging agent (e.g., fluorescein sodium, methylene blue, indocyanine green (ICG)), which can be detected my measuring the emission of light at one or more specific wavelengths using techniques generally known in the art.
  • an imaging agent may be an agent that is detectable using magnetic resonance imaging (MRI), such as, but not limited to, gadoliniumdiethylenetriamine (Gd-DTPA).
  • MRI magnetic resonance imaging
  • an anti-NPMl antibody described herein is conjugated to more than one molecule of an agent (e.g., a drug, an imaging agent), i.e., the antibody and the agent are conjugated together at a ratio greater than 1 : 1.
  • the ratio between the antibody and the agent is between 1 : 1 and 1 : 10.
  • the ratio between the antibody and the agent is 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10.
  • an anti-NPMl antibody described herein is conjugated (linked) to an agent using a chemical linker known in the art.
  • a chemical linker known in the art.
  • conjugated means that two molecules are associated, preferably through a covalent bond or with sufficient affinity that the therapeutic or diagnostic benefit of the association between the two entities is realized.
  • a linker is a cleavable linker.
  • a cleavable linker is capable of releasing a conjugated moiety in response to a stimulus.
  • the stimulus is a physiological stimulus.
  • Non-limiting examples of stimuli include the presence of an enzyme, acidic conditions, basic conditions, or reducing conditions.
  • cleavable linkers include peptide linkers, P-glucuronide linkers, glutathione-sensitive linkers (or disulfide linkers) and pH-sensitive linkers.
  • a pH-sensitive linker is cleaved at a pH between 5.0 and 6.5 or between a pH of 4.5 and 5.0.
  • a pH-sensitive linker is not cleaved when the pH is between 7 and 7.5. In some embodiments, a pH-sensitive linker is not cleaved when the pH is between 7.3 and 7.5. In some embodiments, a cleavable linker is a protease-sensitive linker.
  • cleavable linkers examples include N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), Sulfo-SPDB, valine-citrulline dipeptide (Val-Cit), acetyl butyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal. See, e.g., Donaghy, mAbs. 2016 May-Jun;8(4):659-71, incorporated herein by reference.
  • a linker is non-cleavable.
  • a non-cleavable linker is a linker that is not cleaved within systemic circulation in a subject.
  • a non-cleavable linker is a linker that is resistant to protease cleavage.
  • Non- cleavable linkers include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate (SMCC), maleimidomethyl cyclohexane- 1 -carboxylate (MCC) and MC-VC-PAB.
  • the ADCs comprising an anti-NPMl antibody conjugated to a drug are advantageous to use therapeutically, in part because the drugs (e.g., chemotherapeutic drugs) are toxic and can be targeted to particular cell types expressing cell surface NPM1 (e.g., NPM1 -expressing cancer cells).
  • the toxicity of the ADC may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, compared to the drug in its free from.
  • Conjugates comprising an anti-NPMl antibody conjugated to an imaging agent are advantageous as they are specific for particular cells and tissues expressing cell surface NPM1 (e.g., NPM1 -expressing cancer cells), enabling greater specificity of the imaging agent for the cell or tissue than could be achieved by the free imaging agent.
  • Such conjugates are useful for detecting the presence and abundance of cells and tissues expressing cell surface NPM1 (e.g., NPM1 -expressing cancer cells) and may be administered, for example, prior to, during, or after any course of treatment.
  • the agent to which an anti-NPMl antibody is linked is a radioisotope.
  • the radioisotope is a radioisotope that is useful for the treatment of a cancer (i.e., a therapeutic radioisotope), such as, for example a radioisotope that is useful for the treatment of acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, or myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • Radioisotopes that are useful for treatment of cancer include, but are not limited to, Iodine-131 ( 131 I), Rhenium-188 ( 188 Re), Yttrium-90 ( 90 Y), Bismuth-213 ( 213 Bi), and Actinium-225 ( 225 Ac).
  • Examples of radioisotope-linked antibodies and techniques for producing such antibodies are also well known in the art (see, e.g., Rosenblat TL, et al. “Sequential cytarabine and alpha-particle immunotherapy with bismuth-213-lintuzumab (HuM195) for acute myeloid leukemia”. Clin Cancer Res .
  • Antibodies comprising a radioisotope are advantageous to use therapeutically, in part because the radioisotopes are toxic and can be targeted to particular cell types expressing cell surface NPM1 (e.g., NPM1 -expressing cancer cells).
  • NPM1 e.g., NPM1 -expressing cancer cells.
  • the toxicity of the radioisotope may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, compared to the radioisotope in its free from.
  • compositions comprising an antibody that binds to NPM1 provided herein (e.g., an antibody that binds to WT NPM1 or mutant NPM1).
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a “pharmaceutically acceptable carrier” may be a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agents from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the patient (e.g., physiologically compatible, sterile, physiologic pH, etc.).
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as
  • compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy.
  • unit dose when used in reference to a pharmaceutical composition of the present disclosure refers to physically discrete units suitable as unitary dosage for administration to a subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the formulation of the pharmaceutical composition may dependent upon the route of administration to a subject.
  • injectable preparations suitable for parenteral administration or intraperitoneal, intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oleaginous suspensions and may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 propanediol or 1,3 butanediol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P.
  • injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir or an emulsion.
  • compositions e.g., pharmaceutical compositions administered to a subject
  • sterile filtration membranes e.g., 0.2 micron membranes
  • preservatives can be used to prevent the growth or action of microorganisms.
  • Various preservatives are well known and include, for example, phenol and ascorbic acid.
  • the pharmaceutical composition ordinarily will be stored in lyophilized form or as an aqueous solution if it is highly stable to thermal and oxidative denaturation.
  • the pH of the preparations typically will be about from 6 to 8, although higher or lower pH values can also be appropriate in certain instances.
  • the present disclosure further provides for the administration of an anti-NPMl antibody described herein, a conjugate comprising an anti-NPMl antibody described herein, or a composition thereof (e.g., a pharmaceutical composition) to a subject.
  • a method is provided for treating a disease associated with cell surface expression of NPM1 (e.g., a NPM1 -expressing cancer) by administering an anti-NPMl antibody described herein, a conjugate comprising an anti-NPMl antibody described herein, or a composition thereof (e.g., a pharmaceutical composition) to a subject.
  • a method for evaluating the presence of a disease associated with cell surface expression of NPM1 (e.g., a NPM1 -expressing cancer) by administering an anti-NPMl antibody described herein, a conjugate comprising an anti-NPMl antibody described herein, or a composition thereof (e.g., a pharmaceutical composition) to a subject.
  • NPM1 e.g., a NPM1 -expressing cancer
  • the present disclosure provides a method for treating a NPM1- expressing cancer in a subject, comprising administering an effective amount of an anti-NPMl antibody described herein, a conjugate comprising an anti-NPMl antibody that is conjugated to an agent (e.g., a drug, a radioisotope) described herein, or a composition thereof (e.g., a pharmaceutical composition), to a subject in need thereof.
  • an agent e.g., a drug, a radioisotope
  • a composition thereof e.g., a pharmaceutical composition
  • administer refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing an agent described herein (e.g., an antibody, a conjugate), or a composition thereof (e.g., a pharmaceutical composition), in or on a subject.
  • treatment refers to the application or administration of an agent described herein (e.g., an antibody, a conjugate), or a composition thereof (e.g., a pharmaceutical composition), to a subject in need thereof for the purpose of reducing the severity of a disease (e.g., a cancer) in the subject.
  • a “subject in need thereof’ refers to an individual that has a disease, a symptom of the disease, or a predisposition toward the disease.
  • a method for treating a disease may encompass administering to a subject an agent described herein (e.g., an antibody, a conjugate), or a composition thereof (e.g., a pharmaceutical composition) with the intention to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, a symptom of the disease, or predisposition toward the disease in the subject.
  • a method for treating a disease may encompass prophylaxis, wherein an agent is administered to the subject for the purpose of preventing development of the disease, for example, in a subject that is not known to have the disease, but may develop or be at risk of developing the disease in the future.
  • a “therapeutically effective amount” or “effective amount” refers to the amount of an agent (e.g., an antibody or conjugate described herein) that is sufficient to elicit the desired biological response in the subject, for example, alleviating one or more symptoms of the disease (e.g., a cancer).
  • a therapeutically effective amount may be an amount that is either administered to the subject alone or in combination with one or more other agents.
  • an effective dose is lower than the highest dose that can be safely administered to the subject. It will be understood by those of ordinary skill in the art, however, that a subject or health practitioner may select a lower dose (e.g., the minimum effective dose) in order to mitigate any potential risks of treatment, such as side effects of the treatment.
  • doses ranging from about 0.01 to 1000 mg/kg of an agent (e.g., an antibody or conjugate described herein) may be administered. In some embodiments, the dose is between 1 to 200 mg.
  • an agent e.g., an antibody or conjugate described herein
  • the particular dosage regimen i.e., the dose, timing, and repetition, will depend on the particular subject and that subject's medical history, as well as the properties of the agent (such as the pharmacokinetics of the agent) and other consideration well known in the art.
  • Treating a disease may include delaying the development or progression of the disease or reducing disease severity. Treating the disease does not necessarily require curative results.
  • a method that delays the development of a disease, or delays the onset of the disease is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and/or reduces extent of the symptoms in a given time frame, as compared to the absence of such a method. Comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.
  • development refers to initial manifestations and/or ensuing progression of the disease in a subject. Development of a disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression may refer to the development or progression of symptoms of a disease.
  • development includes the occurrence, recurrence, and onset of a disease. As used herein “onset” or “occurrence” of a disease includes the initial onset of a disease, as well as recurrence of the disease (i.e., in a subject who has had the disease previously).
  • a “subject” to which administration is contemplated refers to a human (i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or a non-human animal.
  • a human i.e., male or female of any age group, e.g., pediatric subject (e.g., infant, child, or adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) or a non-human animal.
  • the non-human animal is a mammal (e.g., rodent, e.g., mouse or rat), a primate (e.g., cynomolgus monkey or rhesus monkey), a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog), or a bird (e.g., commercially relevant bird, such as chicken, duck, goose, or turkey).
  • the non-human animal may be a male or female at any stage of development and may be a juvenile animal or an adult animal.
  • the non-human animal may be a transgenic animal or genetically engineered animal.
  • the subject is a companion animal (e.g., a pet or service animal).
  • a companion animal refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
  • the subject is a research animal. Non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.
  • the subject has, is suspected of having, or is at risk for a NPM1- expressing cancer.
  • NPM1 -expressing cancer refers to a cancer that is characterized by expression of NPM1 (e.g., enhanced expression of NPM1, as compared to a noncancerous cell), which may be further characterized by a change in the subcellular localization of NPM1, such as, for example, an increase of NPM1 present on the cell surface.
  • An NPM1 -expressing cancer may express WT NPM1 and/or mutant NPM1.
  • a NPM1 -expressing cancer may express WT NPM1 and/or mutant NPM1 on the cell surface.
  • a NPM1 -expressing cancer may also refer to a cancer which is not initially characterized by expression of NPM1, that expresses NPM1 (e.g., cell surface WT NPM1 and/or mutant NPM1) in response to administration of or treatment with an agent (e.g., a chemotherapeutic drug).
  • a NPM1 -expressing cancer is a solid (tissue) or liquid (biological fluid, e.g., blood) cancer.
  • a NPM1- expressing cancer is a cancer selected from a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer.
  • a NPM1- expressing cancer is acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • a NPM1 -expressing cancer is a metastatic cancer.
  • a NPM1 -expressing cancer is a therapy -related cancer or a secondary malignancy.
  • a therapy-related cancer or a secondary malignancy may be a cancer, such as a leukemia, carcinoma, or a lymphoma, resulting from a previous treatment with a chemotherapeutic or a radioisotope.
  • a NPM1 -expressing cancer is therapy-related AML (t- AML) or a secondary malignancy of non-
  • the subject has been treated previously for a NPM1 -expressing cancer.
  • the subject has a NPM1 -expressing cancer that is resistant toward one or more treatments (e.g., treatment with one or more chemotherapeutic drugs).
  • a NPM1- expressing cancer is said to be resistant toward a treatment (e.g., treatment with one or more chemotherapeutic drugs) if the treatment cannot effectively kill and/or inactivate cells of the cancer, for example, due to genetic and/or epigenetic changes occurring in the cancer that result in inactivation and/or efflux of one or more drugs of the treatment, or inhibition of one or more downstream effects of the treatment, especially if the treatment was previously effective for killing and/or inactivating cells of the cancer.
  • a NPM1 -expressing cancer that is resistant toward one or more treatments expresses NPM1 on the surface of cancer cells as a result of a previous treatment (e.g., a previous treatment with one or more chemotherapeutic drugs) to which the cancer is or has become resistant to.
  • the level of NPM1 on the surface of a NPM1 -expressing cancer that is resistant toward one or more treatments is increased as a result of a previous treatment (e.g., a previous treatment with one or more chemotherapeutic drugs) to which the cancer is or has become resistant to.
  • the antibody, conjugate, or composition thereof can be administered via any conventional route, e.g., administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, intraperitoneal, or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraperitoneal, intrathecal, intralesional, and intracranial injection or infusion techniques.
  • the antibody, conjugate, or composition thereof is administered via intravenous injection or infusion.
  • the antibody, conjugate, or composition thereof can be administered to the subject via injectable depot routes of administration such as using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods.
  • the antibody, conjugate, or composition thereof e.g., a pharmaceutical composition
  • the injection is intravenous injection or intratumoral injection.
  • the administration occurs more than once. In some embodiments, the administration occurs once per day, once per 2 days, once per 3 days, once per 4 days, once per 5 days, once per 6 days, once per week, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year.
  • the administration results in reduced growth of NPM1 -expressing cancer cells of the subject, as compared to growth of NPM1 -expressing cancer cells of the subject in the absence of the administration.
  • reduced growth refers to a reduction in the rate of cell division (mitosis) occurring in NPM1 -expressing cancer cells of the subject.
  • the administration results in reduced growth of NPM1- expressing cancer cells of the subject by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7- fold, up to 8-fold, up to 9-fold, or up to 10-fold, as compared to growth of NPM1 -expressing cancer cells in the absence of the administration.
  • the administration results in increased cell death of NPM1- expressing cancer cells of the subject, as compared to cell death of NPM1 -expressing cancer cells of the subject in the absence of the administration.
  • cell death refers to an increase in the rate of cell death occurring in NPM1 -expressing cancer cells of the subject via any pathway by which cells cease to be viable, such as, but not limited to, apoptosis, autophagy, necrosis, and entosis.
  • the administration results in increased cell death of NPM1 -expressing cancer cells of the subject by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, or up to 10-fold, as compared to cell death of NPM1- expressing cancer cells of the subject in the absence of the administration.
  • the present disclosure further provides a method for evaluating the presence of (detecting) a NPM1 -expressing cancer in a subject, comprising administering an effective amount of an anti-NPMl antibody described herein, a conjugate comprising an anti- NPM1 antibody that is conjugated to an imaging agent described herein, or a composition thereof (e.g., a pharmaceutical composition), to a subject in need thereof.
  • a method for evaluating the presence of (detecting) a NPM1 -expressing cancer in a subject comprising administering an effective amount of an anti-NPMl antibody described herein, a conjugate comprising an anti- NPM1 antibody that is conjugated to an imaging agent described herein, or a composition thereof (e.g., a pharmaceutical composition), to a subject in need thereof.
  • such a method comprises administering to a subject in need thereof an effective dose of an anti-NPMl antibody described herein, a conjugate between an anti-NPMl antibody described herein and an imaging agent, or a composition thereof (e.g., a pharmaceutical composition), imaging the antibody or conjugate, and determining the presence of a NPM1 -expressing cancer in the subject based on the level and position of the antibody or conjugate imaged in the subject.
  • the effective amount will vary, as recognized by those skilled in the art, depending on the particular condition to be detected, the severity of the condition, the individual subject parameters including age, physical condition, size, gender and weight, the duration of the detection, the specific route of administration and like factors within the knowledge and expertise of the health practitioner.
  • the NPM1 -expressing cancer is a cancer in which WT NPM1 and/or mutant NPM1 is expressed on the surface of cancer cells.
  • a NPM1 -expressing cancer is a solid (tissue) or liquid (biological fluid, e.g., blood) cancer.
  • a NPM1 -expressing cancer is a cancer selected from a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer.
  • a NPM1 -expressing cancer is acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), nonHodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • a NPMl- expressing cancer is a metastatic cancer.
  • a NPM1 -expressing cancer is a therapy-related cancer or a secondary malignancy.
  • a NPM1 -expressing cancer is therapy -related AML (t-AML) or a secondary malignancy of non-Hodgkin’s lymphoma.
  • the imaging conducted is luminescent imaging or fluorescent imaging.
  • the method of imaging may be selected based on the imaging agent administered to the subject, for example, the type of imaging agent conjugated to or binding to an anti-NPMl antibody administered to the subject.
  • the imaging is magnetic resonance imaging (MRI).
  • a method of evaluating the presence of (detecting) an NPM1- expressing cancer in a subject further comprises administering an effective amount of an anti- NPM1 antibody, a conjugate comprising an anti-NPMl antibody and an agent (e.g., a drug, a radioisotope) described herein, or a composition thereof (e.g., a pharmaceutical composition) to the subject, for example, if a NPM1 -expressing cancer is determined to be present in the subject.
  • an agent e.g., a drug, a radioisotope
  • an anti-NPMl antibody, a conjugate comprising an anti-NPMl antibody and an agent e.g., a drug, a radioisotope
  • a composition thereof e.g., a pharmaceutical composition
  • the effective amount of an anti-NPMl antibody, a conjugate comprising an anti-NPMl antibody and an agent (e.g., a drug, a radioisotope), or a composition thereof (e.g., a pharmaceutical composition) to be administered to the subject may depend on a variety of factors that are within the knowledge and expertise of the health practitioner to determine, including the amount and location of the NPM1 -expressing cancer that is determined to be present in the subject.
  • the present disclosure provides ex vivo methods for evaluating the presence of (detecting) a NPM1 -expressing cancer in a subject.
  • some aspects of the present disclosure relate to a method for evaluating the presence of (detecting) a NPM1- expressing cancer in a subject, comprising collecting a biological sample from a subject in need thereof (or providing a biological sample previously obtained from such a subject), contacting the biological sample with an anti-NPMl antibody or a conjugate between an anti-NPMl antibody and an imaging agent described herein, analyzing binding between the antibody or conjugate and NPM1 -expressing cancer cells in the biological sample, and determining the presence of a NPM1 -expressing cancer in the subject, based on the level of binding between the conjugate and NPM1 -expressing cancer cells analyzed in the sample.
  • the NPM1 -expressing cancer is a cancer in which WT NPM1 and/or mutant NPM1 is expressed on the surface of cancer cells.
  • a NPM1 -expressing cancer is a solid (tissue) or liquid (biological fluid, e.g., blood) cancer.
  • a NPM1 -expressing cancer is a cancer selected from a hematological cancer, a lung cancer, a breast cancer, a brain cancer, a gastrointestinal cancer, a liver cancer, a kidney cancer, a bladder cancer, a pancreatic cancer, an ovarian cancer, a testicular cancer, a prostate cancer, an endometrial cancer, a muscle cancer, a bone cancer, a neuroendocrine cancer, a connective tissue cancer, a head or neck cancer, or a skin cancer.
  • a NPM1 -expressing cancer is acute myeloid leukemia (AML), acute promyeloid leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • APL acute promyeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MDS myelodysplastic syndrome
  • a NPM1 -expressing cancer is a metastatic cancer.
  • a NPM1 -expressing cancer is a therapy -related cancer or a secondary malignancy.
  • a NPM1 -expressing cancer is therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin’s lymphoma.
  • the biological sample collected from the subject comprises a tissue biopsy, a blood sample, a serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a fecal sample, a lymphatic fluid sample, a synovial fluid sample, a cerebrospinal fluid sample, or an interstitial fluid sample.
  • a tissue biopsy is a tumor biopsy.
  • the biological sample collected from the subject is a blood sample, a serum sample, or a plasma sample.
  • the analysis conducted is luminescent analysis or fluorescent analysis.
  • the method of analysis may be selected based on the type of imaging agent conjugated to an anti-NPMl antibody described herein.
  • the anti-NPMl antibody is conjugated to a luminescent or fluorescent imaging agent.
  • the analysis is performed via flow cytometry.
  • binding between an antibody or a conjugate described herein and NPM1 -expressing cancer cells in the sample may alternately be analyzed by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • binding between an antibody or a conjugate described herein and NPM1 -expressing cancer cells in the sample is analyzed by cell-based ELISA using techniques that are well known in the art (see, e.g., Molnar E., “Cell-Based Enzyme-Linked Immunosorbent Assay (Cell-ELISA) Analysis of Native and Recombinant Glutamate Receptors.” Methods Mol Biol.
  • the total level of NPM1 (WT or mutant NPM1) expressed by cells in the sample (e.g., cancer cells) and/or the level of NPM1 (WT or mutant NPM1) localized on the surface of cells in the sample (e.g., cancer cells) may be further analyzed using techniques that are well known in the art, such as, for example, fractionation of cellular membranes followed by immunoblotting for NPM1, e.g., by using one or more anti- NPMl antibodies described herein.
  • a method of evaluating the presence of (detecting) an NPM1- expressing cancer in a subject further comprises administering an effective amount of an anti- NPM1 antibody, a conjugate comprising an anti-NPMl antibody and an agent (e.g., a drug, a radioisotope) described herein, or a composition thereof (e.g., a pharmaceutical composition) to the subject, for example, if a NPM1 -expressing cancer is determined to be present in the subject.
  • an agent e.g., a drug, a radioisotope
  • an anti-NPMl antibody, a conjugate comprising an anti-NPMl antibody and an agent e.g., a drug, a radioisotope
  • a composition thereof e.g., a pharmaceutical composition
  • the effective amount of an anti-NPMl antibody, a conjugate comprising an anti-NPMl antibody and an agent (e.g., a drug, a radioisotope), or a composition thereof (e.g., a pharmaceutical composition) to be administered to the subject may depend on a variety of factors that are within the knowledge and expertise of the health practitioner to determine, including the amount and location of the NPM1 -expressing cancer that is determined to be present in the subject.
  • Example 1 NPM1 is localized on the surface of human leukemia cells.
  • nucleophosmin 1 Various human leukemia cell lines were evaluated for cell surface expression of nucleophosmin 1 (NPM1). Briefly, K562, Kasumi, 0CI-AML2, 0CI-AML3, M0LM13, Jekol, Nalm6, Jurkat, and SupTl cells were cultured in vitro with RPM1 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen/Strep). Cellular membrane and cytosolic fractions were collected using established techniques (see, e.g., Flynn et al., Cell. 2021; 184(12):3109-3124.
  • FBS fetal bovine serum
  • Pen/Strep penicillin-streptomycin
  • NPM1-WT (Santa Cruz Biotechnology # sc-32256), NPMl-Mut (Thermo Scientific # PA1- 46356), beta-actin (Santa Cruz Biotechnology # sc-47778), and RPN1 (Santa Cruz Biotechnology # sc-48367).
  • WT wild-type
  • Mut mutant
  • NPM1 neutrophil-associated cytotoxic payloads to NPM1 -expressing cancers in patients, such as, for example, in patients with acute myeloid leukemia (AML), non-Hodgkin’s lymphoma, and myelodysplastic syndrome (MDS).
  • AML acute myeloid leukemia
  • MDS myelodysplastic syndrome
  • PB peripheral blood
  • BM bone marrow
  • NPM1 cell surface expression was relatively low, but was increased in cells expressing mutant NPM1 (FIGs. 1C and ID). These results were confirmed by FACS analysis of human BM cells, in which an antibody specific for WT NPM1 did not effectively bind human BM cells (FIG. IE). These results strongly suggest that an antibody specific for NPM1 could be used to effectively treat NPM1 -expressing cancers.
  • 0CI-AML3 cells are a model for human acute myeloid leukemia (AML). Briefly, approximately 150,000 cells were cultured in RPMI media with 10% FBS and 1% Pen/Strep. Streptavidin-Saporin (Strep-ZAP) was bound to biotinylated antibodies at 2.6 pg Strep-ZAP to 1 pg IgG for 30 minutes on ice. Strep-ZAP was bound to either biotinylated Abl anti-NPMl antibody (FIG.
  • NPM1 antibodies could be used to target endogenous NPM1 on the surface of non-human cell types, due to the high degree of conservation of NPM1.
  • the amino acid sequence of WT NPM1 is approximately 95% identical between humans and mice (FIG. 4A), and greater than 99% identical between humans and other primates (FIG. 4B).
  • the high degree of conservation also suggests that results obtained by testing the effect of anti-NPMl antibodies or ADCs in, for example, murine cells or in mice, would be expected to extend to humans.
  • Example 3 Antibodies detect WT and mutant NPM1 on the surface of human and murine models
  • NPM1 antibodies could be used to target NPM1 on cancer cells
  • the level of NPM1 on the surface of various human and murine cancer cells was explored using FACS analysis.
  • live cells were assayed to observe cell surface localized NPM1.
  • fixed cells were assayed to observe intracellular and cell surface levels of NPM1.
  • Significant levels of NPM1 were observed on the surface of live 0CI-AML3 cells, however NPM1 was not robustly observed in significant quantities on M0LM13 cells (FIG. 5 A).
  • FIGs. 5B and 5C Similar to 0CI-AML3 cells, significant levels ofNPMl were observed on the surface of various primary murine AML cell lines (FIGs. 5B and 5C).
  • ADCs comprising either of these anti-NPMl antibodies could be used to selectively direct chemotherapeutic agents to cancer cells, with low risk of toxicity against noncancerous tissue.
  • These antibodies could further be used in other applications, including, for example, diagnostic agents by instead conjugating the antibodies with an imaging agent, which can in turn be used to locate and measure the relative abundance of cancer cells in a patient.
  • NPM1 is a cell surface protein.
  • nucleophosmin 1 confocal imaging was performed on 0CI-AML3 (human cell line). Both intracellular and cell surface staining was performed. Intracellular staining was achieved by fixing and permeabilizing the cells prior to staining. Comparatively, cell surface staining was performed by staining the cells prior to fixation. The results of the staining show that NPM1 is found both inside the cells and on the cell surface. Inside the cells, NPM1 is nucleolar and on the cell surface, NPM1 forms clusters (FIG. 9A).
  • RNA Binding Proteins include Heterogeneous nuclear ribonucleoprotein U (HNRNPU), Nucleolar RNA helicase 2 (DDX21), Dolichyl-diphosphooligosaccharide protein glycosyltransferase subunit 1 (RPN1), and RIO Kinase 1 (RI0K1).
  • HNRNPU Heterogeneous nuclear ribonucleoprotein U
  • DDX21 Nucleolar RNA helicase 2
  • RPN1 Dolichyl-diphosphooligosaccharide protein glycosyltransferase subunit 1
  • RI0K1 RIO Kinase 1
  • NPM1 primary murine acute myeloid leukemia
  • NPM1 The cell surface localization of NPM1 was also tested using western blot.
  • anti-NPMl captures full length NPM1 from cellular membrane fractions (FIG. 11 A).
  • Cell surface NHS- biotinylation (only surface proteins) followed by anti-NPMl IP from membrane fractions selectively isolates a biotinylated band at the molecular weight of NPM1 (FIG. 11B).
  • NPM1 is a cell surface protein in both human and murine cell lines. Additionally, the super resolution microscopy demonstrated that on the cell surface NPM1 forms regular nanoclusters on various human cancer cell lines, including, acute myeloid leukemia and pancreatic carcinoma.
  • Example 6 Ab2.2 antibody targets NPM1.
  • Ab2.2 An antibody having the Heavy chain (Ab2.2) (SEQ ID NO: 44) and Light chain (Ab2.2) (SEQ ID NO: 45), hereinafter “Ab2.2”, was generated and used to target NPM1 on the surface of human and non-human cell types.
  • Ab2.2 The ability of Ab2.2 to bind to NPM1 on the cell surface of cancer cells (0CI-AML3 cells) was tested using both live cell microscopy and microscopy of fixed and permeabilized cells.
  • Ab2.2 was compared to commercially available NPM1 antibodies.
  • the commercially available NPM1 antibody used was Santa Cruz (SC) Anti-NPM1-AF647.
  • SC Santa Cruz
  • the live cell microscopy shows that both commercially available NPM1 antibodies and Ab2.2 stain surface puncta. Therefore, Ab2.2 performs similarly to commercially available NPM1 antibodies on the cell surface (FIG. 13).
  • the microscopy of the fixed and permeabilized cells shows that commercially available NPM1 antibodies results in nucleolar and cytoplasmic, whereas the Ab2.2 antibody results in nucleolar and more major cytoplasmic staining. Therefore, Ab2.2 detects more cytoplasmic NPM1 in mutant 0CI-AML3 cells compared to commercially available NPM1 antibodies (FIG. 13).
  • the ability of Ab2.2 to bind to NPM1 on the cell surface of healthy, non-cancerous, cells was also tested using flow cytometry.
  • the cells measured for Ab2.2 binding were leukocytes (CD45+ cells), myeloid cells (CD33+ cells), and hematopoietic stem cells (“HSCs”; CD34+ cells).
  • HSCs hematopoietic stem cells
  • the Ab2.2 antibody was further validated using western blot and compared to commercially available antibodies.
  • the commercially available antibody was supplied from Santa Cruz (SC FC8791). Two sources of lysate were evaluated, WCE and crude membrane (Mem). Both Ab2.2 and the commercially available antibody resulted in a band around 38kB. Therefore, commercially available and Ab2.2 show near-identical banding pattern to NPM1 (FIG. 14B)
  • Example 7 Ab2.2 targets NPM1 in cancer cells in vitro.
  • Ab2.2 binds best to LSCs in NPMlc patient marrow (FIGs. 18D-18E).
  • the results from the 12 AML patients in FIG. 15 indicate that Ab2.2 strongly stains the blasts, agnostic to mutational status, disease state, or prior treatment.
  • the results from the 15 AML patients in FIG. 17 show that Ab2.2 strongly stains the blasts.
  • NPMlc patients who’s LSCs are CD34-, Ab2.2 strongly stains and therefore Ab2.2 will target leukemia initiating cells.
  • Example 8 Ab2.2 does not cause toxicity in mice.
  • WT wild-type mice received weekly treatment of Ab2.2. There were 5 mice per group.
  • the Target group received Ab2.2 administered by IP injection at a dose of 2.5 mg/kg, 5mg/kg, or lOmg/kg.
  • the control group was administered 5mg/kg of IgG (FIG. 19A).
  • Four doses total were delivered, one dose per week.
  • Weekly bleeds were performed for sample collection on days (D) 1, 7, 14, 20 and 27 of treatment.
  • WBC white blood cells
  • PHT platelets
  • HGB hemoglobin
  • Example 9 Ab2.2 treatment improves survival in mouse model of AML and does not affect healthy mice.
  • mice were subjected sub-lethal irradiation, followed by transplantation of primary murine AML cells.
  • the mice receiving the primary murine AML were administered weekly antibody treatments of Ab2.2 at a dose of 5mg/kg. Administration occurred by IP injection. Weekly bleeds were performed and the overall survival of mice, analysis of bone marrow (BM) and spleen and molecular phenotyping were performed (FIG. 20A).
  • the primary murine cells used were NPMlc/Flt3-ITD AML and a Syngeneic AML mouse model was used.
  • the control group received 5mg/kg dose (IgG) and the target group received 5mg/kg dose (Ab2.2).
  • mice were subjected sub-lethal irradiation, followed by transplantation of primary murine AML cells.
  • the mice receiving the primary murine AML was administered weekly antibody treatments of Ab2.2 at a dose of 5mg/kg. Administration occurred by IP injection. Weekly bleeds were performed and the overall survival of mice, and molecular phenotyping were performed (FIG. 21A).
  • the primary murine cells used for transplantation were MLL-AF9/Flt3-ITD AML.
  • a syngeneic AML mouse model was used.
  • the control group received 5mg/kg dose (IgG) and the target group received 5mg/kg dose (Ab2.2).
  • a total of 4 doses of Ab2.2 were administered. Antibody binding was observed (FIG. 21B).
  • the mean survival of the control group was about 20 days, compared to about 55 days in the target group (FIG. 21C). Therefore, Ab2.2 improves overall survival in AML model with lower surface levels of NPM1. Further, these results indicate that any amount of surface expression of NPM1 will lead to survival benefit with Ab2.2 treatment.
  • Efficacy model 2 was also used to assess the LSC targeting of Ab2.2 in secondary recipients (FIG. 22A).
  • the transplanted primary murine cells were MLL-AF9/Flt3-ITD AML and a syngeneic AML mouse model was used.
  • the control group received 5mg/kg dose (IgG) and the target group received 5mg/kg dose (Ab2.2).
  • One dose of Ab2.2 was administered before secondary recipients received transplantation.
  • Secondary recipients in the control group received transplanted cells from IgG treated mouse.
  • the target group received transplanted cells from Ab2.2 treated mouse. The same number of cells were transplanted, and a survival assay, flow cytometry, examination of engraftment and LSC functional measurement were performed.
  • the results of the survival assay demonstrate that the mean survival of the control group was less than 30 days, compared to about 50 days in the target group (FIG. 22C).
  • the extension of life in the Ab2.2 treated mice demonstrate that fewer stem cells were present in Ab2.2 treated mice.
  • Flow cytometry analysis showed that Ab2.2 stains the stem cell compartment fractionally better than the bulk tumor (FIG. 22D) and MLL-AF9 model has low expression of NPM1 on the surface (FIG. 22E).
  • WBC count was decreased in Ab2.2 treated mice and PLT was increased (FIG. 22F). Further analysis of the WBC over time demonstrated that WBC remain lower in Ab2.2 treated mice compared to IgG treated over time (FIG. 22G).
  • the spleens of Ab2.2 treated secondary recipients weighed less than the IgG treated mice (FIG. 22H). These results indicate that Ab2.2 treatment results in robust reduction of organ weight, indicating tumor clearance.
  • the bone marrow (BM) and peripheral blood (PB) were assessed for percentage (%) of AML. In both the BM and PB there was a reduction in AML % in Ab2.2 treated groups compared to IgG controls (FIG. 221). These results indicate that Ab2.2 targets tumors after only one dose.
  • Wild type mice receiving sub-lethal irradiation were tested for effects of Ab2.2 treatment.
  • WT C57BL/6J mice were subjected to sub-lethal irradiation and four treatments of Ab2.2.
  • Ab2.2 treatments occurred weekly via IP injection and weekly bleeds were performed.
  • the control group received 5mg/kg of IgG and Ab2.2 treated groups received lOmg/kg of Ab2.2.
  • Regular blood counts, animal phenotyping and measurement of adverse/toxic effects were performed (FIG. 25A).
  • the results show that between day (D) 1 and D27 there was no significant difference observed between the weight, WBC count, HGB levels or PLT levels of IgG treated and Ab2.2 treated mice (FIG. 25B). Therefore, WT mice do not exhibit an observable effect of Ab2.2 treatment.
  • Example 10 Ab2.2 effect on survival depends on immune system.
  • the immune mediated killing ability of Ab2.2 was assessed using efficacy model 3.
  • efficacy model 3 NSG mice receiving transplantation of primary murine AML cells were given weekly Ab2.2 treatments and assessed for overall survival (FIG. 23A).
  • the transplanted primary murine cells were MLL-AF9/Flt3-ITD AML.
  • the mouse model used was an immunocompromised AML mouse model (NSG).
  • the control group received 5mg/kg dose (IgG) and the target group received 5mg/kg dose (Ab2.2).
  • a total of four doses of Ab2.2 were administered.
  • the results of the survival assay demonstrate that the mean survival of the control group and the Ab2.2 treated group were about 30 days (FIG. 23B). Therefore, Ab2.2 does not provide survival benefit without an intact immune system.
  • Example 11 Ab2.2 treatment reduces tumor volume in vivo.
  • the solid tumor activity of Ab2.2 was assessed in vivo using efficacy model 4.
  • mice that had received transplantation were subjected to weekly treatment of Ab2.2 and assessed for overall survival, calculation of tumor burden and molecular phenotyping (FIG. 24A).
  • the transplanted cells were from a MC38 mouse colorectal adenocarcinoma.
  • a syngeneic mouse model was used.
  • the control group received lOmg/kg dose (IgG) and the target group received lOmg/kg dose (Ab2.2).
  • a total of three doses (1 per week) were administered.
  • the results of the calculation of tumor burden demonstrate that on day 10 and day 13 Ab2.2 reduces tumor volume (FIGs. 24B-24C).
  • Example 12 Ab2.2 present on pre-cancerous cells.
  • DNMT3a mutations are directly associated with clonal hematopoiesis of indeterminate potential (CHIP) and pre-leukemia.
  • CHIP indeterminate potential
  • DNMT3a mutations are directly associated with clonal hematopoiesis of indeterminate potential (CHIP) and pre-leukemia.
  • I)mnl3a p ' 22211 and DmnlSa 1 ' 22211 ⁇ /Npmlc cells were stained with either IgG control or Ab2.2. The results show that there is high surface detection of Npmlc even on single Dmnt3a R882H mutant cells (FIG. 27).
  • Example 13 Ab2.2 binds to human tumors in vitro.
  • the tumor models assessed for Ab2.2 binding were murine melanoma, lung carcinoma (human), Laryngeal carcinoma, colorectal (human and murine), Ewing sarcoma (Human), Pharyngeal carcinoma (Human), Pancreatic carcinoma (Human), Oesophageal cancer (Human), Osteosarcoma (Human), Neuroblastomas (Human), Brain tumors (Human), Hematological malignancies, Fibrosarcoma, Prostate cancers, and Pancreatic adenocarcinoma (Murine) (FIGs. 26A-26O). The results show that Ab2.2 binds to a diverse set of human tumor models in vitro.
  • Example 14 Ab2.2-Saporin ADC exhibits in vitro activity.
  • OCI-AML3 cells were treated with either a negative control, isotype Saporin conjugate or Ab2.2-Saporin conjugate. Cells were treated over 24, 48 and 72 hours. The number of live cells per pL were assessed at those timepoints. At 72 hours, the number of live cells were about 30% decreased in Ab2.2-Saporin conjugate treated cells compared to negative control (FIG. 28). Therefore, NPM1 can serve as a ADC target in at least some models.
  • Example 15 Ab2.2 treatment extends lifespan of mice engrafted with human AML cell line.
  • the ability of Ab2.2 to extend lifespan in mouse models xenografted with human AML cell line was assessed.
  • SCID CB17 mice received transplantation of 0CI-AML3 cells (human AML).
  • engrafted mice received weekly treatment of Ab2.2 (target group) or IgG (control group).
  • the target group received lOmg/kg dose of Ab2.2.
  • Mice received a total of 4 doses. Mice were assessed for overall survival, and molecular phenotyping (FIG. 29A).
  • FIG. 29C Therefore, lifespan extension is observed in mice engrafted with human AML cell line and with natural killer (NK) cells and complement that are treated with Ab2.2.
  • Example 16 A62.2 treatment extends lifespan of mice engrafted with human AML-PDX cell line.
  • Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
  • the disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
  • URL addresses are provided as non-browser-executable codes, with periods of the respective web address in parentheses.
  • the actual web addresses do not contain the parentheses.
  • any particular embodiment of the present disclosure may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the disclosure, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Engineering & Computer Science (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Botany (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/US2023/066299 2022-04-27 2023-04-27 Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease Ceased WO2023212646A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US18/860,335 US20250313647A1 (en) 2022-04-27 2023-04-27 Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease
JP2024563536A JP2025514288A (ja) 2022-04-27 2023-04-27 ヒト疾患の診断および治療標的としての突然変異体および誤局在細胞表面ヌクレオフォスミン１
CN202380050132.2A CN119487077A (zh) 2022-04-27 2023-04-27 突变体和错误定位的细胞表面核仁磷蛋白1作为人疾病的诊断和治疗靶标
EP23797543.8A EP4514858A2 (en) 2022-04-27 2023-04-27 Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GR20220100348 2022-04-27
GR20220100348 2022-04-27

Publications (2)

Publication Number Publication Date
WO2023212646A2 true WO2023212646A2 (en) 2023-11-02
WO2023212646A3 WO2023212646A3 (en) 2023-11-30

Family

ID=88519861

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2023/066299 Ceased WO2023212646A2 (en) 2022-04-27 2023-04-27 Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease

Country Status (5)

Country Link
US (1) US20250313647A1 (enExample)
EP (1) EP4514858A2 (enExample)
JP (1) JP2025514288A (enExample)
CN (1) CN119487077A (enExample)
WO (1) WO2023212646A2 (enExample)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11896619B2 (en) * 2020-03-10 2024-02-13 Massachusetts Institute Of Technology Compositions and methods for immunotherapy of NPM1c-positive cancer

Also Published As

Publication number Publication date
EP4514858A2 (en) 2025-03-05
CN119487077A (zh) 2025-02-18
JP2025514288A (ja) 2025-05-02
WO2023212646A3 (en) 2023-11-30
US20250313647A1 (en) 2025-10-09

Similar Documents

Publication Publication Date Title
US11739158B2 (en) Antibody binding specifically to MUC1 and use thereof
JP2020532509A (ja) 活性化可能抗ｃｄ１６６抗体およびその使用方法
JP2022512920A (ja) 活性化可能抗ｃｄ１６６抗体およびその使用方法
JP6577016B2 (ja) 治療及び診断のための抗コラーゲン抗体
TWI844215B (zh) 醫藥組合
JP2010528056A (ja) がん細胞の細胞殺作用を媒介するヒト化抗ｔｒｏｐ−２抗体とキメラ抗ｔｒｏｐ−２抗体
JP2017505107A (ja) Ｃｃｒ９に対する抗体およびその用途
JP2015509948A (ja) 抗ｓｅｚ６抗体及び使用方法
US20220280653A1 (en) Intercellular adhesion molecule 1 (icam1) antibody drug conjugate and uses thereof
KR20180017088A (ko) B-세포 악성종양의 치료를 위한 수단 및 방법
PT1904102E (pt) Anticorpos anti-receptor ccr7 para o tratamento do cancro
US20250313647A1 (en) Mutant and mislocalized cell surface nucleophosmin 1 as a diagnostic and therapeutic target of human disease
US20250325717A1 (en) Anti-nucleophosmin 1 antibody and antibody conjugate combination therapies
JP2024507275A (ja) 細胞間接着分子１（ｉｃａｍ１）抗体薬物抱合体およびそれらの使用
JP2021527646A (ja) 医薬組合せ
TWI855692B (zh) 抗cd200r1抗體
TWI904701B (zh) 抗ceacam5/6雙結合抗體
US20250264470A1 (en) Diagnosis, prognosis and treatment of malignant neoplasms
WO2021258140A1 (en) Cd83 binding protein conjugates for treating lymphoma
EA046736B1 (ru) Фармацевтические комбинации
EA042216B1 (ru) Фармацевтическая комбинация, содержащая антитело к ly75
EA047995B1 (ru) Фармацевтическая комбинация, содержащая антитело к ly75 и ибрутиниб, и способы ее применения
BR112017018521B1 (pt) Uso de conjugados de fármacos compreendendo anticorpos anticldn18.2, conjugado anticorpo-fármaco e formulação farmacêutica compreendendo o referido conjugado anticorpo-fármaco

Legal Events

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

Ref document number: 23797543

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2024563536

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2023797543

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2023797543

Country of ref document: EP

Effective date: 20241127

WWE Wipo information: entry into national phase

Ref document number: 202380050132.2

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 202380050132.2

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 18860335

Country of ref document: US