Anti-niphospinin 1 antibody and antibody

Abstract

Provided herein is an antibody having specificity for nucleophosprotein 1 (NPM1), which is capable of binding to nucleophosprotein 1 located on the surface of a cell; and antibody-drug conjugates (ADCs) comprising such antibodies are provided. Also provided herein are methods of treating a cancer in which NPM1 is expressed on the surface of a cancer cell by administering to a subject an NPM1-binding antibody or antibody conjugate as described herein and a chemotherapeutic drug.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to Greek Application No. 20220100349, filed on April 27, 2022, entitled “ANTI-NUCLEOPHOSMIN 1 ANTIBODY AND ANTIBODY CONJUGATE COMBINATION THERAPIES”, the contents of which are incorporated herein by reference in their entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic Sequence Listing (C123370245WO00-SEQ-VLJ.xml; size: 47,000 bytes; and creation date: April 21, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Nucleophosmin 1 (NPM1) is a protein that is normally present in the nucleus and cytosol of cells. However, in certain cancers, such as acute myeloid leukemia (AML), NPM1 can be mislocalized to the cell surface. Some cancers are also characterized by mutations in NPM1, which result in the translation of a mutant NPM1 (called NPM1c) that is mislocalized to the cell surface in even greater amounts than wild-type NPM1. NPM1c mutations are present in a significant subset of patients with AML. Summary of the invention

[0006] The present disclosure is based on the identification of antibodies that specifically bind to nucleolar phosphoprotein 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 can be used to bind cytotoxic payloads and optionally target cytotoxic payloads to cells with cell surface expression of WT and / or mutant NPM1. The efficacy of these antibodies or antibody-drug conjugates (ADCs) derived from these antibodies can be enhanced by administering chemotherapeutic drugs before, simultaneously with, and / or after them.

[0007] Thus, some aspects of the present disclosure relate to methods of treating cancers that express NPM1, comprising administering to a subject in need thereof an effective amount of an antibody or ADC that binds to NPM1 and a chemotherapeutic drug.

[0008] In some aspects, the disclosure relates to methods of treating a cancer expressing NPM1, comprising administering to a subject in need thereof an effective amount of an antibody or ADC that binds to NPM1, wherein the subject is receiving or has received treatment with a chemotherapeutic drug.

[0009] In some aspects, the disclosure relates to methods of treating a cancer expressing NPM1 comprising administering an effective amount of a chemotherapeutic drug to a subject in need thereof, wherein the subject is receiving or has received treatment with an antibody or ADC that binds to NPM1.

[0010] In some embodiments, the antibody binds to WT NPM1. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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); the light chain variable region comprises 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). In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 29.

[0011] In some embodiments, the antibody binds to mutant NPM1. In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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); the light chain variable region comprises 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). In some embodiments, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 31.

[0012] In some embodiments, the antibody is a full-length antibody or an antigen-binding fragment thereof.

[0013] In some embodiments, the antibody is a full-length antibody selected from the group consisting of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), and immunoglobulin M (IgM). In some embodiments, the antibody is IgG.

[0014] In some embodiments, the antibody is an antigen-binding fragment selected from the group consisting 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.

[0015] In some embodiments, the antibody is a human antibody or a humanized antibody.

[0016] In some embodiments, the antibody further comprises a heavy chain constant region. In some embodiments, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:32 or SEQ ID NO:46.

[0017] In some embodiments, the antibody further comprises a light chain constant region. In some embodiments, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:33, SEQ ID NO:34 or SEQ ID NO:47.

[0018] In some embodiments, 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.

[0019] In some embodiments, 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.

[0020] In some embodiments, the antibody is conjugated to an agent.

[0021] In some embodiments, the agent is a drug. In some embodiments, the drug is selected from the group consisting of auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E ... D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAD), monomethyl auristatin F (MMAF),auristatin E, MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin Lomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, campathecin, chaetocin, chaetoglobosin, chlamydocin, cinerubin B, cladribine, colchicine, combretastatin A1, combretastatin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin B Lucin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, englerin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanespimycin (17-AAG), glucopiericidin A, gramicidin A, herboxidiene, 9-hydroxyellipticine, hydroxyurea, hygrolidin, hypothemycin, idarubicin, ilimaquinone, isatropoloneA, isofistularin-3, ixabepilone, JW55, lactacystin, luisol A, maytansinol, mertansine (DM1), maytansine DM3, ravtansine (DM4), maytansinoid AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxin A, myoseverin, mytoxin B, nelarabine, nemorubicin, nocuolin A, okilactomycin, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortataxel, tesetaxel, phallacidin, phalloidin, phytosphingosine, piericidin A, pironetin, podophyllotoxin, polyketomycin, prednisone, pseudolaric acid B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinaldopeptin, rachelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinarine, saporin, sinefungin, taltobulin, telomestatin, 6-thioguanine, thiocolchicine, tolytoxin, tripolin A, triptolide, tubastatin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine. In some embodiments, the drug is saporin, daunorubicin, or venetoclax.

[0022] In some embodiments, the antibody is conjugated to the drug via a linker.

[0023] In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a pH sensitive linker, a glutathione sensitive linker or a protease cleavable linker. In some embodiments, the cleavable linker is selected from: N-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio) butyrate (SPDB), sulfo-SPDB, valine-citrulline (Val-cit), acetobutyrate, CL2A, maleimidocaproyl (MC) and Mal-EBE-Mal.

[0024] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is selected from: N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), MC-VC-PAB.

[0025] In some embodiments, the ratio of antibody to drug is 1: 1 to 1: 10. In some embodiments, the ratio of antibody to agent is 1:4.

[0026] 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.

[0027] In some embodiments, a cancer expressing NPM1 is a cancer in which NPM1 is expressed on the surface of cancer cells. In some embodiments, a cancer expressing NPM1 is a cancer in which NPM1 is expressed on the surface of cancer cells due to administration of or treatment with a chemotherapeutic drug. In some embodiments, a cancer expressing NPM1 is a cancer in which wild-type NPM1 and / or mutant NPM1 is expressed on the surface of cancer cells.

[0028] In some embodiments, the cancer is a solid cancer or a liquid cancer selected from: hematological cancer, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, muscle cancer, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer. In some embodiments, the cancer is selected from: acute myeloid leukemia (AML), acute promyeloid leukemia (acute promyeloidleukemia, APL), acute lymphoblastic leukemia (acute lymphoblastic leukemia, ALL), non-Hodgkin lymphoma (non-Hodgkin lymphoma) and myelodysplastic syndrome (myelodysplastic syndrome, MDS). In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a treatment-related cancer or a secondary malignancy. In some embodiments, the cancer is therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin's lymphoma.

[0029] In some embodiments, the chemotherapeutic drug is selected from the group consisting of auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, adoxirubicin, agrochelin, ansatriene, ansamitocin P-3, aphidicolin, apoptin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, camptothecin, chaetocin, globochaetin, chlamydocin , ember erythromycin B, cladribine, colchicine, compretin A1, compretin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, Engelin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanspiramycin (17-AAG), glucocorticoidin A, gramicidin A, oxadiene, 9-hydroxyellipticine, hydroxyurea, oxadiene, paramycin, idarubicin, ilimacin, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisol A, maytansinol, maytansine (DM1), maytansine DM3, ravtansine (DM4), maytansine AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxinA, myoblastic protein, mytoxin B, nelarabine, nemorubicin, nocuolin A, cholinesterase, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortasel, tesetaxel, phalloidin, phalloidin, phytosphingosine, picrotoxin A, bilonisone, podophyllotoxin, polyketidemycin, prednisone, phlorothenic acid B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinadol, rachelmin, rabekamycin, Ro 5-3335, safranin B, sandromycin, sanguinarine, saporin, sinefungin, tatoprin, trostatin, 6-thioguanine, thiocolchicine, monofilamentous algae toxin, tripolin A, triptolide, tobramycin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax and vincristine. In some embodiments, the chemotherapeutic drug is saporin, daunorubicin, venetoclax or azacitidine.

[0030] In some embodiments, the chemotherapeutic drug is a chemotherapeutic drug to which the cancer is resistant.

[0031] In some embodiments, administration is performed systemically or locally. In some embodiments, administration is performed by injection. In some embodiments, injection is intravenous, subcutaneous, intraperitoneal, or intratumoral. In some embodiments, administration is performed orally.

[0032] In some embodiments, administration is performed more than once. In some embodiments, administration is performed from once a day to once every six months.

[0033] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0034] In some embodiments, the administration results in increased binding between the antibody and the NPM1-expressing cancer cells of the subject compared to administration of the antibody alone.

[0035] In some embodiments, the administration results in reduced growth of NPM1-expressing cancer cells in the subject compared to administration of the antibody alone.

[0036] In some embodiments, the administration results in increased cell death of NPM1-expressing cancer cells in the subject compared to administration of the antibody alone.

[0037] Other aspects of the disclosure relate to methods for treating a disease associated with cells expressing cell surface nucleolar phosphoprotein 1 (NPM1), the method comprising administering to a subject in need thereof an effective amount of an antibody or conjugate described herein that binds to NPM1.

[0038] Other aspects of the disclosure relate to compositions for treating cancer expressing NPM1, the treatment comprising administering to a subject in need thereof a composition comprising an antibody or antibody conjugate that binds to NPM1 and a chemotherapeutic drug.

[0039] Other aspects of the disclosure relate to compositions for treating cancer expressing NPM1, the treatment comprising administering the composition to a subject in need thereof, wherein the composition comprises an antibody or antibody conjugate that binds to NPM1 and the subject is receiving or has received treatment with a chemotherapeutic drug.

[0040] Other aspects of the disclosure relate to compositions for treating cancer expressing NPM1, the treatment comprising administering the composition to a subject in need thereof, wherein the composition comprises a chemotherapeutic drug and the subject is receiving or has received treatment with an antibody or antibody conjugate that binds to NPM1. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The accompanying drawings are not intended to be drawn to scale. In the accompanying drawings, each identical or nearly identical component shown in different figures is represented by the same numeral. For the purpose of clarity, not every component may be labeled in every drawing.

[0042] In the attached picture:

[0043] Figures 1A to 1E NPM1 is shown to be localized on the surface of cancer cell types. Figure 1A Shown are immunoblots of WT NPM1 and mutant NPM1 in cytosolic and membrane fractions collected from various human leukemia cell lines. Figure 1B Shown is FACS analysis of NPM1 surface expression in human leukemia cell lines. Figure 1C and 1D Relatively low levels of NPM1 were shown to be expressed on the surface of murine bone marrow (BM) or peripheral blood (PB) cells. Figure 1E FACS analysis showed that NPM1 is not stably expressed on healthy human BM cells. FACS analysis was performed using an isolated anti-NPM1 antibody, such as Figure 2A and Figure 2B As indicated, and commercial anti-NPM1 antibody (Santa Cruz Biotechnology anti-WT NPM1 #sc-32256) was used.

[0044] Figure 2A and 2B The preparation of anti-NPM1 antibodies is shown. Figure 2A Isolation of antibodies binding to WT NPM1 is shown. Figure 2B Isolation of antibodies binding to mutant NPM1 is shown.

[0045] Figure 3A and 3B Anti-NPM1-ADC was shown to be toxic to human cancer cells. Figure 3ACytotoxicity of ADCs containing antibodies specific for WT NPM1 on OCI-AML3 cells is shown. Streptavidin-saporin was bound to biotinylated anti-WT NPM1 or anti-mouse IgG, after which the complexes were added to OCI-AML3 cells and incubated for 24 hours. After incubation, cells were washed and cell killing was analyzed by FACS. Figure 3B Shown with Figure 3A Same conditions, but with a 48 h incubation.

[0046] Figure 4A and 4B It was shown that NPM1 is highly conserved among mammalian species. Figure 4A Pairwise alignments depicting conservation between human and murine NPM1 amino acid sequences (SEQ ID NOs: 39 to 40) are shown. Figure 4B Pairwise alignments depicting conservation between human and Rhesus macaque NPM1 amino acid sequences (SEQ ID NOs: 39 and 41) are shown.

[0047] Figures 5A to 5C It was shown that NPM1 is present on the surface of human AML cell lines and primary murine AML cells. Figure 5A Binding of anti-NPM1 antibody (Merck / Sigma anti-B23 #B0556) to NPM1 located on the surface of human OCI-AML3 cells and MOLM13 cells is shown. Binding to NPM1 was determined in both live and fixed cells. OCI-AML3 cells express mutant NPM1, while MOLM13 cells express relatively low amounts of WT NPM1. Figure 5B and Figure 5C Anti-NPM1 antibody (Merck / Sigma anti-B23 #B0556) is shown to bind to the fixed primary mouse MLL-AF4 / FLT3 ltd / + 、MLL-AF9 / FLT3 ltd / + 、MLL-ENL / FLT3 ltd / + and Npm1c / FLT3 ltd / + Binding of NPM1 on the surface of AML cells.

[0048] Figures 6A to 6F The presence of NPM1c (mutant NPM1) on the surface of a human myeloid leukemia cell line is shown. Fig. 6A Anti-TY1 tag antibody (Diagenode TY1 #C15200054) is shown binding to the surface of human K562 myeloid leukemia cells expressing TY1-tagged WT NPM1 or TY1-tagged NPM1c. Figure 6B Will Fig. 6AThe data in are shown as separate graphs, where the percentage of cells positively bound by TY1 antibody and anti-NPM1 antibody (Merck / Sigma anti-B23 #B0556) is quantified. Figure 6C Binding of anti-NPM1 antibodies (Diagenode TY1#C15200054; Merck / Sigma anti-B23#B0556) to intracellular WT NPM1 and mutant NPM1 as well as cell surface WT NPM1 and mutant NPM1 is shown. Relevant cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type lentiviral vector or TY1-tagged NPM1c lentiviral vector. Fig.6D Binding of anti-NPM1 antibodies (Diagenode TY1#C15200054; Merck / Sigma anti-B23#B0556) to intracellular WT NPM1 and mutant NPM1 is shown. Relevant cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type lentiviral vector or TY1-tagged NPM1c lentiviral vector. Fig. 6E Binding of anti-NPM1 antibodies (Diagenode TY1#C15200054; Merck / Sigma anti-B23#B0556) to WT NPM1 and mutant NPM1 on the surface of K562 cells or MOLM13 cells (negative control) is shown. The cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type lentiviral vector, or TY1-tagged NPM1c lentiviral vector. Fig. 6F Immunofluorescence depicting the binding of anti-NPM1 antibodies (Diagenode TY1#C15200054; Merck / Sigma anti-B23#B0556) to WT NPM1 and mutant NPM1 on the surface of K562 cells or MOLM13 cells (negative control) is shown. The relevant cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type lentiviral vector or TY1-tagged NPM1c lentiviral vector.

[0049] Fig. 7A and 7B Anti-NPM1 antibodies are shown to bind to NPM1 on the surface of primary murine AML cells. Fig. 7A Binding of isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 on the surface of primary murine MLL-rearranged (MLL-r) AML cells is shown. Figure 7B Shown is the binding of isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 on the surface of primary murine NPM1c AML cells.

[0050] Figures 8A to 8D Anti-NPM1 antibodies were shown to bind to NPM1 on the surface of human AML patient-derived xenografts (PDXs) implanted in a murine model. Fig. 8A Binding between isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 located on the surface of PDX-1 cells (MLL-r) is shown. In contrast to PDX-1 cells, host (mouse) bone marrow cells (lower panel) showed relatively low binding. Figure 8B Shown is the binding of isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 located on the surface of PDX-2 (NPM1c) cells. Figure 8C Shown is the binding of isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 located on the surface of PDX-3 (MLL-r, BCOR) cells. Fig.8D Binding of isolated WT anti-NPM1 antibodies and mutant anti-NPM1 antibodies to NPM1 on the surface of PDX-4 (DNMT3A, N / KRAS) cells is shown.

[0051] Figures 9A to 9D Synergy between chemotherapeutic drugs used to treat AML and antibodies targeting cell surface NPM1 is shown. Fig. 9A It is shown that the binding between anti-NPM1 antibody (Merck / Sigma anti-B23 #B0556) and NPM1 on the surface of the venetoclax-resistant AML cell line OCI-AML3 is increased after treatment with 10 nM daunorubicin or 40 nM venetoclax, especially after 8 days after treatment. Fig. 9B Shown are 8 days after treatment with 10 nM daunorubicin or 40 nM venetoclax. Fig. 9A Individual replicates of FACS analysis are shown. Fig. 9C It is shown that after treatment with 10 nM daunorubicin or 40 nM venetoclax, especially after 8 days after treatment, the isolated anti-NPM1 antibody ( Figure 2A and Figure 2B ) and NPM1 on the surface of OCI-AML3. Fig.9D Shown is the expression of anti-NPM1 antibody ((Merck / Sigma anti-B23#B0556)) or isolated anti-NPM1 antibody ( Figure 2A and Figure 2B ) and NPM1 on the surface of OCI-AML3.

[0052] Figures 10A to 10BIntracellular and cell surface staining of isoforms and NPM1 from the cell line OCI-AML3 is shown ( Fig. 10A ) and denatured protein gels and western blotting (WB) of biochemical fractions (cytosol and membrane fractions) from four cell lines ( Fig. 10B ).

[0053] Figures 11A to 11B The expression of anti-NPM1 antibody (AF647 signal) in cultured human suspension cell lines ( Fig.11A ) and primary mouse cell lines ( Fig. 11B ) were used to perform live cell staining.

[0054] Figures 12A to 12B Shown are western blots of cells after cell surface biotinylation with a cell-impermeable biotinylation reagent. Western blots detected NPM1 in the membrane fraction of these cells, and NPM1 IP was able to more robustly enrich NPM1 from membrane lysates ( Fig. 12A Examination of the biotin signal from these fractions (cell surface exposed proteins) revealed the isolation of a single band in the NPM1 IP ( Fig. 12B ), indicating that full-length NPM1 is exposed on the surface of living cells.

[0055] Figures 13A to 13B is for both HL-60 and OCI-AML3 ( Fig.13A ) and the adherent cell line PANC1( Fig. 13B Diffraction limited (DL) and super-resolution reconstruction (SR) of anti-NPM1 staining on the cell surface of ). Cell surface NPM1 appears as distinct clusters.

[0056] Fig.14 Fluorescence images of live (top) or fixed and permeabilized (bottom) OCI-AML3 cells stained with Ab2.2 or commercially available NPM1 antibody.

[0057] Figures 15A to 15B Three healthy donor bone marrow samples stained with Ab2.2 and sorted for various markers of the hematopoietic system are shown. Ab2.2 partially binds to CD33+ cells, but no binding to CD34+ (HSC) cells is observed. Fig. 15B Binding of commercial anti-NPM1 antibody and binding of Ab2.2 to protein lysate samples were shown by western blot. The banding patterns were identical.

[0058] Fig.16is a table describing 12 AML patient samples.

[0059] Figures 17A to 17B Flow cytometric analysis of 12 AMLs is shown.

[0060] Fig.18 is a table describing samples collected from 15 AML patients.

[0061] Figures 19A to 19E Shown is flow cytometric analysis of 15 AML patients.

[0062] Figures 20A to 20B It is shown how the toxicity of Ab2.2 was assessed in vivo. Fig. 20A is a schematic diagram of the experimental scheme. Fig. 20B are weight results, as well as results from blood sample analysis of WBC, PLT, and HGB.

[0063] Figures 21A to 21G It is shown how to assess the efficacy of Ab2.2 treatment in vivo. Fig.21A is a schematic diagram of the experimental design. Fig. 21B Antibodies binding to this AML model are shown. Fig. 21C WBC counts are shown. Fig.21D are the results from qPCR analysis of AML alleles. Fig.21E Shown are spleens and spleen weights of IgG and Ab2.2 treated mice. Fig.21F It is the result of HGB and HCT analysis of blood samples. Figure 21G Results from survival assays are shown.

[0064] Figures 22A to 22C It is shown how mice are sublethally irradiated and transplanted with primary murine AML cells to assess efficacy (Efficacy Model 2). Fig.22A is a schematic diagram of the experimental design. Fig. 22B Antibody binding is shown. Fig. 22C Results from survival assays are shown.

[0065] Figures 23A to 23I Treatment with Ab2.2 was shown to reduce tumor burden in the transplantation model. Fig.23A Shown is a schematic diagram of the experimental design. Fig. 23B are the weights of spleen, lung and liver of mice treated with IgG or Ab2.2. Fig.23C Results from survival assays are shown. Figures 23D to 23E Flow cytometry results are shown. Fig.23F are WBC counts and PLT levels from blood samples. Figure 23G The changes in WBC over time are shown. Fig.23HShown are images of spleens and graphs of spleen weights from mice treated with IgG or Ab2.2. Fig.23I The % AML in BM and PB are shown.

[0066] Figures 24A to 24B The immune dependence of the tumor killing activity of Ab2.2 is shown; it relies on an intact immune system. Fig.24A The experimental design is shown. Fig. 24B Shown are the survival curves of mice treated with Ab2.2 or IgG.

[0067] Figures 25A to 25C Solid tumor activity of Ab2.2 is shown. Fig.25A The experimental design is shown. Fig.25B Tumor volumes at days 10 and 13 are shown. Fig.25C The changes in tumor volume over time from day 7 to day 13 are shown.

[0068] Figures 26A to 26B It is shown that wild-type healthy mice treated with sublethally irradiated and Ab2.2 exhibited no observable effects of Ab2.2 treatment. Fig.26A The experimental design is shown. Fig.26B Weight and WBC count, HGB and PLT analysis from blood samples are shown.

[0069] Figures 27A to 27O In vitro models of various cancer cell types using flow cytometry analysis to assess the ability of Ab2.2 to bind to human or murine tumors are shown.

[0070] Fig.28 High surface detection of Npm1c on single Dmnt3a R882H mutant cells (preleukemic cells) is shown.

[0071] Fig.29 Shown how NPM1 can serve as an ADC target in some models.OCI-AML3 cells were treated with negative control, isotype saporin conjugates or Ab2.2-saporin conjugates to assess the in vitro activity of Ab2.2 with ADCs.

[0072] Figures 30A to 30C Ab2.2 was shown to extend lifespan in a transplantation model of a human AML cell line. Fig. 30A is a schematic diagram of the experimental design. Fig. 30B Antibody binding using flow cytometry is shown. Fig. 30C It is a graph showing the results of the survival assay.

[0073] Figures 31A to 31C Ab2.2 was shown to extend lifespan in a transplantation model of a human AML-PDX cell line. Fig.31A is a schematic diagram of the experimental design. Fig.31B Antibody binding using flow cytometry is shown. Fig.31C It is a graph showing the results of the survival assay.

[0074] Fig.32 Shown is how OCI-AML3 cells were exposed to control or low-dose chemotherapy treatment.

[0075] Fig.33 An in vitro model of non-transformed cell types (HPC7, HOXB8) exposed to low doses of chemotherapeutic agents (daunorubicin, venetoclax) is shown. Cells were analyzed using flow cytometry at 48 and 72 hours. DETAILED DESCRIPTION

[0076] Some aspects of the present disclosure are based on the discovery of antibodies specific for nucleolar phosphoprotein 1 (NPM1), also known as nucleolar phosphoprotein B23 or numatrin, which can be used to target substances (e.g., chemotherapeutic drugs, radioisotopes) to certain cell types (e.g., cancer cells) in a subject. Without wishing to be bound by theory, NPM1 is a multifunctional protein that normally binds to single-stranded and double-stranded nucleic acids and is present in the nucleus and cytosol of cells. However, when certain cell types are exposed to cellular stress (e.g., cancerous transformation), large amounts of NPM1 may instead be mislocalized to the cell surface where they are exposed to the extracellular environment. Although an increase in cell membrane-localized NPM1 can occur in any type of cancer, this increase is particularly common in leukemias (e.g., acute myeloid leukemia (AML)) due to mutations in the gene encoding NPM1. As a result of this mutation, many patients' leukemia (e.g., AML) cells begin to express a mutant variant of NPM1, called NPM1c, which is mislocalized from the nucleus to the cytoplasm, where it is transported to the cell membrane and exposed to the extracellular environment. NPM1c is also known to occur only in cancer cells because it drives malignant transformation in AML. In principle, antibodies or other substances specific for wild-type NPM1 or mutant NPM1 (NPM1c) can be conjugated with a cytotoxic payload and used to target cancer cells. In addition, NPM1-specific antibodies themselves, when bound to cancer cells, may be sufficient to stimulate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP) without being conjugated with a cytotoxic payload. However, although antibodies specific for NPM1 are commercially available, there is a lack of antibodies that can effectively bind to wild-type NPM1 and / or mutant NPM1 on the surface of living cells. In addition, most commercially available anti-NPM1 antibodies are polyclonal, and there are currently no available monoclonal antibodies that bind to NPM1c. Therefore, new antibodies have been developed that are able to effectively and specifically bind to wild-type NPM1 and mutant NPM1 on the surface of cells, including cancer cells. These antibodies and their molecular conjugates (e.g., antibody-drug conjugates) can be used to detect and treat cancers that express NPM1 on their cell surfaces, and can be used to treat subjects suffering from cancers for which there is no known effective treatment.

[0077] Antibodies that bind to NPM1

[0078] The present disclosure provides antibodies that bind to NPM1, e.g., wild-type (WT) NPM1 or mutant NPM1. Such antibodies may have a higher affinity for WT NPM1 than for mutant NPM1, or may have a higher affinity for mutant NPM1 than for WT NPM1.

[0079] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind to a target (called an "antigen", such as, but not limited to, a protein or peptide) through at least one recognition site on the antigen. The term "antibody" as used herein includes both full-length immunoglobulin molecules (immunoglobulins with two heavy chains and two light chains, such as monoclonal or polyclonal full-length antibodies) and antigen-binding fragments thereof, such as, but not limited to, chimeric antibodies, double antibodies, linear antibodies, nano antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), or other antigen-binding fragments that retain specific binding to antigens. "Immunoglobulin (Ig)" is a large Y-shaped protein produced mainly by plasma cells, which is used by the immune system to neutralize foreign substances (e.g., pathogens, such as bacteria and viruses). "Antibody fragment" includes any antigen-binding fragment (i.e., "antigen binding portion") or a single chain thereof. In some embodiments, "antibody" refers to a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (heavy, H) chains and two light (light, L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as V H ) and the heavy chain constant region. The heavy chain constant region consists of three domains C H 1. C H 2 and C H Each light chain consists of a light chain variable region (abbreviated as V L ) and the light chain constant region. The light chain constant region consists of a domain C L The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V LIt is composed of three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In some embodiments, the antibody is an immunoglobulin (Ig) monomer. The antibody can be a polyclonal antibody or a monoclonal antibody.

[0080] In some embodiments, antibodies are heterotetrameric glycoproteins composed of two identical L chains and two H chains (IgM antibodies are composed of 5 basic heterotetrameric units and an additional polypeptide called a J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form multivalent aggregates containing 2 to 5 basic 4-chain units and J chains). In the case of IgG, the 4-chain unit is usually about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, while the two H chains are connected 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 a variable domain (V domain) at the N-terminus. H ), followed by three constant domains (C H ) and the four Cs for the μ and ε isoforms H Each L chain has a variable domain (V L ), followed by a constant domain (C L ). L With V H Aligned, and C L Aligned with the first constant domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. H and V L The pairing together forms a single antigen binding site. For the structure and properties of some non-limiting examples of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page 71 and Chapter 6, which are incorporated herein by reference. In some embodiments, the antibody is an IgG.

[0081] The L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. H ) amino acid sequences, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. Based on C H Relatively minor differences in sequence and function further divide the gamma and alpha classes into subclasses, for example, humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0082] The V domain mediates antigen binding and defines the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V region consists of relatively constant stretches of 15 to 30 amino acids called framework regions (FRs), which are separated by extremely variable shorter regions called "hypervariable regions" of 9 to 12 amino acids each. The variable domains of native heavy and light chains each contain four FRs connected by three hypervariable regions connected by loops, which primarily adopt a β-sheet configuration and in some cases form a portion of a β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chains, contribute to the formation of the antigen-binding site of the antibody (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is incorporated herein by reference). The constant domains are not directly involved in the binding of an antibody to an antigen, but exhibit a variety of effector functions, such as enabling the antibody to participate in antibody-dependent cellular toxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0083] In some embodiments, the antibody is a monoclonal antibody. "Monoclonal antibody" is an antibody obtained from a group of substantially homogeneous antibodies, that is, except for the naturally occurring mutations that may be present in small amounts, the group comprising a single antibody is identical. Monoclonal antibodies are highly specific and are directed to a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibodies available in the present invention can be prepared by the hybridoma method originally described by Kohler et al., Nature, 256:495 (1975), or can be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plant cells (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries, for example, using the techniques described by Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), which are incorporated herein by reference.

[0084] The monoclonal antibodies described herein encompass "chimeric" antibodies, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). The chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0085] In some embodiments, the antibody is a polyclonal antibody. A "polyclonal antibody" is a mixture of different antibody molecules that react with more than one immunogenic determinant of an antigen. Polyclonal antibodies can be isolated or purified from the blood, secretions or other fluids of a mammal or from an egg. Polyclonal antibodies can also be recombinant. Recombinant polyclonal antibodies are polyclonal antibodies produced using recombinant technology. Recombinantly produced polyclonal antibodies typically contain high concentrations of different antibody molecules, all or most of which (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99% or more) show the desired binding activity to an antigen consisting of more than one epitope.

[0086] In some embodiments, antibodies are "humanized" for use in humans (e.g., as therapeutic agents). The "humanized" form of a non-human (e.g., rodent) antibody is a chimeric antibody containing minimal sequences derived from a non-human antibody. A humanized antibody is a human immunoglobulin (acceptor antibody), wherein residues from the hypervariable region of the acceptor are replaced by residues from the hypervariable region of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) with desired antibody specificity, affinity, and capacity. In some cases, the framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the acceptor antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally speaking, a humanized antibody will include substantially all of at least one and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all of the FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0087] Some aspects of the present disclosure relate to antibodies that bind to NPM1. Such antibodies may preferentially bind to WT NPM1 or mutant NPM1. The term "preferential binding" refers to binding to a specific antigen that occurs more frequently, more rapidly, for a longer duration, and / or with higher affinity than binding to other antigens. The terms "preferential binding" and "specific binding" are used interchangeably. The terms "preferential binding" and "specific binding" do not necessarily mean exclusive binding, i.e., an antibody that preferentially binds to an antigen may or may not bind to one or more other antigens. In some embodiments, the antibodies described herein may or may not bind to other forms (variants) of a specific antigen. In some embodiments, an antibody that binds to WT NPM1 may or may not bind to an NPM1 variant comprising one or more amino acid insertions, deletions, or substitutions (i.e., mutant NPM1), and an NPM1 variant comprising one or more amino acid insertions, deletions, or substitutions (i.e., mutant NPM1) may or may not bind to WT NPM1.

[0088] As used herein, "wild-type NPM1" or "WT NPM1" refers to an NPM1 protein that comprises an amino acid sequence identical to a recognized reference sequence of a complete, fully functional NPM1. For example, WT NPM1 can be an NPM1 isoform (e.g., NCBI reference sequence: NP 001341935.1) expressed by a gene encoding WT NPM1 (NCBI reference sequence: NG_016018.1). "Mutant NPM1" as used herein refers to an NPM1 protein that comprises an amino acid sequence comprising one or more amino acid insertions, deletions, or substitutions relative to the WT NPM1 sequence. For example, relative to the WT NPM1 sequence, the mutant NPM1 can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions, or substitutions. Compared to WT NPM1, the mutant NPM1 can have reduced activity and / or altered subcellular localization. The WT NPM1 or mutant NPM1 described herein can be mammalian WT NPM1 or mammalian mutant NPM1. In some embodiments, the WT NPM1 or mutant NPM1 described herein is human WT NPM1 or human mutant NPM1.

[0089] In some embodiments, the antibody bound to NPM1 is a full-length antibody. The full-length antibody described herein can be any antibody class based on the amino acid sequence of its heavy chain constant region. For example, the full-length antibody bound to NPM1 can be immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE) and immunoglobulin M (IgM) or its subclass (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the antibody described herein is an antigen binding fragment. The antigen binding fragment can be in the form 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 double antibody, a single domain heavy chain antibody, and a single domain light chain antibody. The antibodies described herein can be of mouse, rat, human, or any other origin. In some embodiments, the antibodies described herein are human antibodies or humanized antibodies.

[0090] In some embodiments, the antibody that binds to NPM1 (e.g., WT NPM1 or mutant NPM1) comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region and the light chain variable region each comprise a set of complementary determining region (CDR) sequences that determine substrate specificity. The CDR sequences can be determined using any numbering scheme commonly known in the art (e.g., Kabat, Chothia, Contact, IGMT).

[0091] In some embodiments, the 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). In some embodiments, the 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).

[0092] In some embodiments, 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). In some embodiments, 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).

[0093] Table 1 provides amino acid sequences of heavy chain CDRs and light chain CDRs of exemplary antibodies ("Ab1" and "Ab2") specific for NPM1 (WT and / or mutant NPM1). Antibodies having the same heavy chain CDR or light chain CDR sequences as provided in Table 1 are within the scope of the present disclosure as determined using any numbering scheme generally known in the art (e.g., Kabat, Chothia, Contact, IGMT).

[0094] Table 1: CDR sequences of anti-NPM1 antibodies

[0095]

[0096] In some embodiments, an antibody comprising one or more heavy chain CDR and / or light chain CDR sequences represented as "Ab1" in Table 1 binds to (e.g., preferentially binds to) WT NPM1. In some embodiments, an antibody comprising one or more heavy chain CDR and / or light chain CDR sequences represented as "Ab2" in Table 1 binds to (e.g., preferentially binds to) mutant NPM1 (NPM1c). In some embodiments, an antibody comprising one or more heavy chain CDR and / or light chain CDR sequences represented as "Ab2.2" in Table 1 binds to (e.g., preferentially binds to) mutant NPM1 (NPM1c).

[0097] In some embodiments, the antibody that binds to NPM1 comprises the heavy chain variable region (V H ) sequence and / or light chain variable region (V L ) sequence. In some embodiments, the antibody that binds to NPM1 further comprises a heavy chain constant region (C H ) sequence and / or light chain variable region (C LIn some embodiments, the antibody that binds to NPM1 comprises the heavy chain sequence and / or the light chain sequence provided in Table 2.

[0098] Table 2: Heavy and light chain sequences of anti-NPM1 antibodies

[0099]

[0100]

[0101] In some embodiments, the antibodies that bind to NPM1 can be cleaved relative to the CDRs, V H 、V L , C H , C L , heavy chain or light chain sequence comprises one or more mutations (e.g., amino acid insertions, deletions or substitutions). In some embodiments, the antibody that binds to NPM1 may be provided in Table 1 or Table 2 with respect to the CDRs, V H 、V L , C H , C L , heavy chain or light chain sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations (e.g., amino acid insertions, deletions or substitutions). In some embodiments, the antibody that binds to NPM1 can be with the CDR, V H 、V L , C H , C L , heavy chain or light chain sequences have at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity.

[0102] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the Kabat definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the Kabat definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 10 (according to the Kabat definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 11 (according to the Kabat definition system).

[0103] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 15 (according to the Kabat definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 16 (according to the Kabat definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Kabat definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Kabat definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Kabat definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Kabat definition system).

[0104] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:4 (according to the IMGT definition system), a heavy chain complementary determining region 5 (CDR-H2) of SEQ ID NO:2 (according to the Chothia definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:3 (according to the Chothia definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:9 (according to the Chothia definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:10 (according to the Chothia definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:11 (according to the Chothia definition system).

[0105] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO: 18 (according to the Chothia definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO: 19 (according to the Chothia definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Chothia definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Chothia definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Chothia definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Chothia definition system).

[0106] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:6 (according to the IMGT definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO:7 (according to the Contact definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:8 (according to the Contact definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:12 (according to the Contact definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:13 (according to the Contact definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:14 (according to the Contact definition system).

[0107] In some embodiments, an antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:20 (according to the Contact definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO:48 (according to the Contact definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:21 (according to the Contact definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:25 (according to the Contact definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:26 (according to the Contact definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:27 (according to the Contact definition system).

[0108] In some embodiments, an antibody of the present disclosure comprises a heavy chain variable region (VH) comprising no more than 25 amino acid variations in the framework region (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) compared to a VH comprising the amino acid sequence of SEQ ID NO: 28. Alternatively or additionally (e.g., supplementally), an antibody of the present disclosure comprises a light chain variable region (VL) comprising 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 variations) in the framework regions compared to the VL comprising the amino acid sequence of SEQ ID NO: 29.

[0109] In some embodiments, an antibody of the present disclosure comprises a heavy chain variable region (VH) comprising no more than 25 amino acid variations in the framework region (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) compared to a VH comprising the amino acid sequence of SEQ ID NO: 30. Alternatively or additionally (e.g., supplementally), an antibody of the present disclosure comprises a light chain variable region (VL) comprising 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 variations) in the framework region compared to the VL comprising the amino acid sequence of SEQ ID NO: 31.

[0110] In some embodiments, an antibody of the present disclosure comprises a VH comprising in the framework region an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VH comprising the amino acid sequence of SEQ ID NO: 28. Alternatively or additionally (e.g., supplementing), in some embodiments, an antibody of the present disclosure comprises a VL comprising in the framework region an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VL comprising the amino acid sequence of SEQ ID NO: 29.

[0111] In some embodiments, an antibody of the present disclosure comprises a VH comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VH comprising the amino acid sequence of SEQ ID NO: 30. Alternatively or additionally (e.g., supplementing), in some embodiments, an antibody of the present disclosure comprises a VL comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VL comprising the amino acid sequence of SEQ ID NO: 31.

[0112] In some embodiments, an antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 28. Alternatively or additionally (e.g., supplementally), in some embodiments, an antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 29.

[0113] In some embodiments, an antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 30. Alternatively or additionally (e.g., supplementally), in some embodiments, an antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 31.

[0114] In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 35. Alternatively or in addition (e.g., supplementing), the antibodies of the present disclosure comprise a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 37. Alternatively or additionally (e.g., supplementally), an antibody of the present disclosure comprises a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to the amino acid sequence of SEQ ID NO:37.

[0115] In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 36. In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 36. Alternatively or additionally (e.g., supplementing), an antibody of the present disclosure comprises a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 38. Alternatively or additionally (e.g., supplementally), an antibody of the present disclosure comprises a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to the amino acid sequence of SEQ ID NO:38.

[0116] In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 44. Alternatively or in addition (e.g., supplementing), an antibody of the present disclosure comprises a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to the amino acid sequence of SEQ ID NO: 45. Alternatively or additionally (e.g., supplementally), an antibody of the present disclosure comprises a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to the amino acid sequence of SEQ ID NO:45.

[0117] In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 37. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0118] In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 38. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0119] In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibodies of the present disclosure comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 45. Alternatively or additionally (e.g., supplemented), the antibodies of the present disclosure comprise a light chain comprising the amino acid sequence of SEQ ID NO: 45.

[0120] Preparation of anti-NPM1 antibody

[0121] Antibodies capable of binding to NPM1 as described herein can be prepared 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, anti-NPM1 antibodies are prepared by recombinant technology.

[0122] For example, hybridoma cell lines can be prepared from lymphocytes and immortalized myeloma cells expressing antibodies described herein using techniques widely 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). Generally speaking, the production of hybridomas involves the use of a fusing agent (e.g., polyethylene glycol or electric current) to fuse myeloma cells with lymphoid cells to produce immortalized cell lines capable of producing and secreting the desired antibodies. Hybridomas can be cultured in a culture medium under conditions known in the art.

[0123] Alternatively, nucleic acids encoding the heavy and light chains of the anti-NPM1 antibodies as described herein may be cloned into one or more vectors using recombinant techniques known in the art, wherein each nucleotide sequence is operably linked to a suitable promoter (e.g., a constitutive or inducible promoter known in the art). The vector may be an expression vector by which the polypeptide sequence of the anti-NPM1 antibody can be expressed in a specific cell type. One or more expression vectors contain one or more promoters for driving the expression of operably linked genes (usually 3' to the promoter sequence). Each nucleotide sequence encoding the heavy and light chains may be operably linked to different promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains may be operably linked to the same promoter (e.g., within the same expression vector). The choice of expression vector and / or promoter depends on the type of host cell used to produce the antibody. If necessary, an internal ribosomal entry site (IRES) may be inserted between the heavy chain coding sequence and the light chain coding sequence to enhance recombinant expression. An expression vector encoding an anti-NPM1 antibody can be inserted into a cell for expression using any technique known in the art (e.g., chemical transfection, viral transfection). The cell can be a cell that can be cultured using techniques known in the art. The cell can be any cell type capable of expressing a heterologous antibody sequence, such as, for example, a human cell, a mouse cell, a rat cell, a non-human primate cell, or an insect cell that can be cultured in vitro. After culturing such cells under conditions sufficient for expression of the antibody (e.g., under temperature and growth medium conditions sufficient for expression), the anti-NPM1 antibody can be isolated from the culture medium (if the antibody is secreted) or from the cells after lysis. Techniques for isolating antibodies from cells and / or culture medium are generally known in the art, but generally include affinity chromatography.

[0124] Anti-NPM1 antibody conjugate

[0125] The present disclosure also provides molecular conjugates comprising an anti-NPM1 antibody described herein (eg, an antibody that binds to WT NPM1 or mutant NPM1) linked to an agent via a chemical linker.

[0126] In some embodiments, the conjugates described herein comprise an anti-NPM1 antibody connected to a drug. A conjugate comprising an anti-NPM1 antibody conjugated to a drug may be referred to as an "ADC". In some embodiments, the drug is a small molecule. The term "small molecule" refers to a molecule with a relatively low molecular weight, whether naturally occurring or artificially created (e.g., by chemical synthesis). Generally speaking, a small molecule is an organic compound (e.g., it comprises carbon). A small molecule may comprise multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, and heterocycles, etc.). In certain embodiments, the molecular weight of the small molecule is no greater than about 1,000 g / mol, no greater than about 900 g / mol, no greater than about 800 g / mol, no greater than about 700 g / mol, no greater than about 600 g / mol, no greater than about 500 g / mol, no greater than about 400 g / mol, no greater than about 300 g / mol, no greater than about 200 g / mol, or no greater than about 100 g / mol. In certain embodiments, the molecular weight of the 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.

[0127] In some embodiments, the drug is a cytotoxic drug (e.g., a cytotoxic small molecule). In some embodiments, the drug is a cytostatic drug (e.g., a cytostatic small molecule). In some embodiments, the drug is a chemotherapeutic drug. Some 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, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, adoxirubicin, agrochelin, ansatriene, ansamitocin P-3, aphidicolin, apoptin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, camptothecin, chaetocins, chaetomycin, chlamyd ocin, ember erythromycin B, cladribine, colchicine, compretin A1, compretin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, Engelin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanspiramycin (17-AAG), glucocorticoidin A, gramicidin A, oxadiene, 9-hydroxyellipticine, hydroxyurea, oxadiene, paramycin, idarubicin, ilimacin, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisol A, maytansinol, maytansine (DM1), maytansine DM3, ravtansine (DM4), maytansine AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxin A, myotropin, mytoxin B, nelarabine, nemorubicin, nocuolin A, cholinesterase, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortataxel, tesetaxel, phalloidin, phalloidin, phytosphingosine, picrotoxin A, bilonisone, podophyllotoxin, polyketidemycin, prednisone, phlorotoxin B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinadol, rachelmin, rabekamycin, Ro 5-3335, safranin B, sandromycin, sanguinarine, saporin, sinefungin, tatoprin, trostatin, 6-thioguanine, thiocolchicine, monofilamentous algae toxin, tripolin A, triptolide, tobramycin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax and vincristine. The ADC described herein may contain another drug (e.g., chemotherapeutic drug) known in the art to be cytotoxic or cytostatic to a specific cell type (e.g., cancer cell).

[0128] In some embodiments, the anti-NPM1 antibodies described herein are conjugated to more than one agent (e.g., drug) molecule, i.e., the antibody and the agent are conjugated together at a ratio greater than 1: 1. In some embodiments, the ratio between the antibody and the agent is 1: 1 to 1: 10. In some embodiments, 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.

[0129] In some embodiments, the anti-NPM1 antibodies described herein are conjugated (linked) to agents using chemical linkers known in the art. The terms "conjugated," "linked," and "attached" as used herein mean that two molecules are associated, preferably by a covalent bond or with sufficient affinity, to achieve the therapeutic or diagnostic benefit of the association between the two entities.

[0130] In some embodiments, the joint is a cleavable joint. The cleavable joint used herein can release the conjugated part in response to stimulation. In some embodiments, the stimulation is a physiological stimulation. Some non-limiting examples of stimulation include the presence of an enzyme, acidic conditions, alkaline conditions or reducing conditions. For example, cleavable joints include peptide joints, β-glucuronide joints, glutathione sensitive joints (or disulfide joints) and pH sensitive joints. In some embodiments, the pH sensitive joint is cut at a pH of 5.0 to 6.5 or a pH of 4.5 to 5.0. In some embodiments, when the pH is 7 to 7.5, the pH sensitive joint is not cut. In some embodiments, when the pH is 7.3 to 7.5, the pH sensitive joint is not cut. In some embodiments, the cleavable joint is a protease sensitive joint. Some examples of cleavable linkers include N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butyrate (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, which is incorporated herein by reference.

[0131] In some embodiments, the linker is non-cleavable. In some embodiments, a non-cleavable linker is a linker that is not cleaved within the systemic circulation in a subject. In some embodiments, a non-cleavable linker is a linker that is resistant to cleavage by proteases. Non-cleavable linkers include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidomethylcyclohexane-1-carboxylate (MCC), and MC-VC-PAB.

[0132] Any of the conjugates described herein can be synthesized using methods known in the art. See, for example, Yao et al., Int J Mol Sci. 2016 Feb 2; 17(2).

[0133] ADCs comprising anti-NPM1 antibodies conjugated to drugs are advantageous for therapeutic use, in part because drugs (e.g., chemotherapeutic drugs) are toxic and can be targeted to specific cell types expressing cell surface NPM1 (e.g., cancer cells expressing NPM1). By conjugating the drug to the anti-NPM1 antibody, the toxicity of the ADC can 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 form.

[0134] In some embodiments, the agent attached to the anti-NPM1 antibody is a radioactive isotope. In some embodiments, the radioactive isotope is a radioactive isotope that can be used to treat cancer (i.e., a therapeutic radioisotope), such as a radioisotope that can be used to treat acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma, or myelodysplastic syndrome (MDS). Radioisotopes that can be used to treat cancer are well known in the art and 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). Some 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. 2010 Nov 1; 16(21): 5303-11, which is incorporated herein by reference). Antibodies containing radioisotopes are advantageous for therapeutic use, in part because the radioisotopes are toxic and can be targeted to specific cell types expressing cell surface NPM1 (e.g., cancer cells expressing NPM1). By conjugating a radioisotope to an anti-NPM1 antibody, the toxicity of the radioisotope can 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 form.

[0135] Composition

[0136] The present disclosure also provides compositions comprising antibodies that bind to NPM1 provided herein (e.g., antibodies that bind to WT NPM1 or mutant NPM1). In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response or other problems or complications that are commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. A "pharmaceutically acceptable carrier" can be a pharmaceutically acceptable material, composition or carrier that involves transporting or transporting a subject agent from one organ or part of the body to another organ or part of the body, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" (e.g., physiologically compatible, sterile, physiological pH, etc.) in the sense of being compatible with the other ingredients of the formulation and harmless to the patient's tissues. The term "carrier" refers to a natural or synthetic organic or inorganic component that is combined with an active ingredient to facilitate application. The components of the pharmaceutical composition are also capable of being co-mixed with the molecules of the present disclosure and with each other in a manner such that there is no interaction that would significantly impair the desired drug efficacy. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants 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 propylene glycol; (11) polyols , such as glycerol, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) fillers, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives and antioxidants may also be present in the formulation.

[0137] The pharmaceutical 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. The term "unit dosage" when used with respect to the pharmaceutical compositions of the present disclosure refers to physically discrete units suitable as unitary dosages 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).

[0138] The preparation of the pharmaceutical composition may depend on the route of application to the subject. Injectable preparations suitable for parenteral administration or intraperitoneal, intratumoral, peritumoral, intralesional or perilesional administration include, for example, sterile injectable aqueous or oily suspensions, and can be prepared using suitable dispersants or wetting agents and suspending agents according to known techniques. Injectable preparations can also be sterile injectable solutions, suspensions or emulsions in nontoxic parenteral acceptable diluents or solvents, such as solutions in 1,3-propylene glycol or 1,3-butylene glycol. In available carriers and solvents, water, Ringer's solution, USP and isotonic sodium chloride solution can be used. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. In addition, fatty acids (such as oleic acid) can be used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacteria-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.

[0139] Compositions suitable for oral administration may be presented as discrete units (eg, capsules, tablets, lozenges) each containing a predetermined amount of an anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids, such as syrups, elixirs or emulsions.

[0140] In some embodiments, the compositions provided herein (e.g., pharmaceutical compositions applied to objects) must be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (e.g., 0.2 micron membrane). Alternatively, a preservative can be used to prevent the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. If the pharmaceutical composition is highly stable to thermal denaturation and oxidative denaturation, the pharmaceutical composition is usually stored in a lyophilized form or as an aqueous solution. The pH of the preparation will usually be about 6 to 8, although higher or lower pH values ​​may also be suitable in some cases.

[0141] Administration of anti-NPM1 conjugates

[0142] The present disclosure also provides for administering to a subject an anti-NPM1 antibody described herein, a conjugate comprising an anti-NPM1 antibody described herein, or a composition thereof (e.g., a pharmaceutical composition). In some embodiments, a method is provided for treating a disease associated with cell surface expression of NPM1 (e.g., a cancer expressing NPM1) by administering to a subject an anti-NPM1 antibody described herein, a conjugate comprising an anti-NPM1 antibody described herein, or a composition thereof (e.g., a pharmaceutical composition).

[0143] In some aspects, the present disclosure provides methods for treating a cancer expressing NPM1 in a subject, comprising administering to a subject in need thereof an effective amount of an anti-NPM1 antibody described herein, a conjugate comprising an anti-NPM1 antibody conjugated to an agent described herein (e.g., a drug, a radioisotope), or a composition thereof (e.g., a pharmaceutical composition).

[0144] As used herein, the term "administering" and variations thereof refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing an agent (e.g., an antibody, a conjugate) or a composition thereof (e.g., a pharmaceutical composition) described herein into or on a subject. As used herein, the term "treating" and variations thereof refer to applying or administering an agent (e.g., an antibody, a conjugate) or a composition thereof (e.g., a pharmaceutical composition) described herein to a subject in need thereof for the purpose of reducing the severity of a disease (e.g., cancer) in a subject. "Subjects in need thereof" refer to individuals suffering from a disease, having symptoms of a disease, or having a tendency to a disease. Methods for treating a disease may encompass administering an agent (e.g., an antibody, a conjugate) or a composition thereof (e.g., a pharmaceutical composition) described herein to a subject for the purpose of curing, treating, alleviating, alleviating, altering, rescuing, modifying, improving or affecting a disease, a symptom of a disease, or a tendency to a disease in a subject. Methods for treating a disease may encompass prevention, wherein for the purpose of preventing the occurrence of a disease, for example, in a subject who is not aware of having a disease, but may develop the disease in the future or is at risk of developing the disease, an agent is administered to the subject.

[0145] As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of an agent (e.g., an antibody or conjugate described herein) sufficient to induce a desired biological response in a subject (e.g., to alleviate one or more symptoms of a disease (e.g., cancer)). A therapeutically effective amount can be an amount administered to a subject alone or in combination with one or more other agents. As recognized by those skilled in the art, the effective amount varies according to factors such as the desired biological endpoint, the pharmacokinetics of the administered agent, the specific condition or disease being treated, the severity of the condition or disease, the individual parameters of the subject (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of any other concurrent treatments, the specific route of administration, and similar factors determined within the knowledge and expertise of a health practitioner. These factors are well known to those of ordinary skill in the art and can be resolved without exceeding routine experimentation. It is generally preferred to use the maximum dose of a single agent (e.g., an antibody or conjugate described herein) or any combination thereof described herein, which is at most the highest dose that can be safely administered to a subject based on reasonable medical judgment. Preferably, the effective dose is lower than the highest dose that can be safely administered to a subject. However, one of ordinary skill in the art will appreciate that a subject or health practitioner may select a lower dose (eg, a minimum effective dose) to mitigate any potential treatment risks, such as side effects of the treatment.

[0146] In some embodiments, for an adult subject of normal weight, a dose of 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 1 to 200 mg. The specific dosage regimen (i.e., dose, timing, and repetition) will depend on the specific subject and the subject's medical history, as well as the characteristics of the agent (e.g., the pharmacokinetics of the agent), and other considerations known in the art.

[0147] Treatment of a disease (e.g., cancer) may include delaying the occurrence or progression of a disease, or reducing the severity of the disease. Treatment of a disease does not necessarily require a curative outcome. The occurrence of a "delayed" disease as used herein means delaying, hindering, slowing down, delaying, stabilizing and / or postponing the progression of a disease in an object. Delaying the progression of a disease may include delaying or preventing the occurrence of disease spread in an object, such as delaying or preventing the occurrence of cancer to one or more organs or tissues that have not yet been affected by the cancer in an object. This delay may be a different length of time, depending on the medical history and / or the individual being treated. A "delayed" disease occurrence or a method for delaying the onset of a disease is a method that reduces the likelihood of one or more disease symptoms occurring and / or reduces the degree of symptoms within a given time frame compared to the absence of such a method. Comparisons are typically based on clinical studies using many objects sufficient to give statistically significant results.

[0148] "Occurrence" or "progression" of a disease (e.g., cancer) refers to the initial manifestation and / or subsequent progression of a disease in a subject. The occurrence of a disease can be detected and assessed using standard clinical techniques known in the art. However, occurrence also refers to undetectable progression. For the purposes of this disclosure, occurrence or progression may refer to the occurrence or progression of disease symptoms. The term "occurrence" includes disease occurrence, recurrence, and onset. "Onset" or "occurrence" of a disease as used herein includes the initial onset of a disease as well as the recurrence of a disease (i.e., in a subject who previously had the disease).

[0149] A "subject" contemplated for administration refers to a human (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, child, or adolescent) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a rodent, e.g., a mouse or a rat), a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, such as a chicken, a duck, a goose, or a turkey). The non-human animal can be male or female at any stage of development, and can be a juvenile animal or an adult animal. The non-human animal can be a transgenic animal or a genetically modified animal.

[0150] In some embodiments, the subject is a companion animal (e.g., a pet or service animal). As used herein, "companion animal" refers to pets and other domestic animals. Some 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. In some embodiments, the subject is a research animal. Some non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0151] In some embodiments, the subject has, is suspected of having, or is at risk of a cancer that expresses NPM1. The term "cancer that expresses NPM1" refers to a cancer characterized by expression of NPM1 (e.g., enhanced expression of NPM1 compared to non-cancerous cells), which may also be characterized by a change in the subcellular localization of NPM1, such as an increase in NPM1 present on the surface of a cell. Cancers that express NPM1 may express WT NPM1 and / or mutant NPM1. Cancers that express NPM1 may express WT NPM1 and / or mutant NPM1 on the surface of a cell. Cancers that express NPM1 may also refer to cancers that were not initially characterized by expression of NPM1, but express NPM1 (e.g., cell surface WT NPM1 and / or mutant NPM1) in response to administration of an agent (e.g., a chemotherapeutic drug) or treatment with an agent (e.g., a chemotherapeutic drug). In some embodiments, cancers that express NPM1 are solid (tissue) or liquid (biological fluid, such as blood) cancers. In some embodiments, the cancer expressing NPM1 is a cancer selected from the following: hematological cancer, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, myoma, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer. In some embodiments, the cancer expressing NPM1 is acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). In some embodiments, the cancer expressing NPM1 is a metastatic cancer. In some embodiments, the cancer expressing NPM1 is a treatment-related cancer or a secondary malignancy. The treatment-related cancer or secondary malignancy can be a cancer, such as a leukemia, carcinoma, or lymphoma, caused by a previous treatment with a chemotherapeutic agent or a radioactive isotope. In some embodiments, the cancer expressing NPM1 is therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin's lymphoma.

[0152] In some embodiments, the subject has previously been treated for a cancer that expresses NPM1. In some embodiments, the subject has a cancer that expresses NPM1 that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs). A cancer that expresses NPM1 is considered resistant to treatment (e.g., treatment with one or more chemotherapeutic drugs) if the treatment is not effective in killing and / or inactivating cancer cells, for example, due to genetic and / or epigenetic changes that occur in the cancer that result in inactivation and / or efflux of one or more therapeutic drugs, or inhibition of downstream effects of one or more treatments, especially if the treatment was previously effective in killing and / or inactivating cancer cells. In some embodiments, a cancer that expresses NPM1 that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs) expresses NPM1 on the surface of cancer cells as a result of a previous treatment (e.g., previous treatment with one or more chemotherapeutic drugs) to which the cancer is resistant or has become resistant. In some embodiments, a cancer expressing NPM1 that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs) has increased levels of NPM1 on its surface as a result of a prior treatment (e.g., prior treatment with one or more chemotherapeutic drugs) to which the cancer is or has become resistant.

[0153] Depending on the type of disease to be treated (e.g., cancer) or the site of the disease (e.g., cancer), antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) can be administered to a subject using conventional methods known to those of ordinary skill in the medical field. Antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) can be administered systemically (i.e., throughout the body) or locally (i.e., in one or more specific organs, tissues, or locations in the body). Antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) can be administered by any conventional route, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, intraperitoneally, or by implanted reservoirs. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraperitoneal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) are administered by intravenous injection or infusion. In addition, the antibody, conjugate, or a composition thereof (e.g., a pharmaceutical composition) can be administered to a subject via an injectable reservoir administration route, such as using a 1 month, 3 month, or 6 month reservoir injectable or biodegradable material and method. In some embodiments, the antibody, conjugate, or a composition thereof (e.g., a pharmaceutical composition) is administered by injection. In some embodiments, the injection is intravenous or intratumoral.

[0154] In some embodiments, administration is performed more than once. In some embodiments, administration is performed once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once a year.

[0155] In some aspects, the present disclosure provides a method for treating a cancer expressing NPM1 in a subject, the method comprising administering to a subject in need thereof an effective amount of a combination comprising an anti-NPM1 antibody described herein, a conjugate containing an anti-NPM1 antibody conjugated to an agent described herein (e.g., a drug, a radioisotope), or a composition thereof (e.g., a pharmaceutical composition) and a chemotherapeutic drug or a composition thereof (e.g., a pharmaceutical composition). In some embodiments, the intended combination comprises an antibody or conjugate described herein and a single chemotherapeutic drug described herein. In some embodiments, the intended combination comprises an antibody or conjugate described herein and more than one chemotherapeutic drug described herein.

[0156] In some embodiments, the subject to whom the combination is administered has, is suspected of having, or is at risk of a cancer expressing NPM1. Cancers expressing NPM1 may express WT NPM1 and / or mutant NPM1. Cancers expressing NPM1 may express WT NPM1 and / or mutant NPM1 on the surface of cancer cells. In some embodiments, cancers expressing NPM1 are solid (tissue) or liquid (biological fluid, such as blood) cancers. In some embodiments, cancers expressing NPM1 are cancers selected from the following: hematological cancers, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, myoma, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer. In some embodiments, cancers expressing NPM1 are acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). In some embodiments, the cancer expressing NPM1 is a metastatic cancer. In some embodiments, the cancer expressing NPM1 is a therapy-related cancer or a secondary malignancy. In some embodiments, the cancer expressing NPM1 is a therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin's lymphoma.

[0157] In some embodiments, the subject to whom the combination is administered has previously been treated for a cancer expressing NPM1. In some embodiments, the subject suffers from a cancer expressing NPM1 that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs). As exemplified herein (see Example 4), treating cancer cells with chemotherapeutic drugs (e.g., daunorubicin, venetoclax, 5-azacytidine) can increase the level of NPM1 on and / or in cancer cells (e.g., on leukemia cancer cells, such as AML). Without wishing to be bound by theory, pre-dosing cancer cells (including chemotherapeutic-resistant cancers) with a low dose of chemotherapeutic drugs strengthens (enhances) the binding between cancer cells and the antibodies or conjugates described herein. By strengthening the binding by the antibody or conjugate in this manner, cancer cells can be more easily treated in a subject. For example, cancer cells that have been treated with a low dose of chemotherapeutic drugs can be effectively treated by administering a lower dose of an antibody or conjugate than would otherwise be possible (i.e., without treatment with a low dose of chemotherapeutic drugs). "Low dose" can refer to a dose of a chemotherapeutic drug that is not sufficient in itself to effectively treat cancer in a subject.

[0158] In principle, any chemotherapeutic drug generally known in the art for treating cancer can be administered to a subject as part of a combination. In some embodiments, administration of a chemotherapeutic drug to a subject results in an increase in the level of WT and / or mutant NPM1 on the surface of cancer cells in the subject. In some embodiments, administration of an effective amount of a chemotherapeutic drug to a subject results in an increase in the level of WT and / or mutant NPM1 on the surface of cancer cells in 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 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times or up to 10 times. In some embodiments, administration of an effective amount of a chemotherapeutic drug to a subject does not increase the level of WT and / or mutant NPM1 on the surface of non-cancerous cells in the subject, or increases the level of WT and / or mutant NPM1 on the surface of non-cancerous cells in the subject to an extent that is less than the level of WT and / or mutant NPM1 on the surface of cancer cells in the subject.

[0159] Some non-limiting examples of chemotherapeutic drugs suitable for combination include auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, adoxirubicin, agrochelin, ansatriene, ansamitocin P-3, aphidicolin, apoptin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, camptothecin, chaetocins, globochaetins, chlamydoc in, ember erythromycin B, cladribine, colchicine, compretin A1, compretin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, Engelin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanspiramycin (17-AAG), glucocorticoidin A, gramicidin A, oxadiene, 9-hydroxyellipticine, hydroxyurea, oxadiene, paramycin, idarubicin, ilimacin, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisolA, maytansinol, maytansine (DM1), maytansine DM3, ravtansine (DM4), maytansine AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxin A, myostrobin, mytoxin B, nelarabine, nemorubicin, nocuolin A, cholinesterase, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortataxel, tesetaxel, phalloidin, phalloidin, phytosphingosine, picrotoxin A, bilonisone, podophyllotoxin, polyketidemycin, prednisone, phlorotoxin B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinadol, rachelmin, rabekamycin, Ro 5-3335, safranin B, sandromycin, sanguinarine, saporin, sinefungin, tatoprin, trostatin, 6-thioguanine, thiocolchicine, monofilamentous algae toxin, tripolin A, triptolide, tobramycin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax and vincristine. In some embodiments, the chemotherapeutic drug included in the combination is saporin, daunorubicin, venetoclax or azacytidine.

[0160] In some embodiments, the combined antibody, conjugate, or its composition (e.g., pharmaceutical composition) and the combined chemotherapeutic drug or its composition (e.g., pharmaceutical composition) are administered to the object simultaneously, i.e., they are administered to the object simultaneously or about simultaneously. In some embodiments, the combined antibody, conjugate, or its composition (e.g., pharmaceutical composition) and the combined chemotherapeutic drug or its composition (e.g., pharmaceutical composition) are administered to the object sequentially, i.e., they are administered to the object at different times. In some embodiments, the combined antibody, conjugate, or its composition (e.g., pharmaceutical composition) is administered before the combined chemotherapeutic drug. In some embodiments, the combined antibody, conjugate, or its composition (e.g., pharmaceutical composition) is administered after the combined chemotherapeutic drug. In some embodiments, the combined antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) and the combined chemotherapeutic drug are administered to a subject 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months apart.

[0161] The combination can be administered to a subject using conventional methods known to those of ordinary skill in the medical field, depending on the type of disease to be treated (e.g., cancer) or the site of the disease (e.g., cancer). The combination can be administered systemically (i.e., throughout the body) or locally (i.e., in one or more specific organs, tissues, or locations in the body). The combination can also be administered by any conventional route, such as orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, intraperitoneally, or by implanted reservoirs. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraperitoneal, intrathecal, intralesional, and intracranial injection or infusion techniques. In some embodiments, one or more parts of the combination (e.g., antibodies or conjugates, chemotherapeutic drugs) are administered by intravenous injection or infusion. In addition, one or more parts of the combination (e.g., antibodies or conjugates, chemotherapeutic drugs) can be applied to the object by an injectable reservoir administration route, such as using 1 month, 3 months or 6 months reservoir injectable or biodegradable materials and methods. In some embodiments, one or more parts of the combination (e.g., antibodies or conjugates, chemotherapeutic drugs) are administered by injection. In some embodiments, injection is intravenous injection or intratumoral injection. In some embodiments, antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) and chemotherapeutic drugs or compositions thereof (e.g., pharmaceutical compositions) are administered to the object by the same administration route. In some embodiments, antibodies, conjugates, or compositions thereof (e.g., pharmaceutical compositions) and chemotherapeutic drugs or compositions thereof (e.g., pharmaceutical compositions) are administered to the object by different administration routes.

[0162] In some embodiments, the combination is administered more than once. In some embodiments, the combination is administered once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, once every 2 weeks, once every 3 weeks, once a month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once a year.

[0163] In some embodiments, the subject to which the combination is administered is a human, i.e., a male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, or a teenager) or an adult subject (e.g., a young adult, a middle-aged adult, or an elderly adult)) or a non-human animal. In some embodiments, the non-human animal is a mammal (e.g., a rodent, e.g., a mouse or a rat), a primate (e.g., a cynomolgus monkey or a rhesus monkey), a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog), or a bird (e.g., a commercially relevant bird, such as a chicken, a duck, a goose, or a turkey). The non-human animal can be male or female at any stage of development, and can be a juvenile animal or an adult animal. The non-human animal can be a transgenic animal or a genetically modified animal.

[0164] In some embodiments, the subject of the combination is a companion animal (e.g., a pet or service animal). Some 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. In some embodiments, the subject of the combination is a research animal. Some non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0165] In some embodiments, the subject has not been previously administered a combined antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) (e.g., treated therewith) nor a combined chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) (e.g., treated therewith). In some embodiments, the subject has previously been administered a combined antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) (e.g., treated therewith) and is subsequently administered a combined chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition). In some embodiments, the subject has previously been administered a combined chemotherapeutic drug, or composition thereof (e.g., pharmaceutical composition) (e.g., treated therewith) and is subsequently administered a combined antibody, conjugate, or composition thereof (e.g., pharmaceutical composition).

[0166] In some embodiments, the administration of the combination to a subject induces an enhanced biological response in the subject compared to the biological response that would be achieved by an equivalent dose of the antibody or conjugate described herein administered in the absence of a chemotherapeutic drug described herein. In some embodiments, the administration of the combination to a subject induces an enhanced biological response in the subject compared to the biological response that would be achieved by an equivalent dose of the chemotherapeutic drug described herein administered in the absence of an antibody or conjugate described herein. In some embodiments, combining an antibody or conjugate described herein with a chemotherapeutic drug enhances the effectiveness of the antibody or conjugate administered to the subject. The enhanced biological response resulting from the combined administration may include, but is not limited to, alleviating one or more symptoms of the disease in the subject, or effectively preventing the onset or recurrence of the disease in the subject.

[0167] In some embodiments, administration of the combination to a subject results in increased binding between the antibody or conjugate of the administered combination and cancer cells expressing NPM1 in the subject, compared to administration of the antibody or conjugate alone. "Increased binding" as used herein refers to an increase in the ratio of binding of an antibody or conjugate administered to a subject to cancer cells expressing NPM1 in the subject (e.g., the ratio of binding of an antibody or conjugate administered to a subject to WT and / or mutant NPM1 on the surface of cancer cells expressing NPM1 in the subject). In some embodiments, administration of the combination to a subject increases binding between the antibody or conjugate of the administered combination and NPM1-expressing cancer cells in 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 compared to administration of the antibody or conjugate alone.

[0168] In some embodiments, administration of the combination to a subject results in a decrease in the growth of cancer cells expressing NPM1 in the subject, compared to administration of the antibody or conjugate of the combination alone. "Growth reduction" as used herein refers to a decrease in the rate at which cancer cells expressing NPM1 in a subject undergo cell division (mitosis). In some embodiments, administration of the combination to a subject reduces the growth of cancer cells expressing NPM1 in 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 times, up to 3 times, up to 4 times, up to 5 times, up to 6 times, up to 7 times, up to 8 times, up to 9 times or up to 10 times, compared to administration of the antibody or conjugate alone.

[0169] In some embodiments, administration of the combination to a subject results in increased cell death of NPM1-expressing cancer cells in the subject compared to administration of the combined antibody or conjugate alone. "Cell death" as used herein refers to an increase in the rate at which NPM1-expressing cancer cells in a subject die by any pathway by which cells are no longer viable (e.g., but not limited to, apoptosis, autophagy, necrosis, and endocytosis). In some embodiments, administration of the combination to a subject increases cell death of NPM1-expressing cancer cells in 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 compared to administration of the antibody or conjugate alone.

[0170] One skilled in the art will recognize that the therapeutic methods described herein may also be adapted to treat other diseases or disorders associated with cells expressing cell surface NPM1, for example, non-cancerous diseases or disorders associated with cell surface expression of WT NPM1 or mutant NPM1.

[0171] Example

[0172] Example 1 - NPM1 is localized on the surface of human leukemia cells.

[0173] Nucleolar phosphoprotein 1 (NPM1) cell surface expression was evaluated for various human leukemia cell lines. Briefly, K562, Kasumi, OCI-AML2, OCI-AML3, MOLM13, Jeko1, Nalm6, Jurkat, and SupT1 cells were cultured in vitro with RPM1 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen / Strep). Cell membrane and cytosolic fractions were collected using established techniques (see, e.g., Flynn et al., Cell. 2021; 184(12): 3109-3124.e22) and immunoblotted with the following commercially available antibodies: NPM1-WT (Santa Cruz Biotechnology #sc-32256), NPM1-Mut (ThermoScientific #PA1-46356), β-actin (Santa Cruz Biotechnology #sc-47778), and RPN1 (Santa Cruz Biotechnology #sc-48367). Although most NPM1 was localized in the cytosol of each cell type, a large amount of wild-type (WT) and mutant (mutant, Mut) NPM1 ( Figure 1A Further fluorescence activated cell sorting (FACS) analysis of live cells identified different levels of NPM1 on the surface of each cell type assayed.

[0174] These results suggest that antibodies specific for WT and / or mutant NPM1 can be used to deliver cytotoxic payloads to cancers that express NPM1 in patients, such as, for example, in patients with acute myeloid leukemia (AML), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). However, such a therapeutic strategy relies on the expression of higher levels of NPM1 on the surface of cancer cells than on the surface of non-cancerous cells, particularly the cells from which the cancer originally originated. Therefore, NPM1 levels on the surface of peripheral blood (PB) and bone marrow (BM) cells were assessed. NPM1 cell surface expression was relatively low in most murine PB and BM cells tested, but was increased in cells expressing mutant NPM1 ( Figure 1C and 1D These results were confirmed by FACS analysis of human BM cells, where antibodies specific for WT NPM1 did not bind effectively to human BM cells ( Figure 1E). These results strongly suggest that antibodies specific for NPM1 could be used to effectively treat cancers that express NPM1.

[0175] Example 2 - Development of multiple antibodies specific for NPM1

[0176] To further evaluate the possibility that antibodies specific for NPM1 could be used to treat cancers expressing NPM1, new antibodies specific for NPM1 were generated. Different antibodies specific for NPM1 were generated ("Ab1" and "Ab2"; Figure 2A and 2B ).

[0177] To evaluate whether the isolated antibody can be used to treat cancer, the Ab1 anti-NPM1 antibody was arranged in an antibody-drug conjugate and used to treat OCI-AML3 cells in vitro. OCI-AML3 cells are a model for human acute myeloid leukemia (AML). Briefly, approximately 150,000 cells were cultured in RPMI medium containing 10% FBS and 1% Pen / Strep. Streptavidin-saponin (Strep-ZAP) was combined with a biotinylated antibody (2.6 μg Strep-ZAP with 1 μg IgG) on ice for 30 minutes. Strep-ZAP was combined with a biotinylated Ab1 anti-NPM1 antibody ( Figure 2A ) or biotinylated anti-mouse isotype control (Strep-ZAP-IgG) (Thermo Scientific #31800) were combined. After binding, the Strep-ZAP-IgG complex was added to the cells and the cells were grown at 37°C for 24 hours or 48 hours. After each time point, the cells were harvested, washed in PBS, stained with Annexin V to detect apoptosis, and stained with DAPI to detect whether the cells were alive. After staining, the cells were washed and analyzed by FACS. After incubation, it was observed that the killing of OCI-AML3 cells was in a dose-dependent manner, with fewer live cells observed after 48 hours at the highest dose ( Figure 3A and 3B Importantly, less apoptosis and cell death were observed in the presence of the same amount of Strep-ZAP lacking cell surface targeting (Strep-ZAP-IgG sample). These results demonstrate the utility of anti-NPM1 antibody-based ADCs for targeting cytotoxic drugs to cancer cells expressing cell surface NPM1.

[0178] Although these studies were performed with human cell lines, it should be noted that NPM1 antibodies can be used to target endogenous NPM1 on the surface of non-human cell types due to the high conservation of NPM1. Notably, the amino acid sequence of WT NPM1 is approximately 95% identical between human and mouse ( Figure 4A ), and has greater than 99% identity between humans and other primates ( Figure 4B The high degree of conservation also suggests that results obtained by testing the effects of anti-NPM1 antibodies or ADCs in, for example, murine cells or mice would be expected to extend to humans.

[0179] Example 3 - Antibodies detect WT and mutant NPM1 on the surface of human and mouse models

[0180] To further confirm that NPM1 antibodies can be used to target NPM1 on cancer cells, the levels of NPM1 on the surface of various human and mouse cancer cells were explored using FACS analysis. In some cases, live cells were assayed to observe cell surface localized NPM1. In other cases, fixed cells were assayed to observe intracellular and cell surface levels of NPM1. Significant levels of NPM1 were observed on the surface of live OCI-AML3 cells, whereas no significant amounts of NPM1 were robustly observed on MOLM13 cells ( Figure 5A Similar to OCI-AML3 cells, significant levels of NPM1 were observed on the surface of multiple primary murine AML cell lines ( Figure 5B and 5C ). To further explore cell surface NPM1 in non-AML cell lines, human K562 cells, which are a model for myeloid leukemia, were also evaluated. In these experiments, exogenous NPM1 was expressed with a TY1 tag, where the exogenous NPM1 was WT NPM1 or mutant NPM1 (NPM1c). Using live and fixed cell flow cytometry, it was found that the TY1 signal was present on the surface of cells expressing WT-NPM1-TY1 or NPM1c-TY1, indicating that the NPM1c protein may also be present on the cell surface ( Figures 6A to 6E ). An orthogonal immunofluorescence detection strategy determined the surface distribution of TY1-labeled NPM1 molecules ( Fig. 6F ).

[0181] Given that a variety of murine and human leukemia cell lines express cell surface WT and / or mutant NPM1, it was then assessed whether the isolated antibodies could also bind to the surface of these cells. As determined by FACS analysis, the isolated Ab1 anti-NPM1 ( Figure 2A ) and Ab2 anti-NPM1 ( Figure 2B ) antibodies were able to bind to primary mouse MLL-rearranged AML cells ( Fig. 7A) and primary mouse NPM1cAML cells ( Figure 7B ). In addition, the antibody bound to NPM1 on human patient-derived xenograft (PDX) cells transplanted into a mouse model, but not to host mouse bone marrow cells ( Figures 8A to 8D ). These results, combined with the above results, provide additional evidence that ADCs comprising one of these anti-NPM1 antibodies can be used to selectively direct chemotherapeutic agents to cancer cells with a lower risk of toxicity to non-cancerous tissues. These antibodies can be further used in other applications, including, for example, by conjugating the antibodies to imaging agents in place of diagnostic agents, which can then be used to locate and measure the relative abundance of cancer cells in patients.

[0182] Example 4 - Chemotherapy for the treatment of AML increases cell surface levels of NPM1

[0183] Next, the effects of currently approved chemotherapeutic agents for the treatment of AML on cell surface NPM1 levels were evaluated. In principle, administration of these drugs to subjects could alter cell surface expression of WT and / or mutant NPM1, which in turn would modulate the efficacy of anti-NPM1 antibodies or ADCs administered to subjects. The effects of two chemotherapeutic agents used to treat AML (daunorubicin and venetoclax) on the binding of an anti-NPM1 antibody (Merck / Sigma anti-B23 #B0556) to NPM1 on the surface of OCI-AML3 cells were evaluated. Unexpectedly, after 48 hours of treatment with 10 nM daunorubicin or 40 nM venetoclax, antibody binding to cells was enhanced ( Fig. 9A This effect was significantly enhanced 8 days after treatment ( Fig. 9A and Fig. 9B Consistent with these results, treatment with daunorubicin or venetoclax also enhanced the anti-WT NPM1 and anti-mutant NPM1 antibodies isolated above ( Figure 2A and Figure 2B ) and NPM1 on the surface of OCI-AML3 cells ( Fig. 9C The same effect was observed when OCI-AML3 cells were treated with an alternative chemotherapeutic agent, 5-azacytidine (5-Aza; azacitidine), which is also used to treat myelodysplastic syndrome (MDS) ( Fig.9D). Notably, the binding between isolated anti-mutant NPM1 and OCI-AML3 cells was significantly enhanced after only 24 hours of treatment with 5-Aza. These data indicate that a variety of chemotherapeutic agents reliably enhance the binding of anti-NPM1 antibodies to the surface of cancer cells. Although the reason for this effect is not immediately clear, it may be that these drugs increase the level of cellular stress in cancer cells, which further transfers NPM1 from the nucleus and cytosol to the cell membrane. These results suggest that combination therapy may be particularly useful for treating certain cancers, where chemotherapeutic agents are administered to a subject not only for the purpose of killing cancer cells, but also to increase the level of NPM1 on the surface of cancer cells, which can be used to bind to the anti-NPM1 antibody or ADC co-administered to the subject. Potentially, the chemotherapeutic agent and the anti-NPM1 antibody or ADC may be administered to the subject simultaneously (e.g., as part of the same composition or as separate compositions) or at different times during the course of treatment.

[0184] Example 5: NPM1 is a cell surface protein.

[0185] To evaluate the subcellular localization of nucleolar phosphoprotein 1 (NPM1), confocal imaging was performed on OCI-AML3 (human cell line). Both intracellular and cell surface staining were performed. Intracellular staining was achieved by fixing the cells and permeabilizing the cells before staining. In contrast, cell surface staining was performed by staining the cells before fixation. The staining results showed that NPM1 was present both inside the cell and at the cell surface. Inside the cell, NPM1 is in the nucleolus, and on the cell surface, NPM1 forms clusters ( Fig. 10A ). To investigate whether the biochemical fractionation was specific, other RNA binding proteins (RBPs) were investigated, including heterogeneous nuclearribonucleoprotein U (HNRNPU), nucleolar RNA helicase 2 (DDX21), dolichol diphosphate oligosaccharide protein glycosyltransferase subunit 1 (RPN1), and RIO kinase 1 (RIOK1). RBPs were measured in the cytoplasm and membrane compartments of various cell lines including 293, A549, K562, and AML3. The results of biochemical fractionation and western blotting (WB) showed that some other RBPs but not all highly abundant cytoplasmic proteins were present in the membrane fractions of various cells ( Fig. 10B ).

[0186] In addition to evaluating the cell surface expression of NPM1 on human cells, primary murine acute myeloid leukemia (AML) cells were also studied using live cell flow cytometry. As with the human OCI-AML3 cell line, cell surface expression of NPM1 was detected on primary murine AML cells ( Figures 11A to 11B ).

[0187] The cell surface localization of NPM1 was also tested using western blotting. Anti-NPM1 captured full-length NPM1 from the cell membrane fraction ( Fig. 12A ). Cell surface NHS-biotinylation (surface proteins only) followed by anti-NPM1 IP from the membrane fraction selectively isolated the biotinylated band at the molecular weight of NPM1 ( Fig. 12B ).

[0188] In addition, to evaluate the ability of NPM1 to form nanoclusters on the cell surface, super-resolution microscopy was performed on HL-60 (a human leukemia cell line) and OCI-AML3 cells. Super-resolution microscopy defined clusters of 120 to 150 nanometers (nm). These NPM1 clusters were present on the cell surface of both HL-60 and OCI-AML3 cell lines. The clustering and non-random pattern of NPM1 markers indicates a highly regulated cell biological process ( Figures 13A to 13B Super-resolution microscopy performed on the PANC1 cell line (pancreatic cancer) also showed that NPM1 forms nanoclusters on the surface of cancer cells ( Fig. 13B ).

[0189] Results from intracellular and cell surface staining, live cell flow cytometry, and western blotting indicate that NPM1 is a cell surface protein in both human and mouse cell lines. In addition, super-resolution microscopy showed that NPM1 forms regular nanoclusters on the cell surface of a variety of human cancer cell lines, including acute myeloid leukemia, leukemia, and pancreatic cancer.

[0190] Example 6: Ab2.2 antibody targets NPM1.

[0191] An antibody having a heavy chain (Ab2.2) (SEQ ID NO:44) and a light chain (Ab2.2) (SEQ ID NO:45) (hereinafter referred to as "Ab2.2") was generated and used to target NPM1 on the surface of human and non-human cell types.

[0192] The ability of Ab2.2 to bind to NPM1 on the cell surface of cancer cells (OCI-AML3 cells) was tested using both live cell microscopy and microscopy of fixed and permeabilized cells. Ab2.2 was compared to a commercially available NPM1 antibody. The commercially available NPM1 antibody used was Santa Cruz (SC) anti-NPM1-AF647. Live cell microscopy showed that both the commercially available NPM1 antibody and maAb2 stained surface spots. Therefore, Ab2.2 behaved similarly to the commercially available NPM1 antibody on the cell surface ( Fig.14). Microscopy of fixed and permeabilized cells showed that the commercial NPM1 antibody resulted in staining of the nucleolus and cytoplasm, whereas the Ab2.2 antibody resulted in nucleolus and more predominant cytoplasm staining. Thus, Ab2.2 detected more cytoplasmic NPM1 in mutant OCI-AML3 cells compared to the commercial NPM1 antibody ( Fig.14 ).

[0193] Ab2.2 was also tested for its ability to bind to NPM1 on the cell surface of healthy non-cancerous cells using flow cytometry. The cells used to measure Ab2.2 binding were leukocytes (CD45+ cells), myeloid cells (CD33+ cells), and hematopoietic stem cells ("HSC", CD34+ cells). Only 3.78% of the cells in donor 1 were CD34+ and Ab2.2 double positive (donors 2 and 3 were less than 3% double positive). Therefore, almost no binding of Ab2.2 to healthy HSCs was observed ( Fig.15A ).

[0194] Ab2.2 antibody was further validated using western blot and compared with a commercial antibody. The commercial antibody was provided by Santa Cruz (SC FC8791). Lysates from two sources, WCE and crude membrane (Mem), were evaluated. Both Ab2.2 and the commercial antibody produced a band of approximately 38 kB. Thus, the commercial and Ab2.2 showed almost identical banding patterns for NPM1 ( Fig. 15B ).

[0195] Example 7: Ab2.2 targets NPM1 in cancer cells in vitro.

[0196] To determine whether Ab2.2 targets NPM1 in cancer cells in vitro, patient samples collected from Dana-Farber Cancer Institute (DFCI) and the United Kingdom (UK) were used. Fig.16 ) and 15 AML patients from the UK ( Fig.18 ) and analyzed using flow cytometry. Figures 17A to 17B and Figures 19A to 19C The results showed that Ab2.2 and AML bone marrow blasts ( Figures 17A to 17B , patient 5) and NPM1c AML bone marrow ( Figures 19D to 19E ). In addition, NPM1c blasts are low in CD34, allowing the detection of a population of LSCs that are highly bound by Ab2.2. Thus, Ab2.2 binds best to LSCs in the bone marrow of NPM1c patients ( Figures 19D to 19E ). The results from DFCI indicate that Ab2.2 strongly stains blasts, regardless of mutation status, disease state, or prior treatment. The results from UK show that Ab2.2 strongly stains blasts. Furthermore, in the NPM1c patient, whose LSCs are CD34-, Ab2.2 strongly stains, and thus Ab2.2 will target leukemia initiating cells.

[0197] Example 8: Ab2.2 does not cause toxicity in mice.

[0198] To evaluate the toxicity of Ab2.2 in vivo, wild-type (WT, C57BL / 6J) mice were treated with Ab2.2 weekly. There were 5 mice in each group. The targeted group received 2.5 mg / kg, 5 mg / kg, or 10 mg / kg doses of Ab2.2 by IP injection. The control group was given 5 mg / kg of IgG ( Fig. 20A ). A total of four doses were delivered, one dose per week. Weekly blood sampling was performed to collect samples on day 1 (D), day 7, day 14, day 20, and day 27 of treatment. Mice were weighed at D1, D7, D14, D20, and D27, and samples were tested for white blood cells (WBC), platelets (PLT), and hemoglobin (HGB). Ab2.2 treatment did not result in observable toxicity in mice ( Fig. 20B ).

[0199] Example 9: Ab2.2 treatment improves survival of AML mouse model and does not affect healthy mice.

[0200] Different models were used to evaluate the efficacy of Ab2.2 treatment. In efficacy model 1, mice were subjected to sublethal irradiation and then transplanted with primary murine AML cells. Mice receiving primary murine AML were treated with Ab2.2 antibody at a dose of 5 mg / kg weekly. Administration was performed by IP injection. Weekly blood sampling was performed and mouse overall survival, bone marrow (BM) and spleen analysis and molecular phenotyping ( Fig.21A ). The primary murine cells used were NPM1c / Flt3-ITD AML, and a syngeneic AML mouse model was used. The control group received a 5 mg / kg dose (IgG), and the targeted group received a 5 mg / kg dose (Ab2.2). Three doses were administered (1 dose per week). Antibody binding was observed, and results from total WBC counts and qPCR showed a reduction in tumor burden in mice treated with Ab2.2 ( Figures 21B to 21D In addition, spleen weight, HGB and hematocrit (HCT) levels indicated that Ab2.2 resolved the hematopoietic defect caused by AML ( Figures 21E to 21FFinally, Ab2.2 treatment improved the overall survival of mice ( Figure 21G The survival study was terminated at day 100 due to regimen rather than toxicity or AML relapse.

[0201] In efficacy model 2, mice were subjected to sublethal irradiation and subsequently transplanted with primary murine AML cells. Mice receiving primary murine AML were treated with Ab2.2 antibody weekly at a dose of 5 mg / kg. Administration was performed by IP injection. Weekly blood sampling was performed and overall survival and molecular phenotyping of mice were performed ( Fig.22A ). The primary mouse cells used for transplantation were MLL-AF9 / Flt3-ITD AML. A syngeneic AML mouse model was used. The control group received a 5 mg / kg dose (IgG) and the targeted group received a 5 mg / kg dose (Ab2.2). A total of 4 doses of Ab2.2 were administered. Antibody binding was observed using flow cytometry ( Fig. 22B The mean survival of the Ab2.2 treated group was significantly higher than that of the IgG treated animals (p=0.0027). The survival of the IgG treated mice was approximately 20 days compared to approximately 55 days in the Ab2.2 treated mice ( Fig. 22C ). Thus, Ab2.2 improves overall survival in AML models with lower NPM1 surface levels. Furthermore, these results suggest that any amount of surface expression of NPM1 will result in a survival benefit with Ab2.2 treatment.

[0202] Spleen, lung and liver samples collected three days after the first dose of Ab2.2 treatment were compared between the control (IgG) and Ab2.2 treated groups. The spleen, lung and liver of Ab2.2 treated mice all weighed less than those of IgG treated mice ( Fig. 23B ). These results indicate that a single dose of Ab2.2 resulted in a robust reduction in organ weights, indicative of tumor clearance.

[0203] Efficacy Model 2 was also used to evaluate LSC targeting of Ab2.2 in secondary recipients ( Fig.23A ). The primary mouse cells transplanted were MLL-AF9 / Flt3-ITD AML, and a syngeneic AML mouse model was used. The control group received a 5 mg / kg dose (IgG), and the targeted group received a 5 mg / kg dose (Ab2.2). A dose of Ab2.2 was administered to the secondary recipients before they received the transplant. The secondary recipients in the control group received transplanted cells from mice treated with IgG. The targeted group received transplanted cells from mice treated with Ab2.2. The same number of cells were transplanted, and survival assays, flow cytometry, transplant examinations, and LSC function measurements were performed. The results of the survival assays showed that the average survival of the control group was less than 30 days ( 50 days) compared to the targeted group. Fig.23CThe extended lifespan of Ab2.2-treated mice suggests that fewer stem cells are present in Ab2.2-treated mice. Flow cytometric analysis showed that Ab2.2 stained the stem cell compartment slightly better than large tumors ( Fig.23D ), and the MLL-AF9 model has low expression of NPM1 on the surface ( Fig.23E ). In mice treated with Ab2.2, WBC counts decreased and PLTs increased ( Fig.23F ). Further analysis of WBC over time showed that WBC remained lower in Ab2.2 treated mice compared to IgG treated mice over time ( Figure 23G The spleen weights of secondary recipients treated with Ab2.2 were lower than those of mice treated with IgG ( Fig.23H These results indicate that Ab2.2 treatment resulted in a robust reduction in organ weights, indicating tumor clearance. The percentage (%) of AML in the bone marrow (BM) and peripheral blood (PB) was assessed. In both BM and PB, the % AML in the Ab2.2 treated group was reduced ( Fig.23I ). These results indicate that Ab2.2 targets tumors after only one dose.

[0204] The effect of Ab2.2 treatment was tested on wild-type mice that had received sublethally irradiated. WT C57BL / 6J mice were subjected to sublethally irradiated and four Ab2.2 treatments. Ab2.2 treatments were performed weekly by IP injection and weekly blood draws were performed. The control group received 5 mg / kg of IgG and the Ab2.2 treated group received 10 mg / kg of Ab2.2. Routine blood cell counts, animal phenotyping, and measurements of adverse / toxic effects were performed ( Fig.26A The results showed that no significant differences were observed in weight, WBC counts, HGB levels, or PLT levels between IgG-treated mice and Ab2.2-treated mice between day 1 (D) and D27 ( Fig.26B ). Thus, WT mice showed no observable effects of Ab2.2 treatment.

[0205] Example 10: The effect of Ab2.2 on survival depends on the immune system.

[0206] The immune-mediated killing ability of Ab2.2 was evaluated using efficacy model 3. In efficacy model 3, NSG mice transplanted with primary murine AML cells were treated weekly with Ab2.2 and overall survival ( Fig.24A). The transplanted primary mouse cells were MLL-AF9 / Flt3-ITD AML. The mouse model used was an immunocompromised AML mouse model (NSG). The control group received a 5 mg / kg dose (IgG), and the targeted group received a 5 mg / kg dose (Ab2.2). A total of four doses of Ab2.2 were administered. The results of the survival assay showed that the average survival of the control group and the targeted group was about 30 days ( Fig. 24B ). Therefore, in the absence of an intact immune system, Ab2.2 does not provide a survival benefit.

[0207] Example 11: Ab2.2 treatment reduces tumor volume in vivo.

[0208] Ab2.2 was evaluated in vivo for solid tumor activity using efficacy model 4. In efficacy model 4, transplanted mice were subjected to weekly Ab2.2 treatments and assessed for overall survival, calculated tumor burden, and molecular phenotyping ( Fig.25A ). The transplanted cells were from MC38 mouse colorectal adenocarcinoma. A syngeneic mouse model was used. The control group received a 10 mg / kg dose (IgG) and the targeted group received a 10 mg / kg dose (Ab2.2). A total of three doses were administered (1 dose per week). Calculation of tumor burden showed that Ab2.2 reduced tumor volume ( Figures 25B to 25C ).

[0209] Example 12: Ab2.2 is present on precancerous cells.

[0210] DNMT3a mutations are directly associated with clonal hematopoiesis of indeterminate potential (CHIP) and preleukemia. To test whether Ab2.2 can detect Npm1c on Dmt3aR882H mutant cells, IgG control or Ab2.2 against Dmnt3a R882H / - and Dmnt3a R882H / - The results showed that even on a single Dmnt3a R882H mutant cell there was high Npm1c surface detection ( Fig.28 ).

[0211] Example 13: Ab2.2 binds to human tumors in vitro.

[0212] To evaluate the ability of Ab2.2 to bind to human tumors, multiple types of in vitro models were studied using flow cytometry. The tumor models used to evaluate Ab2.2 binding were murine melanoma, lung cancer (human), laryngeal cancer, colorectal cancer (human and mouse), Ewing's sarcoma (human), pharyngeal cancer (human), pancreatic cancer (human), esophageal cancer (human), osteosarcoma (human), neuroblastoma (human), brain tumor (human), hematological malignancies, fibrosarcoma, prostate cancer, and pancreatic adenocarcinoma (mouse) ( Figures 27A to 27O ). The results showed that Ab2.2 bound to a variety of human tumor model groups in vitro.

[0213] Example 14: Ab2.2-Saporin ADC exhibits in vitro activity.

[0214] To evaluate the in vitro activity of Ab2.2 in the context of an ADC, OCI-AML3 cells were treated with a negative control, an isotype saporin conjugate, or an Ab2.2-saporin conjugate. The cells were treated for 24, 48, and 72 hours. The number of viable cells per μL was assessed at these time points. At 72 hours, the number of viable cells in cells treated with the Ab2.2-saporin conjugate was reduced by approximately 30% ( 1:100) compared to the negative control. Fig.29 ). Therefore, NPM1 can serve as an ADC target at least in some models.

[0215] Example 15: Ab2.2 treatment extends the lifespan of mice transplanted with human AML cell lines.

[0216] The ability of Ab2.2 to extend lifespan in a mouse model xenografted with a human AML cell line was evaluated. SCIDCB17 mice were transplanted with OCI-AML3 cells (human AML). Next, the transplanted mice were treated weekly with Ab2.2 (targeted group) or IgG (control group). The targeted group received a 10 mg / kg dose of Ab2.2. The mice received a total of 4 doses. The overall survival of the mice was evaluated and molecular phenotyping was performed ( Fig. 30A ).

[0217] Antibody binding was assessed using flow cytometry ( Fig. 30B The results of the survival assay showed that the survival of the transplanted mice treated with Ab2.2 was significantly longer than that of the mice treated with IgG (p=0.0015) for more than 40 days after transplantation. Fig. 30C Thus, lifespan extension was observed in mice transplanted with a human AML cell line and Ab2.2-treated natural killer (NK) cells and complement.

[0218] Example 16: Ab2.2 treatment extends the lifespan of mice transplanted with human AML-PDX cell lines.

[0219] The ability of Ab2.2 to extend lifespan in a mouse model xenografted with human AML-PDX was evaluated. SCIDCB17 mice were transplanted with PDX(MLL-R) cells (human AML-PDX). Next, the transplanted mice were treated with Ab2.2 (targeted group) or IgG (control group) weekly. The targeted group received a 10 mg / kg dose of Ab2.2. The mice received a total of 4 doses. The overall survival of the mice was evaluated and molecular phenotyping was performed ( Fig.31A ).

[0220] Antibody binding was assessed using flow cytometry ( Fig.31B The results of the survival assay showed that the survival of the transplanted mice treated with Ab2.2 survived more than 60 days after transplantation, which was significantly longer than that of the mice treated with IgG (p=0.0023). Fig.31C ). Accordingly, lifespan extension was observed in mice transplanted with a human AML-PDX cell line as well as Ab2.2-treated natural killer (NK) cells and complement.

[0221] Example 17: Low-dose chemotherapy induces cell surface NPM1.

[0222] To determine whether low doses of chemotherapeutic agents (daunorubicin and venetoclax) induce cell surface NPM1, OCI-AML3 cells were exposed to control or low dose chemotherapeutic treatments. OCI-AML3 cells are resistant to BLC2i. Daunorubicin was administered at 10 nanomoles (nm), and venetoclax was administered at 40nm. The administration of daunorubicin increased NPM1 on the cell surface at 48 hours, and maintained high expression for at least 9 days. The administration of venetoclax and 5-Aza alone increased NPM1 at 48 hours, and maintained high expression for at least 7 days ( Fig.32 ). These results suggest that low doses of chemotherapeutic agents induce cell surface NPM1 on AML cells and that Ab2.2 enhances the safety or efficacy of approved drugs.

[0223] The ability of low-dose chemotherapeutic agents (daunorubicin and venetoclax) to induce cell surface NPM1 on non-transformed cells was tested. The normal hematopoietic progenitor cell line HPC7 and the normal myeloid progenitor cell line HOXB8 were exposed to control or low-dose chemotherapeutic treatment. Doses of daunorubicin and venetoclax were administered at 10 nanomolar (nM) and 40 nM, respectively. The antibody binding results measured by flow cytometry showed no change in low-dose chemotherapeutic agents compared to control ( Fig.33 ). Therefore, low doses of chemotherapeutic agents do not induce NPM1 expression in normal, non-transformed cells.

[0224] Equivalent programs and scope

[0225] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to some of the embodiments described herein.The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0226] Unless otherwise indicated or otherwise obvious from the context, a noun without a quantifier may mean one or more than one. Unless otherwise indicated or otherwise obvious from the context, a claim or description containing "or / or" between two or more members of a group is considered to meet the requirements if one, more than one, or all group members are present. The disclosure of a group containing "or / or" between two or more group members provides embodiments in which there is exactly one group member, embodiments in which there are more than one group member, and embodiments in which all group members are present. For brevity, these embodiments are not individually set forth herein, but it is understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0227] It should be understood that the present disclosure covers all variations, combinations and permutations in which one or more limitations, elements, clauses or descriptive terms from one or more claims or from one or more relevant parts of the specification are introduced into another claim. For example, a claim that refers to another claim can be amended to include one or more limitations found in any other claim that refers to the same base claim. In addition, where a composition is recited in the claims, it should be understood that unless otherwise stated or unless it is obvious to a person of ordinary skill in the art that a contradiction or inconsistency will arise, methods of making or using the composition (if any) according to any of the methods disclosed herein or methods known in the art are included.

[0228] In the case where the element is represented by a list (e.g., in the form of a Markush group), it should be understood that each possible subgroup of the element is also disclosed, and any element or subgroup of the element can be removed from the group. It should also be noted that the term "comprising / including" is intended to be open, and allows the inclusion of additional elements or steps. It should be understood that, in general, in the case where an embodiment, product or method is referred to as comprising a specific element, feature or step, an embodiment, product or method consisting of such an element, feature or step or substantially consisting of such an element, feature or step is also provided. For the sake of brevity, these embodiments are not separately set forth herein, but it should be understood that each of these embodiments is provided herein and can be specifically claimed or abandoned for protection.

[0229] In the case of giving a range, endpoints are included. In addition, it should be understood that, unless otherwise stated or otherwise apparent from the context and / or the understanding of those of ordinary skill in the art, the value expressed as a range may be assumed in some embodiments to be one-tenth of the unit of any specific value in the range to the lower limit of the range, unless the context clearly specifies otherwise. For the sake of brevity, the value in each range is not individually set forth herein, but it should be understood that each of these values ​​is provided herein and may be specifically claimed or waived. It should also be understood that, unless otherwise stated or otherwise apparent from the context and / or the understanding of those of ordinary skill in the art, the value expressed as a range may be assumed to be any sub-range within a given range, wherein the endpoint of the sub-range is represented with the same accuracy as one-tenth of the unit of the lower limit of the range.

[0230] In the case of websites, the URL address is provided as non-browser executable code with the period of the corresponding URL in brackets. The actual URL does not contain brackets.

[0231] In addition, it should be understood that any specific embodiment of the present disclosure may be explicitly excluded from any one or more claims. Where a range is given, any value within the range may be explicitly excluded from any one or more claims. Any embodiment, element, feature, application or aspect of the composition and / or method of the present disclosure may be excluded from any one or more claims. For the sake of brevity, all embodiments in which one or more elements, features, purposes or aspects are excluded are not explicitly set forth herein.

Claims

1. A method for treating a cancer expressing nucleolar phosphoprotein 1 (NPM1), the method comprising administering to a subject in need thereof an effective amount of an antibody that binds to NPM1 and a chemotherapeutic drug.

2. A method for treating a cancer expressing NPM1, the method comprising administering to a subject in need thereof an effective amount of an antibody that binds to NPM1, wherein the subject is receiving or has received treatment with a chemotherapeutic drug.

3. A method for treating a cancer expressing NPM1, the method comprising administering to a subject in need thereof an effective amount of a chemotherapeutic drug, wherein the subject is receiving or has received treatment with an antibody that binds to NPM1.

4. The method of any one of claims 1 to 3, wherein the antibody comprises: a heavy chain variable region comprising a heavy chain (HC) complementarity determining region (CDR) 1 comprising the amino acid sequence of NIFVH (SEQ ID NO: 1), a HC CDR2 comprising the amino acid sequence of KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and a HC CDR3 comprising the amino acid sequence of DSSGYDAVDY (SEQ ID NO: 3); and A light chain variable region comprising a light chain (LC) CDR1 comprising the amino acid sequence of RASESVYTYLA (SEQ ID NO: 9), a LC CDR2 comprising the amino acid sequence of NAKTLTE (SEQ ID NO: 10), and a LC CDR3 comprising the amino acid sequence of QHHYGTPYT (SEQ ID NO: 11).

5. The method of claim 4, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

29.

6. The method of any one of claims 1 to 3, wherein the antibody comprises: a heavy chain variable region comprising a heavy chain (HC) complementarity determining region (CDR) 1 comprising the amino acid sequence of SYAMS (SEQ ID NO: 15), a HC CDR2 comprising the amino acid sequence of AISGSGGSTYYADSVKG (SEQ ID NO: 16), and a HC CDR3 comprising the amino acid sequence of WRNNAFDY (SEQ ID NO: 17); and A light chain variable region comprising a light chain (LC) CDR1 comprising the amino acid sequence of QGDSLRSYYAS (SEQ ID NO: 22), a LC CDR2 comprising the amino acid sequence of GKNNRPS (SEQ ID NO: 23), and a LC CDR3 comprising the amino acid sequence of NSSPRLKHRVV (SEQ ID NO: 24).

7. The method of claim 6, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 30; and / or the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:

31.

8. The method of any one of claims 1 to 7, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

9. The method of claim 8, wherein the antibody is a full-length antibody selected from the group consisting of immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), and immunoglobulin M (IgM).

10. The method of claim 9, wherein the antibody is IgG.

11. The method of any one of claims 1 to 7, wherein the antibody is an antigen binding fragment selected from the group consisting 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.

12. The method of any one of claims 1 to 11, wherein the antibody is a human antibody or a humanized antibody.

13. The method of any one of claims 1 to 7, wherein the antibody further comprises a heavy chain constant region.

14. The method of claim 13, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:

32.

15. The method of any one of claims 1 to 7, wherein the antibody further comprises a light chain constant region.

16. The method of claim 15, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:

34.

17. The method of any one of claims 13 to 16, wherein 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.

18. The method of any one of claims 13 to 16, wherein 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.

19. The method of any one of claims 13 to 16, wherein 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.

20. The method of any one of claims 4, 5, and 8 to 17, wherein the antibody preferentially binds to wild-type NPM1.

21. The method of any one of claims 6 to 16 and 18 to 19, wherein the antibody preferentially binds to mutant NPM1.

22. The method of any one of claims 1 to 21, wherein the antibody is conjugated to an agent.

23. The method of claim 22, wherein the agent is a drug.

24. The method of claim 23, wherein the drug is selected from the group consisting of auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, adoxirubicin, agrochelin, ansatriene, ansamitocin P-3, aphidicolin, apoptin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, camptothecin, chaetocin, chaetocrinin, chlamydochinosin, cin, ember erythromycin B, cladribine, colchicine, compretin A1, compretin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, Engelin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanspiramycin (17-AAG), glucocorticoidin A, gramicidin A, oxadiene, 9-hydroxyellipticine, hydroxyurea, oxadiene, paramycin, idarubicin, ilimagone, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisolA, maytansinol, maytansine (DM1), maytansine DM3, ravtansine (DM4), maytansine AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxin A, myostrobin, mytoxin B, nelarabine, nemorubicin, nocuolin A, cholinesterase, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortataxel, tesetaxel, phalloidin, phalloidin, phytosphingosine, picrotoxin A, bilonisone, podophyllotoxin, polyketidemycin, prednisone, phlorotoxin B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinaldotide, rachelmin, rabekamycin, Ro 5-3335, safranin B, sandrocin, sanguinarine, saporin, sinefungin, tatoprolol, trostatin, 6-thioguanine, thiocolchicine, monacolin, tripolin A, triptolide, tobramycin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine.

25. The method of claim 24, wherein the drug is saporin, daunorubicin, venetoclax, or azacitidine.

26. The method of any one of claims 22 to 25, wherein the antibody and the drug are conjugated via a linker.

27. The method of claim 26, wherein the linker is a cleavable linker.

28. The method of claim 27, wherein the linker is a pH-sensitive linker, a glutathione-sensitive linker, or a protease-cleavable linker.

29. The method of claim 27 or 28, wherein the cleavable linker is selected from: N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butyrate (SPDB), Sulfo-SPDB, Valine-Citrulline (Val-cit), Acetobutyrate, CL2A, Maleimidocaproyl (MC) and Mal-EBE-Mal.

30. The method of claim 26, wherein the linker is a non-cleavable linker.

31. The method of claim 30, wherein the non-cleavable linker is selected from: N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), MC-VC-PAB.

32. The method of any one of claims 23 to 31, wherein the ratio of the antibody to the drug is 1:1 to 1:

10.

33. The method of claim 32, wherein the ratio of the antibody to the reagent is 1:

4.

34. The method of claim 22, wherein the agent is a radioisotope.

35. The method of claim 34, wherein the radioisotope is selected from the group consisting of iodine-131, rhenium-188, yttrium-90, bismuth-213, and actinium-225.

36. The method of any one of claims 1 to 35, wherein the NPM1-expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells.

37. The method of any one of claims 1 to 36, wherein the NPM1-expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells as a result of administration of or treatment with the chemotherapeutic drug.

38. The method of claim 36 or 37, wherein the NPM1-expressing cancer is a cancer in which wild-type NPM1 and / or mutant NPM1 is expressed on the surface of cancer cells.

39. The method of any one of claims 1 to 38, wherein the cancer is a solid cancer or a liquid cancer selected from the group consisting of hematological cancer, lung cancer, breast cancer, brain cancer, gastrointestinal cancer, liver cancer, kidney cancer, bladder cancer, pancreatic cancer, ovarian cancer, testicular cancer, prostate cancer, endometrial cancer, muscle cancer, bone cancer, neuroendocrine cancer, connective tissue cancer, head and neck cancer, or skin cancer.

40. The method of any one of claims 1 to 39, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma, and myelodysplastic syndrome (MDS).

41. The method of any one of claims 1 to 40, wherein the cancer is a metastatic cancer.

42. The method of any one of claims 1 to 41, wherein the cancer is a therapy-related cancer or a secondary malignancy.

43. The method of claim 42, wherein the cancer is therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin's lymphoma.

44. The method of any one of claims 1 to 43, wherein the chemotherapeutic drug is selected from the group consisting of auristatin E, auristatin F, monomethyl auristatin D (MMAD), monomethyl auristatin F (MMAF), monomethyl auristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroplysinin, adoxirubicin, agrochelin, ansatriene, ansamitocin P-3, aphidicolin, apoptin, L-asparaginase, azacitidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, camptothecin, chaetocin, globochaetin, chl amydocin, ember erythromycin B, cladribine, colchicine, comprestatin A1, comprestatin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curvulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, duocarmycin SA, duocarmycin TM, duocarmycin MA, duocarmycin DM, doxorubicin, Engelin A, epothilone A, epothilone B, epothilone C, etoposide, fludarabine, fumagillin, geldanamycin, tanspiramycin (17-AAG), glucocorticoidin A, gramicidin A, oxadiene, 9-hydroxyellipticine, hydroxyurea, sphingomyelin, sphingomyelin, idarubicin, ilimacquinone, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisol A, maytansinol, maytansine (DM1), maytansine DM3, ravtansine (DM4), maytansine AP-3, mechercharmycin A, mensacarcin, methotrexate, 6-mercaptopurine, microcolin B, microcystin LR, mitoxantrone, muscotoxin A, myostrobin, mytoxin B, nelarabine, nemorubicin, nocuolinA, cholinesterase, oligomycin A, oligomycin B, paclitaxel, larotaxel, milataxel, ortasel, tesetaxel, phalloidin, phalloidin, phytosphingosine, picrotoxin A, bilonisone, podophyllotoxin, polyketidemycin, prednisone, phlorotoxin B, pseurotin A, puwainaphycin F, pyrrolobenzodiazepine Quinaldotide, rachelmin, rabekamycin, Ro 5-3335, safranin B, sandrocin, sanguinarine, saporin, sinefungin, tatoprolol, trostatin, 6-thioguanine, thiocolchicine, monacolin, tripolin A, triptolide, tobramycin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine.

45. The method of claim 44, wherein the chemotherapeutic drug is saporin, daunorubicin, venetoclax, or azacitidine.

46. ​​The method of any one of claims 1 to 45, wherein the chemotherapeutic drug is one to which the cancer is resistant.

47. The method of any one of claims 1 to 46, wherein the administering is performed systemically or locally.

48. The method of claim 47, wherein the administering is performed orally or by injection.

49. The method of claim 48, wherein the injection is intravenous, subcutaneous, intraperitoneal, or intratumoral.

50. The method of any one of claims 1 to 49, wherein the administering is performed from once a day to once every six months.

51. The method of any one of claims 1 to 50, wherein the subject is a mammal.

52. The method of claim 51, wherein the subject is a human.

53. The method of any one of claims 1 to 52, wherein the administration results in increased binding between the antibody and NPM1-expressing cancer cells in the subject compared to administration of the antibody alone.

54. The method of any one of claims 1 to 53, wherein the administration results in reduced growth of NPM1-expressing cancer cells in the subject compared to administration of the antibody alone.

55. The method of any one of claims 1 to 54, wherein the administration results in increased cell death of NPM1-expressing cancer cells in the subject compared to administration of the antibody alone.

56. A composition for treating a cancer expressing nucleolar phosphoprotein 1 (NPM1), the treatment comprising administering the composition to a subject in need thereof, wherein the composition comprises an antibody or antibody conjugate that binds to NPM1 and a chemotherapeutic drug.

57. A composition for treating a cancer expressing nucleolar phosphoprotein 1 (NPM1), the treatment comprising administering the composition to a subject in need thereof, wherein the composition comprises an antibody or antibody conjugate that binds to NPM1, and wherein the subject is receiving or has received treatment with a chemotherapeutic drug.

58. A composition for treating a cancer expressing nucleolar phosphoprotein 1 (NPM1), the treatment comprising administering the composition to a subject in need thereof, wherein the composition comprises a chemotherapeutic drug, and wherein the subject is receiving or has received treatment with an antibody or antibody conjugate that binds to NPM1.

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