Combination treatment with anti-nucleophosmin 1 antibody and antibody conjugate

Abstract

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

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to Greek Application No. 20220100349, entitled "ANTI-NUCLEOPHOSMIN 1 ANTIBODY AND ANTIBODY CONJUGATE COMBINATION THERAPIES," filed April 27, 2022, the contents of which are incorporated by reference in their entirety herein.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (C123370245WO00-SEQ-VLJ.xml; size: 47,000 bytes; creation date: April 21, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] 2. Background of the Invention Nucleophosmin 1 (NPM1) is a protein that is typically located 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 that lead to the translation of a mutant NPM1, called NPM1c, that is mislocalized to the cell surface in greater amounts than wild-type NPM1. NPM1c mutations are present in a significant subpopulation of patients with AML. Summary of the Invention

[0004] Summary of the Invention The present disclosure is based on the identification of antibodies that specifically bind to nucleophosmin 1 (NPM1). These antibodies are capable of targeting wild-type (WT) and / or mutant NPM1 located on the surface of cells, including cancer cells, and are useful for binding to cells with cell surface expression of WT and / or mutant NPM1, and optionally for targeting cytotoxic payloads to said cells. The efficacy of these antibodies, or antibody-drug conjugates (ADCs) derived from these antibodies, may be enhanced by prior, co-administration, and / or subsequent administration of chemotherapeutic drugs.

[0005] Accordingly, some aspects of the disclosure relate to methods of treating an NPM1-expressing cancer, comprising administering to a subject in need thereof an effective amount of an antibody or ADC that binds to NPM1 and a chemotherapeutic drug.

[0006] In some aspects, the disclosure relates to a method of treating an NPM1-expressing cancer, the method comprising administering to a subject in need thereof an effective amount of an antibody or ADC that binds to NPM1, wherein the subject is undergoing or has undergone treatment with a chemotherapeutic drug.

[0007] In some aspects, the disclosure relates to a method of treating an NPM1-expressing cancer, 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.

[0008] In some embodiments, the antibody binds to WT NPM1. In some embodiments, the antibody 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), and 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 set forth in SEQ ID NO:28 and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:29.

[0009] In some embodiments, the antibody binds to mutant NPM1. In some embodiments, the antibody 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), and 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 set forth in SEQ ID NO: 30, and / or in the light chain the variable region comprises the amino acid sequence set forth in SEQ ID NO: 31.

[0010] In some embodiments, the antibody is a full-length antibody or an antigen-binding fragment thereof. In some embodiments, the antibody is a full-length antibody selected from Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin D (IgD), Immunoglobulin E (IgE), and Immunoglobulin M (IgM). In some embodiments, the antibody is an IgG.

[0011] In some embodiments, the antibody is an antigen-binding fragment selected from a Fab fragment, a F(ab')2 fragment, an Ig monomer, an Fd fragment, a scFv, a scAb, a dAb, an Fv, an affibody, a diabody, a single domain heavy chain antibody, and a single domain light chain antibody.

[0012] In some embodiments, the antibody is a human or humanized antibody. 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 set forth in SEQ ID NO:32 or SEQ ID NO:46.

[0013] 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 set forth in SEQ ID NO:33, SEQ ID NO:34, or SEQ ID NO:47.

[0014] 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.

[0015] In some embodiments, the antibody binds preferentially to wild-type NPM1. In some embodiments, the antibody binds preferentially to mutant NPM1. In some embodiments, the antibody binds to both wild-type and mutant NPM1. In some embodiments, the antibody is conjugated to an agent.

[0016] In some embodiments, the agent is a drug. In some embodiments, the drug is auristatin E, auristatin F, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroprisinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacytidine, bafilomycin A1, bafilomycin A2, bafilomycin B3, bafilomycin C4, bafilomycin D5, bafilomycin E6, bafilomycin F7, bafilomycin E8, bafilomycin F9, bafilomycin E10, bafilomycin F11, bafilomycin F12, bafilomycin F13, bafilomycin F14, bafilomycin F15, bafilomycin F16, bafilomycin F17, bafilomycin F18, bafilomycin F19, bafilomycin F20, bafilomycin F21, bafilomycin F22, bafilomycin F23, bafilomycin F24, bafilomycin F25, bafilomycin F26, bafilomycin F27, bafilomycin F28, bafilomycin F30, bafilomycin F31, bafilomycin F32, bafilomycin F33, bafilomycin F34, bafilomycin F35, bafilomycin F36, bafilomycin F37, cytarabine, dactylic acid, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 20, dolastatin 21, dolastatin 22, dolastatin 23, dolastatin 24, dolastatin 25, dolastatin 26, dolastatin 27, dolastatin 28, dolastatin 29, dolastatin 30, dolastatin 31, dolastatin 32, dolastatin 33, dolastatin 34, dolastatin 35, dolastatin 36, dolastatin 37, dolastatin 38, dolastatin 39, dolastatin 40, dolastatin 41, dolastatin 42, dolastatin 43, dolastatin 44, dolastatin 45, dolastatin 46, dolastatin 47, dolastatin 48, dolastatin 49, dolastatin 50, dolastatin 51, dolastatin 52, dolastatin 53, dolastatin 54, dolastatin 55, dolastatin 56, dolastatin 57, dolastatin 58, dolastatin 59, dolastatin 60, dolastatin 61, dolastatin 62, dolastatin 63, dolastatin 64, dolastatin 65, dolastatin 66, dolastatin 67, dolastatin 68, dolastatin 69, dolastatin 70, dolastatin 71, dolastatin 72, dolastatin 80, dolastatin 90, dolastatin 10, dolastatin 10, dolastatin 10, dolastatin 10 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), Glucopyericidin A, Gramicidin A, Helboxidien, 9-Hydroxyellipticine, Hydroxyurea, Hygrolysine, Hypothemycin, Idarubicin, Irimaquinone, Isatropolone A, Isofistularin-3, Ixabepilone, J W55, lactacystin, luisol A, maytansinol, mertansine (DM1), maytansine DM3, ravtansine (DM4), maytansinoid AP-3, mekelcarmycin A, menthacarcin, methotrexate, 6-mercaptopurine, microcorrin B, microcystin LR, mitoxantrone, muscotoxin A, myoseverin, myotoxin B, nelarabine, nemorubicin, noqualin A, okilactomycin, oligomycin A, oligomycin B, paclitaxel, larotaxel, mirataxel, ortataxel,The drug is selected from the group consisting of tesetaxel, phallacidin, phalloidin, phytosphingosine, piericidin A, pironetin, podophyllotoxin, polyketomycin, prednisone, pseudolaric acid B, xulotin A, pwainaphycin F, pyrrolobenzodiazepine, quinaldopeptin, rachelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinarine, saporin, sinefungin, taltobulin, telomestatin, 6-thioguanine, thiocolchicine, triptoxin, triporin 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.

[0017] In some embodiments, the antibody and the drug are conjugated via a linker. 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 the group consisting of N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), sulfo-SPDB, valine-citrulline (Val-cit), acetylbutyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal.

[0018] In some embodiments, the linker is a non-cleavable linker. In some embodiments, the non-cleavable linker is selected from the group consisting of N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), MC-VC-PAB.

[0019] In some embodiments, the antibody to drug ratio is between 1: 1 and 1: 10. In some embodiments, the antibody to drug ratio is 1:4. In some embodiments, the agent is a radioisotope. In some embodiments, the radioisotope is selected from the group consisting of iodine-131, rhenium-188, yttrium-90, bismuth-213, and actinium-225.

[0020] In some embodiments, NPM1 expressing cancer is cancer that NPM1 is expressed on the surface of cancer cells.In some embodiments, NPM1 expressing cancer is cancer that NPM1 is expressed on the surface of cancer cells as a result of administering chemotherapeutic drugs or treating with chemotherapeutic drugs.In some embodiments, NPM1 expressing cancer is cancer that wild type NPM1 and / or mutant NPM1 is expressed on the surface of cancer cells.

[0021] In some embodiments, the cancer is a solid or liquid cancer selected from the group consisting of blood 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 or neck cancer, or skin cancer. In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is a therapy-related cancer or a secondary malignancy. In some embodiments, the cancer is a therapy-related AML (t-AML) or a secondary malignancy of non-Hodgkin's lymphoma.

[0022] In some embodiments, the chemotherapeutic drug is auristatin E, auristatin F, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroprisinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacytidine, bafilomycin A1, bafilomycin A2, bafilomycin B3, bafilomycin C4, bafilomycin D5, bafilomycin E6, bafilomycin F7, bafilomycin E8, bafilomycin F9, bafilomycin F10, bafilomycin F11, bafilomycin F12, bafilomycin F13, bafilomycin F14, bafilomycin F15, bafilomycin F16, bafilomycin F17, bafilomycin F18, bafilomycin F19, bafilomycin F20, bafilomycin F21, bafilomycin F22, bafilomycin F23, bafilomycin F24, bafilomycin F25, bafilomycin F26, bafilomycin F27, bafilomycin F28, bafilomycin F29, bafilomycin F30, bafilomycin F31, bafilomycin F32, bafilomycin F33, bafilomycin F34, bafilomycin F35, bafilomycin F36, bafilom cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dolastatin 15, dexamethasone, dolastatin 16, dolastatin 17, dolastatin 19, dolastatin 20, dolastatin 21, dolastatin 22, dolastatin 23, dolastatin 24, dolastatin 25, dolastatin 26, dolastatin 27, dolastatin 28, dolastatin 29, dolastatin 30, dolastatin 31, dolastatin 32, dolastatin 33, dolastatin 34, dolastatin 35, dolastatin 36, dolastatin 37, dolastatin 38, dolastatin 39, dolastatin 40, dolastatin 41, dolastatin 42, dolastatin 43, dolastatin 44, dolastatin 45, dolastatin 46, dolastatin 47, dolastatin 48, dolastatin 49, dolastatin 50, dolastatin 51, dolastatin 52, dolastatin 53, dolastatin 54, dolastatin 55, dolastatin 55, dolastatin 56, dolastatin 57, dolastatin 58, dolastatin 59, dolastatin 60, dolastatin 61, dolastatin 62, dolastatin 63, dolastatin 64, dolastatin 65, dolastatin 66, dolastatin 67, dolastatin 68, dolastatin 69, dolastatin 70, dolastatin 71, dolastatin 72, Duocarmycin SA, Duocarmycin TM, Duocarmycin MA, Duocarmycin DM, Doxorubicin, Englerin A, Epothilone A, Epothilone B, Epothilone C, Etoposide, Fludarabine, Fumagillin, Geldanamycin, Tanespimycin (17-AAG), Glucopyericidin A, Gramicidin A, Helboxidien, 9-Hydroxyellipticine, Hydroxyurea, Hygrolysine, Hypothemycin, Idarubicin, Irimaquinone, Isatropolone A, Isofistularin-3, Ixabepilone, JW55, Lactasis Chin, Luisol A, Maytansinol, Mertansine (DM1), Maytansine DM3, Rabtansine (DM4), Maytansinoid AP-3, Mekelcarmycin A, Menthacarcin, Methotrexate, 6-Mercaptopurine, Microcorrin B, Microcystin LR, Mitoxantrone, Muscotoxin A, Myoseverin, Mytoxin B, Nelarabine, Nemorubicin, Noqualin A, Okilactomycin, Oligomycin A, Oligomycin B, Paclitaxel, Larotaxel, Mirataxel, Ortataxel, Tesetaxel, Phalacidine,The chemotherapeutic agent is selected from the group consisting of phalloidin, phytosphingosine, piericidin A, pironetin, podophyllotoxin, polyketomycin, prednisone, pseudodoralic acid B, xulotin A, pwainaphycin F, pyrrolobenzodiazepine, quinaldopeptin, rachelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinarine, saporin, sinefungin, taltobulin, telomestatin, 6-thioguanine, thiocolchicine, triptoxin, triporin A, triptolide, tubastatin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine. In some embodiments, the chemotherapeutic agent is saporin, daunorubicin, venetoclax, or azacitidine.

[0023] In some embodiments, the chemotherapeutic drug is one to which the cancer is resistant. In some embodiments, administration occurs systemically or locally. In some embodiments, administration occurs via injection. In some embodiments, the injection is intravenous, subcutaneous, intraperitoneal, or intratumoral. In some embodiments, administration occurs orally.

[0024] In some embodiments, administration occurs more than once, hi some embodiments, administration occurs between once per day and once per six months. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0025] In some embodiments, administration results in increased binding between the antibody and NPM1-expressing cancer cells of the subject, as compared to administration of the antibody alone. In some embodiments, administration results in reduced growth of NPM1-expressing cancer cells in the subject when compared to administration of the antibody alone. In some embodiments, administration results in increased cell death of NPM1-expressing cancer cells in the subject when compared to administration of the antibody alone.

[0026] Another aspect of the present disclosure relates to a method for treating a disease associated with cells expressing cell surface nucleophosmin 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.

[0027] Another aspect of the present disclosure relates to a composition for use in treating an NPM1-expressing cancer, the treatment comprising administering to a subject in need thereof a composition, wherein the composition comprises an antibody or antibody conjugate that binds to NPM1 and a chemotherapeutic drug.

[0028] Another aspect of the present disclosure relates to a composition for use in treating an NPM1-expressing cancer, the treatment comprising administering a composition to a subject in need thereof, wherein the composition comprises an antibody or antibody conjugate that binds to NPM1, and the subject is undergoing or has undergone treatment with a chemotherapeutic drug.

[0029] Another aspect of the present disclosure relates to a composition for use in treating an NPM1-expressing cancer, the treatment comprising administering the composition to a subject in need thereof, where 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]

[0030] BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component is labeled in every drawing. In the drawing:

[0031] [Figure 1-1]Figures 1A-1E show that NPM1 is localized on the surface of cancerous cell types. Figure 1A shows immunoblotting of WT and mutant NPM1 in cytosolic and membrane fractions collected from various human leukemia cell lines. Figure 1B shows FACS analysis of NPM1 surface expression in human leukemia cell lines. [Figure 1-2] Figure 1B shows FACS analysis of NPM1 surface expression in human leukemia cell lines, and Figures 1C and 1D show relatively low levels of NPM1 expressed on the surface of mouse bone marrow (BM) or peripheral blood (PB) cells. [Figure 1-3] 1C and 1D show relatively low levels of NPM1 expressed on the surface of mouse bone marrow (BM) or peripheral blood (PB) cells. [Figure 1-4] Figure 1E shows FACS analysis indicating that NPM1 is not robustly expressed on healthy human BM cells. FACS analysis was performed with the isolated anti-NPM1 antibodies shown in Figures 2A and 2B, as well as a commercial anti-NPM1 antibody (Santa Cruz Biotechnology anti-WT NPM1 # sc-32256).

[0032] [Diagram 2] Figures 2A and 2B show the preparation of anti-NPM1 antibodies. Figure 2A shows the isolation of an antibody that binds to WT NPM1. Figure 2B shows the isolation of an antibody that binds to mutant NPM1.

[0033] [Figure 3-1] Figures 3A and 3B show that anti-NPM1-ADCs are toxic to human cancer cells. Figure 3A shows the cytotoxicity of ADCs containing antibodies specific for WT NPM1 against OCI-AML3 cells. Streptavidin-saporin was conjugated to either biotinylated anti-WT NPM1 IgG or anti-mouse IgG, and the complexes were then added to OCI-AML3 cells and incubated for 24 hours. After incubation, cells were washed and cell killing was analyzed by FACS. [Figure 3-2]FIG. 3B shows the same as FIG. 3A at 48 hours of incubation.

[0034] [Figure 4-1] Figures 4A and 4B show that NPM1 is highly conserved among mammalian species. Figure 4A shows a pairwise alignment illustrating the conservation between human and mouse NPM1 amino acid sequences (SEQ ID NOs: 39-40). [Figure 4-2] FIG. 4B shows a pairwise alignment illustrating the conservation between human and rhesus monkey NPM1 amino acid sequences (SEQ ID NOs: 39 and 41).

[0035] [Figure 5-1] Figures 5A-5C show that NPM1 is present on the surface of human AML cell lines and primary mouse AML cells. Figure 5A shows binding of anti-NPM1 antibody (Merck / Sigma anti-B23 # B556) to NPM1 localized on the surface of human OCI-AML3 and MOLM13 cells. Binding to NPM1 was assayed in both live and fixed cells. OCI-AML3 cells express mutant NPM1, while MOLM13 cells express relatively lower amounts of WT NPM1. [Figure 5-2] Figures 5B and 5C show binding of anti-NPM1 antibody (Merck / Sigma anti-B23 # B0556) to NPM1 localized on the surface of fixed primary mouse MLL-AF4 / FLT3ltd / +, MLL-AF9 / FLT3ltd / +, MLL-ENL / FLT3ltd / +, and Npm1c / FLT3ltd / + AML cells. [Figure 5-3] Figures 5B and 5C show binding of anti-NPM1 antibody (Merck / Sigma anti-B23 # B0556) to NPM1 localized on the surface of fixed primary mouse MLL-AF4 / FLT3ltd / +, MLL-AF9 / FLT3ltd / +, MLL-ENL / FLT3ltd / +, and Npm1c / FLT3ltd / + AML cells.

[0036] [Figure 6-1] Figures 6A-6F show that NPM1c (mutant NPM1) is present on the surface of human myeloid leukemia cell lines. Figure 6A shows that anti-TY1 tag antibody (Diagenode TY1 # C15200054) binds to the surface of human K562 myeloid leukemia cells expressing TY1-tagged WT NPM1 or TY1-tagged NPM1c. [Figure 6-2] FIG. 6B shows the data from FIG. 6A in separate panels with quantification of the percentage of cells positively bound by TY1 and anti-NPM1 antibodies (Merck / Sigma anti-B23 # B556). [Figure 6-3] Figure 6C shows binding of anti-NPM1 antibodies (Diagenode TY1 # C15200054; Merck / Sigma anti-B23 # B556) to intracellular and cell surface WT and mutant NPM1 in cells expressing empty TY1 lentiviral vector, TY1-tagged NPM1-wildtype lentiviral vector, or TY1-tagged NPM1c lentiviral vector. [Figure 6-4] Figure 6C shows binding of anti-NPM1 antibodies (Diagenode TY1 # C15200054; Merck / Sigma anti-B23 # B556) to intracellular and cell surface WT and mutant NPM1 in cells expressing empty TY1 lentiviral vector, TY1-tagged NPM1-wildtype lentiviral vector, or TY1-tagged NPM1c lentiviral vector. [Figure 6-5] Figure 6D shows binding between anti-NPM1 antibodies (Diagenode TY1 # C15200054; Merck / Sigma anti-B23 # B556) and WT and mutant NPM1 in cells expressing empty TY1 lentiviral vector, TY1-tagged NPM1-wildtype lentiviral vector, or TY1-tagged NPM1c lentiviral vector. [Figure 6-6] Figure 6E shows the binding between anti-NPM1 antibody (Diagenode TY1 # C15200054; Merck / Sigma anti-B23 # B556) and WT and mutant NPM1 on the surface of K562 or MOLM13 cells (negative control). The relevant cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1-wild type lentiviral vector, or TY1-tagged NPM1c lentiviral vector. Figure 6F shows immunofluorescence illustrating the binding between anti-NPM1 antibody (Diagenode TY1 # C15200054; Merck / Sigma anti-B23 # B556) and WT and mutant NPM1 on the surface of K562 or MOLM13 cells (negative control). The relevant cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1-wild type lentiviral vector, or TY1-tagged NPM1c lentiviral vector.

[0037] [Figure 7-1] Figures 7A and 7B show that anti-NPM1 antibodies bind to NPM1 on the surface of primary mouse AML cells. Figure 7A shows the binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of primary mouse MLL-rearranged (MLL-r) AML cells. [Figure 7-2] FIG. 7B shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of primary murine NPM1c AML cells.

[0038] [Figure 8-1]Figures 8A-8D show that anti-NPM1 antibodies bind to NPM1 on the surface of human AML patient-derived xenografts (PDX) implanted in a mouse model. Figure 8A shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-1 cells (MLL-r). In contrast to PDX-1 cells, host (mouse) bone marrow cells (bottom panel) exert relatively low binding. [Figure 8-2] Figure 8A shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-1 cells (MLL-r). In contrast to PDX-1 cells, host (mouse) bone marrow cells (bottom panel) exert relatively low binding. [Figure 8-3] FIG. 8B shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-2 (NPM1c) cells. [Figure 8-4] FIG. 8C shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-3 (MLL-r, BCOR) cells. [Figure 8-5] FIG. 8D shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-4 (DNMT3A, N / KRAS) cells.

[0039] [Figure 9-1] Figures 9A-9D show synergy between chemotherapeutics for the treatment of AML and antibodies targeting cell surface NPM1. Figure 9A shows increased binding between an anti-NPM1 antibody (Merck / Sigma anti-B23 # B0556) and NPM1 on the surface of the venetoclax-resistant AML cell line OCI-AML3 after treatment with 10 nM daunorubicin or 40 nM venetoclax, particularly after 8 days of treatment. [Figure 9-2] FIG. 9B shows individual repeats of the FACS analysis depicted in FIG. 9A after 8 days of treatment with 10 nM daunorubicin or 40 nM venetoclax. [Figure 9-3] Figure 9C shows increased binding between isolated anti-NPM1 antibodies (Figures 2A and 2B) and NPM1 on the surface of OCI-AML3 following treatment with 10 nM daunorubicin or 40 nM venetoclax, particularly after 8 days of treatment. [Figure 9-4] Figure 9D shows increased binding between anti-NPM1 antibody (Merck / Sigma anti-B23 # B0556) or isolated anti-NPM1 antibody (Figures 2A and 2B) and NPM1 on the surface of OCI-AML3 after treatment with 40 nM 5-azacytidine (5-Aza).

[0040] [Figure 10] Figures 10A-10B show intracellular and cell surface staining of isotype and NPM1 from the cell line OCI-AML3 (Figure 10A), as well as denaturing protein gels and western blotting (WB) of biochemical fractions (cytosolic and membrane fractions) from the four cell lines (Figure 10B).

[0041] [Figure 11-1] 11A-11B show results from live cell staining of a human suspension cell line (FIG. 11A) and a primary mouse cell line (FIG. 11B) cultured with anti-NPM1 antibody (AF647 signal). [Figure 11-2] 11A-11B show results from live cell staining of a human suspension cell line (FIG. 11A) and a primary mouse cell line (FIG. 11B) cultured with anti-NPM1 antibody (AF647 signal). [Figure 11-3] 11A-11B show results from live cell staining of a human suspension cell line (FIG. 11A) and a primary mouse cell line (FIG. 11B) cultured with anti-NPM1 antibody (AF647 signal).

[0042] [Figure 12]Figures 12A-12B show Western blots of cells after cell surface biotinylation with a cell-impermeant biotinylation reagent. The Western blots detected NPM1 in the membrane fraction of these cells, and NPM1 IPs were able to enrich NPM1 more robustly from membrane lysates (Figure 12A). Examination of the biotin signal from these fractions (cell surface exposed proteins) demonstrated isolation of a single band in the NPM1 IPs (Figure 12B), indicating that full-length NPM1 is exposed to the surface of live cells.

[0043] [Figure 13-1] Figures 13A-13B are diffraction-limited (DL) and super-resolution (SR) reconstructions of anti-NPM1 staining the cell surface of both HL-60 and OCI-AML3 (Figure 13A) and the adherent cell line PANC1 (Figure 13B). Cell surface NPM1 appears as distinct clusters. [Figure 13-2] Figures 13A-13B are diffraction-limited (DL) and super-resolution (SR) reconstructions of anti-NPM1 staining the cell surface of both HL-60 and OCI-AML3 (Figure 13A) and the adherent cell line PANC1 (Figure 13B). Cell surface NPM1 appears as distinct clusters.

[0044] [Figure 14] FIG. 14 shows fluorescent images of live (top) or fixed and permeabilized (bottom) OCI-AML3 cells stained with either Ab2.2 or the commercial NPM1 antibody.

[0045] [Figure 15-1] 15A-15B show bone marrow samples from three healthy donors stained with Ab2.2 and sorted for various markers of the hematopoietic system. Ab2.2 partially binds to CD33+ cells, however, there is no observable binding to CD34+ (HSC) cells. [Figure 15-2]Figure 15B shows the binding of a commercial anti-NPM1 antibody as well as Ab2.2 binding to protein lysate samples by Western blotting. The banding patterns are identical.

[0046] [Figure 16] FIG. 16 is a table listing 12 AML patient samples.

[0047] [Figure 17-1] 17A-17B show flow cytometry analysis of 12 AML cases. [Figure 17-2] 17A-17B show flow cytometry analysis of 12 AML cases. [Figure 17-3] 17A-17B show flow cytometry analysis of 12 AML cases. [Figure 17-4] 17A-17B show flow cytometry analysis of 12 AML cases. [Figure 17-5] 17A-17B show flow cytometry analysis of 12 AML cases. [Figure 17-6] 17A-17B show flow cytometry analysis of 12 AML cases.

[0048] [Figure 18] FIG. 18 is a table listing samples collected from 15 AML patients.

[0049] [Figure 19-1] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-2] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-3] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-4] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-5]19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-6] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-7] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-8] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-9] 19A-19E show flow cytometry analysis of 15 AML patients. [Figure 19-10] 19A-19E show flow cytometry analysis of 15 AML patients.

[0050] [Figure 20-1] Figures 20A-20B show how the toxicity of Ab2.2 was assessed in vivo. Figure 20A is a schematic of the experimental protocol. Figure 20B shows weight results and results from blood sample analysis of WBC, PLT, and HGB. [Figure 20-2] FIG. 20B shows the weight results and the results from the blood sample analysis of WBC, PLT, and HGB. [Figure 20-3] FIG. 20B shows the weight results and the results from the blood sample analysis of WBC, PLT, and HGB.

[0051] [Figure 21-1] Figures 21A-21G show how the efficacy of Ab2.2 treatment was assessed in vivo. Figure 21A is a schematic of the experimental design. Figure 21B shows antibody binding to this AML model. [Figure 21-2] Figure 21C shows the WBC counts, and Figure 21D shows the results from qPCR analysis of AML alleles. [Figure 21-3] Figure 21E shows the spleens and spleen weights of mice treated with IgG and Ab2.2. Figure 21F shows the results of HGB and HCT analysis of blood samples. [Figure 21-4] FIG. 21G shows the results from the survival assay.

[0052] [Figure 22-1] Figures 22A-22C show how mice were subjected to sublethal irradiation and transplantation of primary murine AML cells to assess efficacy (efficacy model 2). Figure 22A is a schematic of the experimental design. [Figure 22-2] Figure 22B shows antibody binding, and Figure 22C shows the results from a viability assay.

[0053] [Figure 23-1] Figures 23A-23I show that treatment with Ab2.2 reduces tumor burden in a transplant model. Figure 23A shows a schematic of the experimental design. Figure 23B shows the spleen, lung, and liver weights of mice treated with IgG or Ab2.2. [Figure 23-2] FIG. 23C shows the results from the survival assay. [Figure 23-3] Figures 23D-23E show the results of flow cytometry, and Figure 23F shows the WBC count and PLT levels from blood samples. [Figure 23-4] Figures 23D-23E show the results of flow cytometry, and Figure 23F shows the WBC count and PLT levels from blood samples. [Figure 23-5] Figure 23G shows WBC over time. Figure 23H shows images of spleens and a graph of spleen weight in mice treated with IgG or Ab2.2. [Figure 23-6] FIG. 23I shows AML% in BM and PB.

[0054] [Figure 24] Figures 24A-24B show the immune dependence of the tumor killing activity of Ab2.2 versus an intact immune system. Figure 24A shows the experimental design. Figure 24B shows the survival curves of mice treated with either Ab2.2 or IgG.

[0055] [Figure 25-1] Figures 25A-25C show the solid tumor activity of Ab2.2. Figure 25A shows the experimental design. Figure 25B shows the tumor volumes on days 10 and 13. [Figure 25-2] FIG. 25C shows tumor volume over time from days 7 to 13.

[0056] [Figure 26-1] Figures 26A-26B show that wild-type healthy mice treated with sublethally irradiation and Ab2.2 exhibited no observable effect of Ab2.2 treatment. Figure 26A shows the experimental design. [Figure 26-2] FIG. 26B shows the weight, WBC count, HGB analysis, and PLT analysis from the blood samples. [Figure 26-3] FIG. 26B shows the weight, WBC count, HGB analysis, and PLT analysis from the blood samples.

[0057] [Figure 27-1] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-2] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-3] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-4] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-5]Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-6] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-7] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-8] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-9] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 27-10] Figures 27A-27O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors.

[0058] [Figure 28] FIG. 28 shows high surface detection of Npm1c on single Dmnt3a R882H mutant cells (pre-leukemic cells).

[0059] [Figure 29] Figure 29 shows how NPM1 can serve as an ADC target in several models. To assess the in vitro activity of Ab2.2 in ADCs, OCI-AML3 cells were treated with negative control, isotype saporin conjugate, or Ab2.2-saporin conjugate.

[0060] [Figure 30-1] Figures 30A-30C show that Ab2.2 extends life span in a transplant model of a human AML cell line. Figure 30A is a schematic of the experimental design. [Figure 30-2] Figure 30B shows antibody binding using flow cytometry, and Figure 30C is a graph of the results of a viability assay.

[0061] [Figure 31-1] Figures 31A-31C show that Ab2.2 extends life span in a transplant model of human AML-PDX cell line. Figure 31A is a schematic of the experimental design. Figure 31B shows antibody binding using flow cytometry. [Figure 31-2] FIG. 31C is a graph of the results of the viability assay.

[0062] [Diagram 32] FIG. 32 shows how OCI-AML3 cells were exposed to either control or low-dose chemotherapy treatment.

[0063] [Diagram 33] Figure 33 shows an in vitro model of non-transformed cell types (HPC7, HOXB8) exposed to low doses of chemotherapy drugs (daunorubicin, venetoclax). Cells were analyzed over 48 and 72 hours using flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0064] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS Aspects of the present disclosure are based on the discovery that antibodies specific for nucleophosmin 1 (NPM1) (also known as nucleolar phosphoprotein B23 or numatrin) may be used to target agents (e.g., chemotherapy drugs, radioactive isotopes) to certain cell types (e.g., cancer cells) in a subject. Without wishing to be bound by theory, NPM1 is a multifunctional protein that typically binds to single-stranded and double-stranded nucleic acids and is present in the nucleus and cytosol of cells. However, when certain cell types are exposed to cellular stress, such as 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, it is particularly common in leukemias (e.g., acute myeloid leukemia (AML)) that result from mutations in the gene encoding NPM1. As a result of this mutation, leukemia (e.g., AML) cells in many patients begin to express a mutant variant of NPM1, called NPM1c, which is mislocalized from the nucleus to the cytoplasm, where it is trafficked to the cell membrane and exposed to the extracellular environment. NPM1c is also known to occur only in cancer cells, where it drives malignant transformation in AML. In principle, antibodies or other agents specific for wild-type NPM1 or mutant NPM1 (NPM1c) can be conjugated to a cytotoxic payload and used to target cancer cells. Alternatively, an NPM1-specific antibody may itself be sufficient to stimulate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) upon binding to cancer cells, even without conjugation to a cytotoxic payload. However, while antibodies specific for NPM1 are commercially available, there is a lack of antibodies that can effectively bind to wild-type 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 monoclonal antibodies available that bind to NPM1c.Thus, new antibodies have been developed that can effectively and specifically bind to wild-type and mutant NPM1 on the surface of cells, including cancer cells. These antibodies and molecular conjugates thereof (e.g., antibody-drug conjugates) may be useful for detecting and treating cancers that express NPM1 on their cell surface, and may be used to treat subjects with cancers for which there are no known effective treatments.

[0065] Antibody that binds to NPM1 The 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 compared to mutant NPM1, or may have a higher affinity for mutant NPM1 compared to WT NPM1.

[0066] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target, such as, but not limited to, a protein or peptide, referred to as an "antigen", through at least one recognition site on the antigen. As used herein, the term "antibody" encompasses both full-length immunoglobulin molecules (immunoglobulins having two heavy chains and two light chains, e.g., monoclonal or polyclonal full-length antibodies) and antigen-binding fragments thereof, such as, but not limited to, chimeric antibodies, diabodies, linear antibodies, nanobodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), or other antigen-binding fragments that retain specific binding to the antigen. "Immunoglobulins (Ig)" are large, Y-shaped proteins produced primarily by plasma cells, which are used by the immune system to neutralize foreign substances (e.g., pathogens such as bacteria and viruses). "Antibody fragments" encompass any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. In some embodiments, an "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (herein referred to as V H The heavy chain constant region is made up of three domains, C H 1. C H 2, and C H Each light chain is composed of a light chain variable region (V L The light chain constant region consists of one domain, C L It consists of: V H Area and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). H and V Lis made of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In some embodiments, the antibody is an immunoglobulin (Ig) monomer. The antibody may be a polyclonal or monoclonal antibody.

[0067] In some embodiments, antibodies are heterotetrameric glycoproteins made of two identical L chains and two H chains (IgM antibodies consist of five basic heterotetrameric units together with an additional polypeptide called the J chain, thus containing 10 antigen-binding sites, while secreted IgA antibodies can polymerize to form multivalent assemblies containing two to five basic four-chain units together with the J chain). In the case of IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain contains a variable domain (V H ), followed by three constant domains for each of the α and γ chains (C H ), four C for μ and ε isotypes H Each L chain has a variable domain (V L ), followed by a constant domain (C L ) V L V H and C L is aligned with the first constant domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light- and heavy-chain variable domains. H and V LThe pairing of the two together forms a single antigen-binding site. For non-limiting examples of the structures and properties of various classes of antibodies, see, for example, 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 (incorporated herein by reference). In some embodiments, the antibody is an IgG.

[0068] 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 domain. H Depending on the amino acid sequence of the immunoglobulins, they can be assigned to various 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. The γ and α classes are further divided into C H They are divided into subclasses based on relatively minor differences in sequence and function; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0069] The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid length of the variable domain. Instead, the V region consists of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions", each of which is 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs that mostly adopt a β-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and contribute to the formation of the antigen-binding site of antibodies with the hypervariable regions from the other chain (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0070] In some embodiments, the antibody is a monoclonal antibody. A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for the possible presence of small amounts of naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described, e.g., in 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.

[0071] The monoclonal antibodies described herein encompass "chimeric" antibodies, as well as fragments of such antibodies, in which some heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)), so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape, etc.) and human constant region sequences.

[0072] 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 may be isolated or purified from mammalian blood, secretions, or other fluids, or from eggs. Polyclonal antibodies may also be recombinant. Recombinant polyclonal antibodies are polyclonal antibodies produced by the use of recombinant technology. Recombinantly produced polyclonal antibodies often contain a high concentration of different antibody molecules, all or a majority of which (e.g., more than 80%, more than 85%, more than 90%, more than 95%, more than 99%, or more) exert the desired binding activity against an antigen made of more than one epitope.

[0073] In some embodiments, antibodies are "humanized" for use in humans (e.g., as therapeutics). "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate animal, having the desired antibody specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will also optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. For further 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).

[0074] 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 "preferentially binds" refers to binding that occurs more frequently, at a higher rate, for a longer duration, and / or with greater affinity to a specific antigen than other antigens. The terms "preferentially binds" and "specifically binds" may be used interchangeably. The terms "preferentially binds" and "specifically binds" do not necessarily indicate exclusive binding, i.e., an antibody that preferentially binds to an antigen may or may not bind to one or more additional antigens. In some embodiments, the antibodies described herein may or may not bind to other forms (variants) of a particular antigen. In some embodiments, an antibody that binds to WT NPM1 may or may not bind to a variant of NPM1 that includes one or more amino acid insertions, deletions, or substitutions (i.e., mutant NPM1), and a variant of NPM1 that includes one or more amino acid insertions, deletions, or substitutions (i.e., mutant NPM1) may or may not bind to WT NPM1.

[0075] As used herein, "wild-type NPM1" or "WT NPM1" refers to an NPM1 protein that comprises an amino acid sequence identical to that of the commonly accepted reference sequence for intact, fully functional NPM1. For example, WT NPM1 may be an NPM1 isoform (e.g., NCBI reference sequence: NP_001341935.1) expressed from a gene encoding WT NPM1 (NCBI reference sequence: NG_016018.1). As used herein, "mutant NPM1" refers to an NPM1 protein that comprises an amino acid sequence that comprises one or more amino acid insertions, deletions, or substitutions compared to the WT NPM1 sequence. For example, mutant NPM1 may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid insertions, deletions, or substitutions compared to the WT NPM1 sequence. Mutant NPM1 may have reduced activity and / or altered subcellular localization compared to WT NPM1. The WT NPM1 or mutant NPM1 described herein may be a mammalian WT NPM1 or a mammalian mutant NPM1. In some embodiments, the WT NPM1 or mutant NPM1 described herein is a human WT NPM1 or a human mutant NPM1.

[0076] In some embodiments, the antibody that binds to NPM1 is a full-length antibody. The full-length antibody described herein may belong to any antibody class based on the amino acid sequence of its heavy chain constant region. For example, the full-length antibody that binds to NPM1 may be Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin D (IgD), Immunoglobulin E (IgE), and Immunoglobulin M (IgM), or a subclass thereof (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the antibody described herein is an antigen-binding fragment. The antigen-binding fragment may be in the format of Fab fragment, F(ab')2 fragment, Ig monomer, Fd fragment, scFv, scAb, dAb, Fv, affibody, diabody, single domain heavy chain antibody, and single domain light chain antibody. The antibody described herein may belong to mouse, rat, human, or any other origin. In some embodiments, the antibody described herein is a human antibody or a humanized antibody.

[0077] In some embodiments, an 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 complementarity determining region (CDR) sequences that determine substrate specificity. The CDR sequences may be determined using any numbering scheme commonly known in the art (e.g., Kabat, Chothia, Contact, IGMT).

[0078] 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 NIFVH (SEQ ID NO: 1), an HC CDR2 comprising the amino acid sequence KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and an HC CDR3 comprising the amino acid sequence DSSGYDAVDY (SEQ ID NO: 3). In some embodiments, an antibody that binds to NPM1 comprises a light chain comprising a light chain CDR1 comprising the amino acid sequence RASESVYTYLA (SEQ ID NO: 9), an LC CDR2 comprising the amino acid sequence NAKTLTE (SEQ ID NO: 10), and an LC CDR3 comprising the amino acid sequence QHHYGTPYT (SEQ ID NO: 11).

[0079] 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), an HC CDR2 comprising the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 16), and an 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), an LC CDR2 comprising the amino acid sequence GKNNRPS (SEQ ID NO: 23), and an LC CDR3 comprising the amino acid sequence NSSPRLKHRVV (SEQ ID NO: 24).

[0080] Table 1 provides the amino acid sequences of the heavy and light chain CDRs for exemplary antibodies ("Ab1" and "Ab2") specific for NPM1 (WT and / or mutant NPM1). Antibodies having the same heavy or light chain CDR sequences as provided in Table 1, as determined using any numbering scheme commonly known in the art (e.g., Kabat, Chothia, Contact, IGMT), are within the scope of this disclosure.

[0081] Table 1: CDR sequences of anti-NPM1 antibodies [Table 1]

[0082] In some embodiments, an antibody comprising one or more heavy and / or light chain CDR sequences designated in Table 1 as "Ab1" binds (e.g., preferentially binds) to WT NPM1. In some embodiments, an antibody comprising one or more heavy and / or light chain CDR sequences designated in Table 1 as "Ab2" binds (e.g., preferentially binds) to mutant NPM1 (NPM1c). In some embodiments, an antibody comprising one or more heavy and / or light chain CDR sequences designated in Table 1 as "Ab2.2" binds (e.g., preferentially binds) to mutant NPM1 (NPM1c).

[0083] In some embodiments, the antibody that binds to NPM1 has a heavy chain variable region (V H ) sequence and / or the light chain variable region (V L In some embodiments, the antibody that binds to NPM1 further comprises a heavy chain constant region (C H ) sequence and / or the light chain variable region (C L ) sequences. In some embodiments, the antibody that binds to NPM1 comprises the heavy chain and / or light chain sequences provided in Table 2.

[0084] Table 2: Heavy and light chain sequences of anti-NPM1 antibodies [Table 2-1]

[0085] [Table 2-2]

[0086] In some embodiments, the antibody that binds to NPM1 has the CDRs, VDRs, VV ... H , V L , C H , C LIn some embodiments, an antibody that binds to NPM1 may contain one or more mutations (e.g., amino acid insertions, deletions, or substitutions) compared to the CDRs, V, VD, VE, VF ... H , V L , C H , C L In some embodiments, an antibody that binds to NPM1 may contain one, two, three, four, five, six, seven, eight, nine, ten, or more mutations (e.g., amino acid insertions, deletions, or substitutions) compared to the CDRs, V, VD, VE, VF, VFs ... H , V L , C H , C L , the heavy chain sequence, or the light chain sequence may be at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% identical to the antibody, heavy chain sequence, or light chain sequence.

[0087] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO:1 (according to the IMGT definition system), heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO:2 (according to the Kabat definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO:3 (according to the Kabat definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO:9 (according to the Kabat definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO:10 (according to the Kabat definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO:11 (according to the Kabat definition system).

[0088] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 15 (according to the Kabat definition system), heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 16 (according to the Kabat definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Kabat definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Kabat definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Kabat definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Kabat definition system).

[0089] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 4 (according to the IMGT definition system), heavy chain complementarity determining region 5 (CDR-H2) of SEQ ID NO: 2 (according to the Chothia definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the Chothia definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 9 (according to the Chothia definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 10 (according to the Chothia definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 11 (according to the Chothia definition system).

[0090] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 18 (according to the Chothia definition system), heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 19 (according to the Chothia definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 17 (according to the Chothia definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 22 (according to the Chothia definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 23 (according to the Chothia definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 24 (according to the Chothia definition system).

[0091] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 6 (according to the IMGT definition system), heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 7 (according to the Contact definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 8 (according to the Contact definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 12 (according to the Contact definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 13 (according to the Contact definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 14 (according to the Contact definition system).

[0092] In some embodiments, an antibody of the disclosure comprises heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 20 (according to the Contact definition system), heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 48 (according to the Contact definition system), heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 21 (according to the Contact definition system), light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 25 (according to the Contact definition system), light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 26 (according to the Contact definition system), and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 27 (according to the Contact definition system).

[0093] In some embodiments, the antibodies of the disclosure comprise a heavy chain variable region (VH) that contains 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 when compared to a VH comprising the amino acid sequence of SEQ ID NO: 28. Alternatively or additionally (e.g., in addition), the antibodies of the disclosure comprise a light chain variable region (VL) that contains 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 when compared to a VL comprising the amino acid sequence of SEQ ID NO: 29.

[0094] In some embodiments, the antibodies of the disclosure comprise a heavy chain variable region (VH) that contains 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 when compared to a VH comprising the amino acid sequence of SEQ ID NO: 30. Alternatively or in addition (e.g., in addition), the antibodies of the disclosure comprise a light chain variable region (VL) that contains 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 when compared to a VL comprising the amino acid sequence of SEQ ID NO: 31.

[0095] In some embodiments, an antibody of the disclosure comprises a VH comprising an amino acid sequence in a 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: 28. Alternatively or additionally (e.g., in addition), in some embodiments, an antibody of the disclosure comprises a VL comprising an amino acid sequence in a 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: 29.

[0096] In some embodiments, an antibody of the disclosure comprises a VH comprising an amino acid sequence in a 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., in addition), in some embodiments, an antibody of the disclosure comprises a VL comprising an amino acid sequence in a 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.

[0097] In some embodiments, an antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 28. Alternatively or additionally (for example, in addition), in some embodiments, an antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 29.

[0098] In some embodiments, an antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 30. Alternatively or additionally (for example, in addition), in some embodiments, an antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 31.

[0099] In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 35. In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 35. Alternatively or additionally (e.g., in addition), an antibody of the disclosure comprises a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 37. Alternatively or additionally (for example, in addition), the antibodies of the disclosure comprise a light chain comprising an amino acid sequence at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:37.

[0100] In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 36. In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 36. Alternatively or additionally (e.g., in addition), an antibody of the disclosure comprises a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 38. Alternatively or additionally (for example, in addition), the antibodies of the disclosure comprise a light chain comprising an amino acid sequence at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:38.

[0101] In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 44. In some embodiments, an antibody of the disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 44. Alternatively or additionally (e.g., in addition), an antibody of the disclosure comprises a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 45. Alternatively or additionally (for example, in addition), the antibodies of the disclosure include a light chain that includes an amino acid sequence at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:45.

[0102] In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 37.

[0103] In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 36. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 38.

[0104] In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, an antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 44. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45. Alternatively or in addition (for example, in addition), an antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 45.

[0105] Preparation of anti-NPM1 antibody The antibody capable of binding to NPM1 as described herein can be produced 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.

[0106] For example, hybridoma cell lines can be prepared from lymphocytes and immortalized myeloma cells expressing the antibodies described herein using techniques well 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). Typically, the generation of hybridomas involves fusing myeloma cells with lymphoid cells using a fusogen (e.g., polyethylene glycol or electric current) to produce an immortalized cell line capable of producing and secreting the desired antibody. Hybridomas may be cultured in a culture medium under conditions known in the art.

[0107] Alternatively, the nucleic acids encoding the heavy and light chains of the anti-NPM1 antibody as described herein can be cloned into one or more vectors using recombinant techniques known in the art, where each nucleotide sequence is operably linked to a suitable promoter (e.g., a constitutive or inducible promoter, as known in the art). The vector can be an expression vector from which the polypeptide sequence of the anti-NPM1 antibody can be expressed in a specific cell type. The one or more expression vectors contain one or more promoters to drive the expression of a gene operably linked (typically 3' to the promoter sequence). Each of the nucleotide sequences encoding the heavy and light chains can be operably linked to different promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to the same promoter (e.g., in the same expression vector). The choice of expression vector(s) and / or promoter(s) depends on the type of host cell for use in producing the antibody. If necessary, an internal ribosome entry site (IRES) can be inserted between the coding sequences of the heavy and light chains 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 any cell that can be cultured using techniques known in the art. The cell can be any cell type capable of expressing heterologous antibody sequences, such as 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 either 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 typically involve affinity chromatography.

[0108] Anti-NPM1 antibody conjugate The disclosure further provides molecular conjugates that include an anti-NPM1 antibody described herein (eg, an antibody that binds to WT NPM1 or a mutant NPM1) linked to an agent through a chemical linker.

[0109] In some embodiments, the conjugates described herein include an anti-NPM1 antibody linked to a drug. A conjugate including 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 having a relatively low molecular weight, whether naturally occurring or artificially created (e.g., via chemical synthesis). Typically, a small molecule is an organic compound (e.g., it contains carbon). A small molecule may contain multiple carbon-carbon bonds, stereocenters, and other functional groups (e.g., amines, hydroxyls, carbonyls, heterocyclic rings, and the like). In some embodiments, the molecular weight of the small molecule is about 1,000 g / mol or less, about 900 g / mol or less, about 800 g / mol or less, about 700 g / mol or less, about 600 g / mol or less, about 500 g / mol or less, about 400 g / mol or less, about 300 g / mol or less, about 200 g / mol or less, or about 100 g / mol or less. In some 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 about 500 g / mol or less) are also feasible.

[0110] 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. 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, aeroprisinin, aldoxorubicin, agrochelin, ansatrienin, ansamitocin P-3, aphidicolin, apoptrienin, and the like. gin, L-asparaginase, azacytidine, bafilomycin A1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, campatecin, chaetocin, chaetoglobosin, chlamydocin, cinerubin B, cladribine, colchicine, combretastatin A1, combretastatin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curbulin, 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), Glucopyricidin A, Gramicidin A, Herboxy Diene, 9-hydroxyellipticine, hydroxyurea, hygrolysine, hypothemycin, idarubicin, irimaquinone, isatropolone A, isofistularin-3, ixabepilone, JW55, lactacystin, luisole A, maytansinol, mertansine (DM1), maytansine DM3, ravtansine (DM4), maytansinoid AP-3, mekelcarmycin A, menthacarcin, methotrexate, 6-mercaptopurine, microcorrin B, microcystin LR,Mitoxantrone, Muscotoxin A, Myoseverin, Mytoxin B, Nelarabine, Nemorubicin, Noqualin A, Okilactomycin, Oligomycin A, Oligomycin B, Paclitaxel, Larotaxel, Mirataxel, Ortataxel, Tesetaxel, Phalacidin, Phalloidin, Phytosphingosine, Piericidin A, Pironetin, Podophyllotoxin, Polyketomycin, Prednisone, Pseudolaric Acid B, Pseudoleucine A, Pwainaphycin F, Pyrrolobenzodiazepines, Quinaldopeptin, Rachermicin, Rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinarine, saporin, sinefungin, taltoburin, telomestatin, 6-thioguanine, thiocolchicine, triptoxin, triporin A, triptolide, tubastatin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine. The ADCs described herein may also include another drug (e.g., a chemotherapeutic drug) known in the art to be cytotoxic or cytostatic to a particular cell type (e.g., a cancer cell).

[0111] In some embodiments, the anti-NPM1 antibodies described herein are conjugated to more than one molecule of an agent (e.g., a drug), i.e., the antibody and agent are conjugated together in a ratio of greater than 1:1. In some embodiments, the ratio between the antibody and agent is between 1:1 and 1:10. In some embodiments, the ratio between the antibody and agent is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0112] In some embodiments, the anti-NPM1 antibodies described herein are conjugated (linked) to an agent using chemical linkers known in the art. As used herein, the terms "conjugated," "linked," and "attached" mean that two molecules are joined together, preferably through a covalent bond or with sufficient affinity that the therapeutic or diagnostic benefit of the association between the two entities is realized.

[0113] In some embodiments, the linker is a cleavable linker. As used herein, a cleavable linker is capable of releasing a conjugated moiety in response to a stimulus. In some embodiments, the stimulus is a physiological stimulus. Non-limiting examples of stimuli include the presence of an enzyme, acidic conditions, basic conditions, or reducing conditions. For example, cleavable linkers include peptide linkers, β-glucuronide linkers, glutathione-sensitive linkers (or disulfide linkers), and pH-sensitive linkers. In some embodiments, the pH-sensitive linker is cleaved at a pH between 5.0 and 6.5 or between 4.5 and 5.0. In some embodiments, the pH-sensitive linker is not cleaved when the pH is between 7 and 7.5. In some embodiments, the pH-sensitive linker is not cleaved when the pH is between 7.3 and 7.5. In some embodiments, the pH-sensitive linker is not cleaved when the pH is between 7.3 and 7.5. In some embodiments, the cleavable linker is a protease-sensitive linker. Examples of cleavable linkers include N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), sulfo-SPDB, valine-citrulline dipeptide (Val-Cit), acetylbutyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal. See, for example, Donaghy, mAbs. 2016 May-Jun;8(4):659-71 (hereby incorporated by reference).

[0114] In some embodiments, the linker is non-cleavable. In some embodiments, the non-cleavable linker is a linker that is not cleaved in the systemic circulation in a subject. In some embodiments, the non-cleavable linker is a linker that is resistant to protease cleavage. Non-cleavable linkers include N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), maleimidomethylcyclohexane-1-carboxylate (MCC), and MC-VC-PAB. Any of the conjugates described herein may be synthesized using methods known in the art, see, for example, Yao et al., Int J Mol Sci. 2016 Feb 2;17(2).

[0115] In part, ADCs comprising anti-NPM1 antibodies conjugated to drugs are advantageous for therapeutic use because the drugs (e.g., chemotherapeutic drugs) are toxic and can be targeted to specific cell types that express cell surface NPM1 (e.g., NPM1-expressing cancer cells). By conjugating a drug to an anti-NPM1 antibody, the toxicity of the ADC may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the drug in its free form.

[0116] In some embodiments, the agent to which the anti-NPM1 antibody is linked is a radioisotope. In some embodiments, the radioisotope is a radioisotope (i.e., a therapeutic radioisotope) that is useful for the treatment of cancer, such as a radioisotope that is useful for the treatment of acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, or myelodysplastic syndrome (MDS). Radioisotopes that are useful for the treatment of cancer are well known in the art and include iodine-131 ( 131 I), rhenium-188( 188 Re), Yttrium-90( 90 Y), Bismuth-213( 213Bi), and Actinium-225 ( 225 Examples of radioisotope-linked antibodies and techniques for producing such antibodies are also well known in the art (see, for example, 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). Radioisotope-containing antibodies are advantageous for therapeutic use, in part because radioisotopes are toxic and can be targeted to specific cell types that express cell surface NPM1 (e.g., NPM1-expressing cancer cells). By conjugating a radioisotope to an anti-NPM1 antibody, the toxicity of the radioisotope may be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the radioisotope in its free form.

[0117] composition The present disclosure further provides compositions comprising the 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 pharma- ceutically acceptable carrier. "Pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable carrier" may be a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting the subject drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not toxic to the patient's tissues (e.g., physiologically compatible, sterile, at physiological pH, etc.). The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate its application. The components of the pharmaceutical compositions also can be commingled with the molecules of the present disclosure, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.Some examples of materials which may serve as pharma- ceutically 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 wax; (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 propylene glycol; Examples of suitable glycerols include 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) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, 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 materials employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation.

[0118] 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 dose" when used in reference 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.

[0119] The formulation of the pharmaceutical composition may depend on the route of administration 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 may be formulated using suitable dispersing or wetting agents and suspending agents according to known art. The injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3 propanediol or 1,3 butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution (USP), and isotonic sodium chloride solution. In addition, sterile fixed oils are also conventionally employed as solvents or suspending media. For this purpose, any sterile fixed oil may be employed, including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid also find use in the preparation of injectables. The injectable preparations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0120] Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the anti-inflammatory agent. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, or emulsion.

[0121] In some embodiments, the compositions provided herein (e.g., pharmaceutical compositions administered to a subject) must be sterile. Sterility is easily accomplished by filtration through a sterile filtration membrane (e.g., a 0.2 micron membrane). Alternatively, preservatives can be used to prevent the growth or action of microorganisms. Various preservatives are well known, including, for example, phenol and ascorbic acid. The pharmaceutical composition will usually be stored in lyophilized form, or as an aqueous solution if it is highly stable against thermal and oxidative denaturation. The pH of the preparation will typically be about 6 to about 8, although higher or lower pH values ​​may also be appropriate in some instances.

[0122] Administration of anti-NPM1 conjugates The disclosure further provides for administration of an anti-NPM1 antibody described herein, a conjugate comprising an anti-NPM1 antibody described herein, or a composition thereof (e.g., a pharmaceutical composition) to a subject. In some embodiments, a method is provided for treating a disease associated with cell surface expression of NPM1 (e.g., an NPM1-expressing cancer) by administering an anti-NPM1 antibody described herein, a conjugate comprising an anti-NPM1 antibody described herein, or a composition thereof (e.g., a pharmaceutical composition) to a subject.

[0123] In some aspects, the disclosure provides a method for treating an NPM1-expressing cancer in a subject, the method 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 (e.g., a drug, a radioisotope) described herein, or a composition thereof (e.g., a pharmaceutical composition).

[0124] As used herein, the terms "administer", "administering" or "administration" refer to implanting, absorbing, ingesting, injecting, inhaling or otherwise introducing an agent (e.g., antibody, conjugate) or a composition (e.g., pharmaceutical composition) described herein into or onto a subject. As used herein, the terms "treatment", "treat" and "treating" refer to the application or administration of an agent (e.g., antibody, conjugate) or (e.g., pharmaceutical composition) described herein to a subject in need thereof for the purpose of reducing the severity of a disease (e.g., cancer) in the subject. A "subject in need thereof" refers to an individual having a disease, a symptom of a disease, or a predisposition to a disease. Methods for treating a disease may include administering to a subject an agent (e.g., an antibody, a conjugate) or a composition thereof (e.g., a pharmaceutical composition) as described herein with the intent to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, symptoms of the disease, or predisposition to the disease in the subject. Methods for treating a disease may also include prophylaxis, where an agent is administered to a subject for the purpose of preventing the onset of the disease, for example in a subject not known to have the disease but who may or may be at risk of developing the disease in the future.

[0125] As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount of an agent (e.g., an antibody or conjugate described herein) sufficient to induce a desired biological response in a subject, thereby alleviating, for example, one or more symptoms of a disease (e.g., cancer). A therapeutically effective amount may be an amount administered to a subject, either alone or in combination with one or more other agents. As will be recognized by those of skill in the art, an effective amount will vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the agent administered, the specific disease or disorder being treated, the severity of the disease or disorder, 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 particular route of administration, and similar factors within the knowledge and belief of the health practitioner to determine. These factors are well known to those of skill in the art and can be addressed with little routine experimentation. It is generally preferred that the maximum dose of each agent described herein (e.g., antibody or conjugate described herein) or any combination thereof to be used is at most the highest dose that can be safely administered to a subject according to sound medical judgment.Preferably, the effective dose is lower than the highest dose that can be safely administered to a subject.However, it will be understood by those skilled in the art that a subject or medical practitioner may choose a lower dose (e.g., a minimum effective dose) to mitigate any potential risk of treatment, such as side effects of treatment.

[0126] In some embodiments, a dose of an agent (e.g., an antibody or conjugate described herein) may be administered to a normal weight adult subject at a dose ranging from about 0.01 mg / kg to 1000 mg / kg. In some embodiments, the dose is between 1-200 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 (such as the pharmacokinetics of the agent) and other considerations well known in the art.

[0127] Treating a disease (e.g., cancer) may include delaying the onset or progression of the disease or reducing the severity of the disease. Treating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a disease means to defer, hinder, slow, retard, stabilize, and / or postpone the progression of the disease in a subject. Delaying the progression of a disease may also include delaying or preventing the spread of a disease occurring in a subject, such as delaying or preventing the metastasis of a cancer occurring in a subject to one or more organs or tissues not yet affected by the cancer. This delay may consist of various lengths of time, depending on the history of the disease and / or the individual being treated. A method of delaying the onset of a disease or delaying the onset of a disease is a method that reduces the probability of developing one or more symptoms of a disease in a given time frame and / or reduces the severity of symptoms in a given time frame when compared to the absence of such method. The comparison is typically based on clinical studies using a sufficient number of subjects to provide statistically significant results.

[0128] "Onset" or "progression" of a disease (e.g., cancer) refers to the initial signs of disease in a subject and / or subsequent progression. Onset of a disease may be detectable and assessed using standard clinical techniques as known in the art. However, onset also refers to progression that may be undetectable. For purposes of this disclosure, onset or progression may refer to the onset or progression of disease symptoms. The term "onset" includes the occurrence, recurrence, and onset of disease. As used herein, "onset" or "onset" of a disease includes the initial onset of disease, as well as the recurrence of disease (i.e., in a subject who previously had the disease).

[0129] The "subject" to which administration is intended refers to a human (i.e., male or female of any age group, e.g., a pediatric subject (e.g., an infant, a child, or an 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 may be male or female at any stage of development, and may be a juvenile or an adult animal. The non-human animal may be a transgenic or genetically modified animal.

[0130] In some embodiments, the subject is a companion animal (e.g., a pet or a service animal). "Companion animal" as used herein refers to pets and other domestic animals. Non-limiting examples of companion animals include dogs and cats; livestock such as horses, cows, 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. Non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human animals of the primate order.

[0131] In some embodiments, the subject has, is suspected of having, or is at risk of having NPM1-expressing cancer. The term "NPM1-expressing cancer" refers to a cancer characterized by expression of NPM1 (e.g., enhanced expression of NPM1 when compared to non-cancerous cells), which may further be characterized by altered subcellular localization of NPM1, such as increased NPM1 present on the cell surface. NPM1-expressing cancer may express WT NPM1 and / or mutant NPM1. NPM1-expressing cancer may express WT NPM1 and / or mutant NPM1 on the cell surface. NPM1-expressing cancer may also refer to a cancer not initially characterized by expression of NPM1, which expresses NPM1 (e.g., cell surface WT NPM1 and / or mutant NPM1) in response to administration or treatment with a drug (e.g., a chemotherapeutic drug). In some embodiments, NPM1-expressing cancer is a solid (tissue) or liquid (biological fluid, e.g., blood) cancer. In some embodiments, the NPM1 expressing cancer is selected from blood 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 or neck cancer, or skin cancer. In some embodiments, the NPM1 expressing cancer is acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). In some embodiments, the NPM1 expressing cancer is metastatic cancer. In some embodiments, the NPM1 expressing cancer is therapy-related cancer or secondary malignancy. The therapy-related cancer or secondary malignancy may be a cancer, such as leukemia, carcinoma, or lymphoma, that occurs as a result of previous treatment with chemotherapeutic drugs or radioisotopes. In some embodiments, the NPM1-expressing cancer is therapy-related AML (t-AML), or a secondary malignancy of non-Hodgkin's lymphoma.

[0132] In some embodiments, the subject has been previously treated for NPM1-expressing cancer. In some embodiments, the subject has an NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs). An NPM1-expressing cancer is said to be resistant to a treatment (e.g., treatment with one or more chemotherapeutic drugs) when the treatment cannot effectively kill and / or inactivate the cancer cells (e.g., due to genetic and / or epigenetic changes occurring in the cancer that result in the inactivation and / or excretion of one or more drugs of the treatment, or the inhibition of one or more downstream effects of the treatment), especially when the treatment was previously effective in killing and / or inactivating the cancer cells. In some embodiments, an NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs) is a cancer that expresses NPM1 on the surface of cancer cells as a result of a previous treatment (e.g., a previous treatment with one or more chemotherapeutic drugs), but the cancer is resistant or has become resistant to the previous treatment. In some embodiments, the level of NPM1 on the surface of an NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs) is increased as a result of a prior treatment (e.g., prior treatment with one or more chemotherapeutic drugs), but the cancer is resistant to, or has become resistant to, the prior treatment.

[0133] Conventional methods known to those skilled in the art of medicine can be used to administer the antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) to a subject, depending on the type of disease (e.g., cancer) or site of the disease (e.g., cancer) to be treated. The antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) can be administered systemically (i.e., all through the body) or locally (i.e., to one or more specific organs, tissues, or locations in the body). The antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) can be administered via any conventional route, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, intraperitoneally, or via an implanted reservoir. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intraperitoneal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) is administered via intravenous injection or infusion. In addition, the antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) can be administered to a subject via an injectable depot administration route, for example, using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some embodiments, the antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) is administered via injection. In some embodiments, the injection is an intravenous injection or an intratumoral injection.

[0134] In some embodiments, administration occurs more than once. In some embodiments, administration occurs once per day, once per 2 days, once per 3 days, once per 4 days, once per 5 days, once per 6 days, once per week, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year.

[0135] In some aspects, the disclosure provides a method for treating an NPM1-expressing cancer 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 comprising an anti-NPM1 antibody conjugated to an agent (e.g., a drug, a radioisotope) described herein, 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 combination contemplated comprises an antibody or conjugate described herein and a single chemotherapeutic drug described herein. In some embodiments, the combination contemplated comprises an antibody or conjugate described herein and more than one chemotherapeutic drug described herein.

[0136] In some embodiments, the subject to whom the combination is administered has, is suspected to have, or is at risk of having an NPM1-expressing cancer. The NPM1-expressing cancer may express WT NPM1 and / or mutant NPM1. The NPM1-expressing cancer may express WT NPM1 and / or mutant NPM1 on the surface of cancer cells. In some embodiments, the NPM1-expressing cancer is a solid (tissue) or liquid (biological fluid, e.g. blood) cancer. In some embodiments, the NPM1-expressing cancer is a cancer selected from blood 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 or neck cancer, or skin cancer. In some embodiments, NPM1 expressing cancer is acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma and myelodysplastic syndrome (MDS).In some embodiments, NPM1 expressing cancer is metastatic cancer.In some embodiments, NPM1 expressing cancer is therapy-related cancer or secondary malignancy.In some embodiments, NPM1 expressing cancer is therapy-related AML (t-AML) or secondary malignancy of non-Hodgkin's lymphoma.

[0137] In some embodiments, the subject to whom the combination is administered has been previously treated for NPM1-expressing cancer. In some embodiments, the subject has an NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapeutic drugs). As exemplified herein (see Example 4), treatment of cancer cells with chemotherapeutic drugs (e.g., daunorubicin, venetoclax, 5-azacytidine) may increase the level of NPM1 on and / or in cancer cells (e.g., on leukemia cancer cells, e.g., AML). Without wishing to be bound by theory, pre-medicating cancer cells, including chemoresistant cancers, with low doses of chemotherapeutic drugs enhances (enhances) the binding between the cancer cells and the antibodies or conjugates described herein. By enhancing the binding by the antibodies or conjugates in this way, the cancer cells may become easier to treat in the subject. For example, cancer cells that have been treated with a low dose of a chemotherapeutic drug may be effectively treated by administration of a lower dose of the antibody or conjugate than would otherwise be possible (i.e., without treatment with the low dose of the chemotherapeutic drug). A "low dose" may refer to a dosage of a chemotherapeutic drug that is insufficient on its own to effectively treat cancer in a subject.

[0138] In principle, any chemotherapeutic drug for the treatment of cancer generally known in the art may be administered to a subject as part of the combination. In some embodiments, administering 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, administering 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-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. 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 a lesser extent than on the surface of cancer cells in the subject.

[0139] Non-limiting examples of chemotherapeutic drugs suitable for use in the combination include auristatin E, auristatin F, monomethylauristatin D (MMAD), monomethylauristatin F (MMAF), monomethylauristatin E (MMAE), actinomycin, actinomycin X2, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, aeroprisinin, aldoxorubicin, agrochelin, ansatrienins, ansamitocin P-3, aphidicolin, apoptolidin, L-asparaginase, azacytidine, bafilomycin A 1, bafilomycin B1, bafilomycin B2, bafilomycin C1, bafilomycin C2, bafilomycin D, bafilomycin E, calicheamicin, campatecin, chaetocin, chaetoglobosin, chlamydocin, cinerubin B, cladribine, colchicine, combretastatin A1, combretastatin A4, cordycepin, cryptophycin, cucurbitacin B, cucurbitacin E, curbulin, cyclopamine, cyclophosphamide, cytarabine, dactinomycin, daunorubicin, decitabine, dexamethasone, dolastatin 10, dora Statins 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), Glucopyericidin A, Gramicidin A, Helboxidien, 9-Hydroxyellipticine, Hydroxyurea, Hygrolysine, Hypothemycin, Idarubicin, Irimaquinone, Isatropolone A, Isofistularin-3, Ixabepilone, JW5 5, lactacystin, luisol A, maytansinol, mertansine (DM1), maytansine DM3, ravtansine (DM4), maytansinoid AP-3, mekelcarmycin A, menthacarcin, methotrexate, 6-mercaptopurine, microcorrin B, microcystin LR, mitoxantrone, muscotoxin A, myoseverin, myotoxin B, nelarabine, nemorubicin, noqualin A, okilactomycin, oligomycin A, oligomycin B, paclitaxel, larotaxel, mirataxel, ortataxel, tesetaxel,These include phallacidin, phalloidin, phytosphingosine, piericidin A, pironetin, podophyllotoxin, polyketomycin, prednisone, pseudodoralic acid B, xulotin A, pwainaphycin F, pyrrolobenzodiazepine, quinaldopeptin, rachelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinarine, saporin, sinefungin, taltobulin, telomestatin, 6-thioguanine, thiocolchicine, triptoxin, triporin A, triptolide, tubastatin A, tubulysin A, tubulysin M, tubulysin IM-1, tubulysin IM-2, tubulysin IM-3, venetoclax, and vincristine. In some embodiments, the chemotherapeutic drug comprised by the combination is saporin, daunorubicin, venetoclax, or azacitidine. ,

[0140] In some embodiments, the combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and the combination chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) are administered to the subject simultaneously, i.e., administered to the subject at the same time or at about the same time. In some embodiments, the combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and the combination chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) are administered sequentially, i.e., administered to the subject at different times. In some embodiments, the combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) is administered prior to the combination chemotherapeutic drug. In some embodiments, the combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) is administered after the combination chemotherapeutic drug. In some embodiments, the combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and the combination 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.

[0141] Conventional methods known to those skilled in the art of medicine can be used to administer the combination to a subject, depending on the type of disease (e.g., cancer) or the site of the disease (e.g., cancer) to be treated. The combination can be administered systemically (i.e., all through the body) or locally (i.e., to one or more specific organs, tissues, or locations in the body). The combination can also be administered via any conventional route, e.g., orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, intraperitoneally, or via an implanted reservoir. 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 of the parts of the combination (e.g., antibody or conjugate, chemotherapeutic drug) are administered via intravenous injection or infusion. In addition, one or more of the combination parts (e.g., antibody or conjugate, chemotherapeutic drug) may be administered to the subject via an injectable depot administration route, e.g., using 1-, 3-, or 6-month depot injectable or biodegradable materials and methods. In some embodiments, one or more of the combination parts (e.g., antibody or conjugate, chemotherapeutic drug) are administered via injection. In some embodiments, the injection is an intravenous injection or an intratumoral injection. In some embodiments, the antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and the chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) are administered to the subject via the same administration route. In some embodiments, the antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and the chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) are administered to the subject via different administration routes.

[0142] In some embodiments, the combination is administered more than once. In some embodiments, the combination is administered once per day, once per 2 days, once per 3 days, once per 4 days, once per 5 days, once per 6 days, once per week, once per 2 weeks, once per 3 weeks, once per month, once per 2 months, once per 3 months, once per 4 months, once per 6 months, once per 7 months, once per 8 months, once per 9 months, once per 10 months, once per 11 months, or once per year.

[0143] In some embodiments, the "subject" to which the combination is administered refers to a human, i.e., a male or female of any age group, for example a pediatric subject (e.g., an infant, a child, or an 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 may be male or female at any stage of development, and may be a juvenile or an adult animal. The non-human animal may be a transgenic or genetically modified animal.

[0144] In some embodiments, the subject to which the combination is administered is a companion animal (e.g., pet or service animal).Non-limiting examples of companion animals include dogs and cats; livestock animals such as horses, cows, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.In some embodiments, the subject to which the combination is administered is a research animal.Non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human animals of the primate order.

[0145] In some embodiments, the subject has not previously been administered (e.g., treated with) a combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) or a combination chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition). In some embodiments, the subject has previously been administered (e.g., treated with) a combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition) and then administered a combination chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition). In some embodiments, the subject has previously been administered (e.g., treated with) a combination chemotherapeutic drug or composition thereof (e.g., pharmaceutical composition) and then administered a combination antibody, conjugate thereof, or composition thereof (e.g., pharmaceutical composition).

[0146] In some embodiments, administration of the combination to a subject induces an enhanced biological response in the subject than would be achieved by an equivalent dose of the antibody or conjugate described herein when administered in the absence of the chemotherapeutic drug described herein. In some embodiments, administration of the combination to a subject induces an enhanced biological response in the subject than would be achieved by an equivalent dose of the chemotherapeutic drug described herein when administered in the absence of the antibody or conjugate described herein. In some embodiments, combining the antibody or conjugate described herein with a chemotherapeutic drug enhances the efficacy of the antibody or conjugate when administered to a subject. The enhanced biological response resulting from administration of the combination may include, but is not limited to, alleviating one or more symptoms of a disease in a subject, or effectively preventing the onset or recurrence of a disease in a subject.

[0147] In some embodiments, administration of the combination to a subject results in increased binding between the antibody or conjugate of the administered combination and NPM1-expressing cancer cells of the subject, as compared to administration of the antibody or conjugate alone. As used herein, "increased binding" refers to an increase in the rate at which an antibody or conjugate administered to a subject binds to NPM1-expressing cancer cells of the subject (e.g., the rate at which an antibody or conjugate administered to a subject binds to WT and / or mutant NPM1 on the surface of NPM1-expressing cancer cells of the subject). In some embodiments, administration of the combination to a subject increases binding between the administered antibody or conjugate of the combination and NPM1-expressing cancer cells of the subject by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, or up to 10-fold when compared to administration of the antibody or conjugate alone.

[0148] In some embodiments, administering the combination to a subject results in reduced growth of NPM1-expressing cancer cells of the subject when compared to administering the antibody or conjugate of the combination alone. As used herein, "reduced growth" refers to a reduction in the rate of cell division (mitosis) occurring in the NPM1-expressing cancer cells of the subject. In some embodiments, administering the combination to a subject reduces the growth of NPM1-expressing cancer cells of the subject by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, or up to 10-fold when compared to administering the antibody or conjugate alone.

[0149] In some embodiments, administering the combination to a subject results in increased cell death of NPM1-expressing cancer cells of the subject when compared to administering the antibody or conjugate of the combination alone. As used herein, "cell death" refers to an increase in the rate of cell death occurring in NPM1-expressing cancer cells of a subject through any pathway that stops cells from surviving, including but not limited to apoptosis, autophagy, necrosis, and entosis. 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 when compared to administration of the antibody or conjugate alone.

[0150] One of skill in the art will recognize that the methods of treatment described herein may also be suitable for treating other diseases or disorders associated with cells expressing cell surface NPM1, such as non-cancerous diseases or disorders associated with cell surface expression of WT NPM1 or mutant NPM1.

[0151] example Example 1 - NPM1 is localized on the surface of human leukemia cells. Various human leukemia cell lines were assessed for cell surface expression of nucleophosmin 1 (NPM1).Briefly, K562, Kasumi, OCI-AML2, OCI-AML3, MOLM13, Jeko1, Nalm6, Jurkat, and SupT1 cells were cultured in vitro in RPM1 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Pen / Strep). Plasma membrane and cytosol fractions were collected using established techniques (see, for example, Flynn et al., Cell. 2021;184(12):3109-3124.e22) and immunoblotted with the following commercial antibodies: NPM1-WT (Santa Cruz Biotechnology # sc-32256), NPM1-Mut (Thermo Scientific # PA1-46356), beta-actin (Santa Cruz Biotechnology # sc-47778), and RPN1 (Santa Cruz Biotechnology # sc-48367). Although the majority of NPM1 was localized in the cytosol of each cell type, significant amounts of wild-type (WT) and mutant (Mut) NPM1 were identified on the surface of all cell types (Figure 1A). Further fluorescence-activated cell sorting (FACS) analysis of live cells confirmed variable levels of NPM1 on the surface of each cell type assayed.

[0152] These results suggest that antibodies specific for WT and / or mutant NPM1 can be used to deliver cytotoxic payloads to NPM1-expressing cancers in patients, such as those with acute myeloid leukemia (AML), non-Hodgkin's lymphoma, and myelodysplastic syndrome (MDS). However, such treatment strategies depend on the levels of NPM1 expressed on the surface of cancer cells being greater than those expressed on the surface of non-cancerous cells, especially those from which the cancer was initially derived. Therefore, the levels of NPM1 on the surface of peripheral blood (PB) and bone marrow (BM) cells were assessed. In most mouse PB and BM cells tested, NPM1 cell surface expression was relatively low, but was increased in cells expressing mutant NPM1 (Figures 1C and 1D). These results were confirmed by FACS analysis of human BM cells, in which antibodies specific for WT NPM1 did not bind effectively to human BM cells (Figure 1E). These results strongly suggest that antibodies specific for NPM1 can be used to effectively treat NPM1-expressing cancers.

[0153] Example 2 - Development of multiple antibodies specific to NPM1 To further assess the possibility that antibodies specific for NPM1 could be used to treat NPM1-expressing cancers, novel antibodies specific for NPM1 were generated. Separate antibodies with specificity for NPM1 were generated ("Ab1" and "Ab2"; Figures 2A and 2B).

[0154] To assess whether the isolated antibodies could be useful in the treatment of cancer, Ab1 anti-NPM1 antibody was placed into 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 with 10% FBS and 1% Pen / Strep. Streptavidin-saporin (Strep-ZAP) was bound to the biotinylated antibody at 2.6 μg Strep-ZAP to 1 μg IgG for 30 minutes on ice. Strep-ZAP was bound to either the biotinylated Ab1 anti-NPM1 antibody (Figure 2A) or the biotinylated anti-mouse isotype control (Strep-ZAP-IgG) (Thermo Scientific # 31800). After binding, the Strep-ZAP-IgG complex was added to the cells and cells were left to grow at 37° C. for either 24 or 48 hours. After each time point, cells were harvested, washed with PBS, and stained with Annexin V to detect apoptosis and also with DAPI to detect whether the cells remained viable. After staining, cells were washed and analyzed by FACS. After incubation, killing of OCI-AML3 cells was observed in a dose-dependent manner, with fewer viable cells observed at the highest dose after 48 hours (FIGS. 3A and 3B). Critically, less apoptosis and cell death was observed with the same amount of Strep-ZAP lacking cell surface targeting (Strep-ZAP-IgG sample). These results confirm the utility of anti-NPM1 antibody-based ADCs to target cytotoxic drugs to cancer cells expressing cell surface NPM1.

[0155] It should be noted that although these studies were performed in human cell lines, NPM1 antibodies can also be used to target endogenous NPM1 on the surface of non-human cell types due to the high degree of conservation of NPM1. Of note, the amino acid sequence of WT NPM1 is approximately 95% identical between humans and mice (FIG. 4A) and more than 99% identical between humans and other primates (FIG. 4B). The high degree of conservation also suggests that results obtained by testing the effect of anti-NPM1 antibodies or ADCs, for example, in mouse cells or in mice, would be expected to be extended to humans.

[0156] Example 3 – Antibodies detect WT and mutant NPM1 on the surface of humans and mouse models To further establish 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, cell surface-localized NPM1 was observed by assaying live cells. In other cases, intracellular and cell surface levels of NPM1 were observed by assaying fixed cells. Significant levels of NPM1 were observed on the surface of live OCI-AML3 cells, however, significant amounts of NPM1 were not robustly observed on MOLM13 cells (Figure 5A). Similar to OCI-AML3 cells, significant levels of NPM1 were observed on the surface of various primary mouse AML cell lines (Figures 5B and 5C). To further explore cell surface NPM1 in non-AML cell lines, human K562 cells, a model for myeloid leukemia, were also assessed. In these experiments, exogenous NPM1 was expressed with a TY1 tag, where the exogenous NPM1 was either WT NPM1 or mutant NPM1 (NPM1c). TY1 signal was found to be present on the surface of cells expressing either WT-NPM1-TY1 or NPM1c-TY1 using live and fixed cell flow cytometry, confirming that NPM1c protein can also be presented on the cell surface (Figures 6A-6E). An orthogonal immunofluorescence detection strategy confirms the surface distribution of TY1-tagged NPM1 molecules (Figure 6F).

[0157] Given that various mouse and human leukemia cell lines express cell surface WT and / or mutant NPM1, we subsequently assessed whether the isolated antibodies could also bind to the surface of these cells. The isolated Ab1 anti-NPM1 (Figure 2A) and Ab2 anti-NPM1 (Figure 2B) antibodies were each able to bind to surface-localized NPM1 on primary mouse MLL-rearranged AML cells (Figure 7A) and primary mouse NPM1c AML cells (Figure 7B), as determined by FACS analysis. Furthermore, the antibodies bound to NPM1 on human patient derived xenograft (PDX) cells implanted into a mouse model, but not to host mouse bone marrow cells (Figures 8A-8D). These results, combined with the above, provide additional evidence that ADCs containing either of these anti-NPM1 antibodies can be used to selectively target chemotherapeutic agents to cancer cells with low risk of toxicity to noncancerous tissues. These antibodies may also be used in other applications, including, for example, as diagnostic agents by alternatively conjugating the antibodies with imaging agents, which in turn may be used to locate and measure the relative abundance of cancer cells in a patient.

[0158] Example 4 – Chemotherapy drugs for the treatment of AML enhance cell surface levels of NPM1 Next, we assessed the effect of chemotherapeutic drugs currently approved for the treatment of AML on the level of cell surface NPM1. In principle, administration of these drugs to a subject may alter the cell surface expression of WT and / or mutant NPM1, which in turn would modulate the efficacy of anti-NPM1 antibodies or ADCs administered to the subject. We assessed the effect of two chemotherapeutic drugs for the treatment of AML, daunorubicin and venetoclax, on the binding of anti-NPM1 antibody (Merck / Sigma anti-B23 # B0556) to NPM1 on the surface of OCI-AML3 cells. Surprisingly, the binding of the antibody to the cells was enhanced 48 hours after treatment with either 10 nM daunorubicin or 40 nM venetoclax (Figure 9A). This effect was significantly enhanced 8 days after treatment (Figure 9A and Figure 9B). Consistent with these results, treatment with daunorubicin or venetoclax also enhanced the binding between isolated anti-WT NPM1 antibodies and the anti-mutant NPM1 antibodies described above (FIG. 2A and FIG. 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), also used for the treatment of myelodysplastic syndromes (MDS) (FIG. 9D). Notably, the binding between isolated anti-mutant NPM1 and OCI-AML3 cells was significantly enhanced only 24 hours after treatment with 5-Aza. These data indicate that various chemotherapeutic agents reliably enhance the binding of anti-NPM1 antibodies to the surface of cancer cells. The reason for this effect is not immediately clear, but it is likely that these drugs increase the level of cellular stress in cancer cells, which further shifts NPM1 from the nucleus and cytosol to the cell membrane. These results indicate that combination therapy may be particularly useful in the treatment of certain cancers, where a chemotherapy agent is administered to a subject not only to kill the cancer cells, but also to increase the levels of NPM1 on the surface of the cancer cells available for binding by an anti-NPM1 antibody or ADC, which is also co-administered to the subject.Potentially, the chemotherapeutic agent and the anti-NPM1 antibody or ADC can be administered to the subject simultaneously (eg, as part of the same composition or as separate compositions) or at different times during the course of treatment.

[0159] Example 5: NPM1 is a cell surface protein. To assess the subcellular localization of nucleophosmin 1 (NPM1), confocal imaging was performed on OCI-AML3 (a human cell line). Both intracellular and cell surface staining was performed. Intracellular staining was achieved by fixing and permeabilizing the cells prior to staining. In contrast, cell surface staining was performed by staining the cells prior to fixation. The staining results show that NPM1 is found both inside the cells and on the cell surface. The NPM1 inside the cells is in the nucleolar form, while the NPM1 on the cell surface forms clusters (Figure 10A). To investigate whether the biochemical fractionation was specific, other RNA-binding proteins (RBPs) were investigated, including heterogeneous nuclear ribonucleoprotein U (HNRNPU), nucleolar RNA helicase 2 (DDX21), dolichyl-diphosphooligosaccharide protein glycosyltransferase subunit 1 (RPN1), and RIO kinase 1 (RIOK1). RBPs were measured in the cytosolic and membrane compartments of various cell lines, including 293, A549, K562, and AML3. Results from biochemical fractionation and Western blot (WB) indicate that several other RBPs are also found in the membrane fractions of various cells, although not all are highly abundant cytosolic proteins (Figure 10B).

[0160] In addition to assessing cell surface expression of NPM1 on human cells, primary murine acute myeloid leukemia (AML) cells were also examined using live-cell flow cytometry. Like the human OCI-AML3 cell line, cell surface expression of NPM1 was detected on primary murine AML cells (Figures 11A-11B).

[0161] We also examined cell surface localization of NPM1 using Western blot. Anti-NPM1 captures 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 isolates a biotinylated band at the molecular weight of NPM1 (Fig. 12B).

[0162] Furthermore, to assess the ability of NPM1 to form nanoclusters on the cell surface, super-resolution microscopy was performed on HL-60 (human leukemia cell line) and OCI-AML3 cells. Super-resolution microscopy defines clusters of 120-150 nanometers (nm). These NPM1 clusters were present on the cell surface of both HL-60 and OCI-AML3 cell lines. The clustered and non-random pattern of NPM1 markers suggests a highly regulated cell biology process (Figures 13A-13B). Super-resolution microscopy performed on PANC1 cell line (pancreatic cancer) also demonstrated that NPM1 forms nanoclusters on the surface of cancer cells (Figure 13B).

[0163] Intracellular and cell surface staining, live cell flow cytometry, and Western blot results demonstrate that NPM1 is a cell surface protein in both human and mouse cell lines. In addition, super-resolution microscopy demonstrated that NPM1 on the cell surface forms ordered nanoclusters on various human cancer cell lines, including acute myeloid leukemia, leukemia, and pancreatic cancer.

[0164] Example 6: The Ab2.2 antibody targets NPM1. An antibody (hereinafter "Ab2.2") having a heavy chain (Ab2.2) (SEQ ID NO: 44) and a light chain (Ab2.2) (SEQ ID NO: 45) was generated and used to target NPM1 on the surface of human and non-human cell types.

[0165] The binding ability of Ab2.2 to NPM1 on the cell surface of cancer cells (OCI-AML3 cells) was tested using both live cell microscopy and fixed and permeabilized cell microscopy. 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 shows that both the commercially available NPM1 antibody and maAb2 stain surface puncta. Thus, Ab2.2 works similarly to the commercially available NPM1 antibody on the cell surface (Figure 14). Fixed and permeabilized cell microscopy shows that the commercially available NPM1 antibody results in nucleolar and cytoplasmic staining, whereas Ab2.2 antibody results in nucleolar and larger cytoplasmic staining. Thus, Ab2.2 detects more cytoplasmic NPM1 in mutant OCI-AML3 cells compared to the commercial NPM1 antibody (FIG. 14).

[0166] The ability of Ab2.2 to bind to NPM1 on the cell surface of healthy, non-cancerous cells was also tested using flow cytometry. Cells measured for Ab2.2 binding were leukocytes (CD45+ cells), myeloid cells (CD33+ cells), and hematopoietic stem cells ("HSC"; CD34+ cells). Only 3.78% of cells in donor 1 were double positive for CD34+ and Ab2.2 (donors 2 and 3 had less than 3%). Thus, little to no binding of Ab2.2 to healthy HSC was observed (Figure 15A).

[0167] The Ab2.2 antibody was further validated using Western blot and compared to a commercial antibody. The commercial antibody was supplied by Santa Cruz (SC FC8791). Two sources of lysate were evaluated: WCE and crude membrane (Mem). Both Ab2.2 and the commercial antibody yielded a band at approximately 38 kB. Thus, the commercial and Ab2.2 show nearly identical banding patterns to NPM1 (Figure 15B).

[0168] Example 7: Ab2.2 targets NPM1 in cancer cells in vitro. To determine whether Ab2.2 targets NPM1 in cancer cells in vitro, patient samples from Dana-Farber Cancer Institute (DFCI) and the United Kingdom (UK) were used. Cells were isolated from bone marrow of 12 acute myeloid leukemia (AML) patients from DFCI (Figure 16) and 15 AML patients from the UK (Figure 18) and analyzed using flow cytometry (Figures 17A-17B and Figures 19A-19C). Results show that Ab2.2 binds strongly to AML bone marrow blasts (Figures 17A-17B, patient 5) and NPM1c AML bone marrow (Figures 19D-19E). Furthermore, NPM1c blasts are CD34-low, allowing examination of the LSC population that is highly bound by Ab2.2. Thus, Ab2.2 binds best to LSC in bone marrow of NPM1c patients (Figures 19D-19E). Results from DFCI indicate that Ab2.2 strongly stains blasts without knowledge of mutation status, disease state, or prior treatment. Results from the UK indicate that Ab2.2 strongly stains blasts. Ab2.2 also strongly stains in NPM1c patients whose LSCs are CD34-, and thus Ab2.2 may target leukemia-initiating cells.

[0169] Example 8: Ab2.2 does not cause toxicity in mice. To assess the toxicity of Ab2.2 in vivo, wild-type (WT; C57BL / 6J) mice were subjected to weekly treatments of Ab2.2. There were 5 mice per group. Target groups received Ab2.2 administered at doses of 2.5 mg / kg, 5 mg / kg, or 10 mg / kg by IP injection. The control group received 5 mg / kg IgG (Figure 20A). One dose per week was delivered for a total of 4 doses. Weekly blood draws were performed on days 1, 7, 14, 20, and 27 (D) of treatment for sample collection. Mice were weighed on 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 (Figure 20B).

[0170] Example 9: Ab2.2 treatment improves survival in a mouse model of AML and has no effect in healthy mice. The efficacy of treatment with Ab2.2 was assessed using different models. In efficacy model 1, mice were subjected to sublethal irradiation followed by transplantation of primary mouse AML cells. Mice receiving primary mouse AML were administered weekly antibody treatment of Ab2.2 at a dose of 5 mg / kg. Administration occurred by IP injection. Weekly blood sampling was performed to analyze overall mouse survival, bone marrow (BM) and spleen, and molecular phenotyping (Figure 21A). The primary mouse 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 target group received a 5 mg / kg dose (Ab2.2). Three doses (one per week) were administered. Antibody binding was observed. Results from total WBC counts and qPCR demonstrate reduced tumor burden in Ab2.2-treated mice (Figures 21B-21D). Spleen weight, HGB, and hematocrit (HCT) levels also demonstrated that Ab2.2 reversed the hematopoietic defect caused by AML (Figures 21E-21F). Finally, Ab2.2 treatment improved the overall survival of mice (Figure 21G). The survival study was terminated at day 100 due to protocol-related, not toxicity or AML relapse.

[0171] In efficacy model 2, mice were subjected to sublethal irradiation followed by transplantation of primary mouse AML cells. Mice receiving primary mouse AML were given weekly antibody treatment with Ab2.2 at a dose of 5 mg / kg. Administration occurred by IP injection. Weekly blood sampling was performed to examine overall survival and molecular phenotype of the mice (Figure 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 target group received a 5 mg / kg dose (Ab2.2). A total of four doses of Ab2.2 were administered. Antibody binding was observed using flow cytometry (Figure 22B). The mean survival rate of the Ab2.2-treated group was significantly higher than that of the IgG-treated animals (p=0.0027). Survival was approximately 20 days in IgG-treated mice compared with approximately 55 days in Ab2.2-treated mice (FIG. 22C). Thus, Ab2.2 improves overall survival in an AML model with lower surface levels of NPM1. Furthermore, these results indicate that any amount of surface expression of NPM1 will translate into a survival benefit with Ab2.2 treatment.

[0172] Spleen, lung, and liver samples taken 3 days after the first dose of Ab2.2 treatment were compared between the control (IgG) group and the Ab2.2 treatment group. The spleens, lungs, and livers of Ab2.2-treated mice all weighed less than those of IgG-treated mice (Figure 23B). These results indicate that a single dose of Ab2.2 results in a robust reduction in organ weights, indicative of tumor clearance.

[0173] We also assessed LSC targeting of Ab2.2 in secondary recipients by using efficacy model 2 (Figure 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 target group received a 5 mg / kg dose (Ab2.2). One dose of Ab2.2 was administered before the secondary recipients received transplants. Secondary recipients in the control group received transplanted cells from IgG-treated mice. The target group received transplanted cells from Ab2.2-treated mice. Equal numbers of cells were transplanted and survival assays, flow cytometry, engraftment tests, and LSC function measurements were performed. The survival assay results demonstrate that the average survival of the control group was less than 30 days compared to approximately 50 days for the target group (Figure 23C). The extended survival of Ab2.2-treated mice demonstrates that fewer stem cells were present in Ab2.2-treated mice. Flow cytometry analysis showed that Ab2.2 stained the stem cell compartment only slightly better than the bulk tumor (Figure 23D) and the MLL-AF9 model had low expression of NPM1 on the surface (Figure 23E). WBC counts were decreased in Ab2.2-treated mice and PLTs were increased (Figure 23F). Further analysis of WBC over time demonstrated that WBC remained lower in Ab2.2-treated mice compared to IgG treatment over time (Figure 23G). Spleens of Ab2.2-treated secondary recipients weighed less than IgG-treated mice (Figure 23H). These results indicate that Ab2.2 treatment results in a robust reduction in organ weight, indicative of tumor clearance. Bone marrow (BM) and peripheral blood (PB) were assessed for percentage (%) of AML. In both the BM and PB, there was a reduction in AML% in the Ab2.2 treatment group compared to the IgG control (Figure 23I). These results indicate that Ab2.2 targets the tumor after only one dose.

[0174] Sublethally irradiated wild-type mice were tested for the effect of Ab2.2 treatment. WT C57BL / 6J mice were subjected to sublethally irradiation and four treatments of Ab2.2. Ab2.2 treatment occurred via weekly IP injections, and weekly blood sampling was performed. The control group received 5 mg / kg IgG, and the Ab2.2-treated group received 10 mg / kg Ab2.2. Regular blood cell counts, phenotyping of animals, and measurement of adverse / toxic effects were performed (Figure 26A). The results show that no significant differences were observed in weight, WBC count, HGB levels, or PLT levels between IgG-treated and Ab2.2-treated mice between day 1 (D) and D27 (Figure 26B). Thus, WT mice do not exhibit observable effects of Ab2.2 treatment.

[0175] Example 10: The effect of Ab2.2 on survival is immune system dependent. The immune-mediated killing ability of Ab2.2 was assessed using efficacy model 3. In efficacy model 3, NSG mice undergoing transplantation of primary mouse AML cells were given weekly Ab2.2 treatment and assessed for overall survival (Figure 24A). The transplanted primary mouse cells were MLL-AF9 / Flt3-ITD AML. The mouse model used was the immunocompromised AML mouse model (NSG). The control group received a 5 mg / kg dose (IgG) and the target 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 demonstrate that the average survival of the control and target groups was approximately 30 days (Figure 24B). Thus, Ab2.2 does not provide a survival benefit without an intact immune system.

[0176] Example 11: Ab2.2 treatment reduces tumor volume in vivo. The solid tumor activity of Ab2.2 was assessed in vivo using efficacy model 4. In efficacy model 4, implanted mice were subjected to weekly treatments of Ab2.2 and assessed for overall survival, tumor burden calculations, and molecular phenotyping (Figure 25A). Implanted 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 target group received a 10 mg / kg dose (Ab2.2). A total of 3 doses (1 per week) were administered. Tumor burden calculation results demonstrate that Ab2.2 reduces tumor volume on days 10 and 13 (Figures 25B-25C).

[0177] Example 12: Ab2.2 present on precancerous cells. DNMT3a mutations are directly associated with clonal hematopoiesis without cytopenias (CHIP) and preleukemia. To test whether Ab2.2 could detect Npm1c on Dmt3a R882H mutant cells, we R882H / - and Dmnt3a R882H / - / Npm1c cells were stained with either IgG control or Ab2.2. The results show high surface detection of Npm1c even on Dmnt3a R882H single mutant cells (Figure 28).

[0178] Example 13: Ab2.2 binds to human tumors in vitro. To assess the binding ability of Ab2.2 to human tumors, various types of in vitro models were investigated using flow cytometry. The tumor models assessed for Ab2.2 binding were mouse melanoma, lung cancer (human), laryngeal cancer, colon (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-27O). The results show that Ab2.2 binds to a range of diverse human tumor models in vitro.

[0179] Example 14: Ab2.2-saporin ADCs are active in vitro. To assess the in vitro activity of Ab2.2 in ADCs, OCI-AML3 cells were treated with either negative control, isotype saporin conjugate, or Ab2.2-saporin conjugate. Cells were treated for 24, 48, and 72 hours. The number of viable cells per μL was assessed at those time points. At 72 hours, the number of viable cells was reduced by about 30% in Ab2.2-saporin conjugate treated cells compared to the negative control (FIG. 29). Thus, NPM1 may serve as an ADC target in at least some models.

[0180] Example 15: Ab2.2 treatment extends survival of mice engrafted with human AML cell lines. The ability of Ab2.2 to extend survival in a mouse model xenografted with human AML cell lines was assessed. SCID CB17 mice were transplanted with OCI-AML3 cells (human AML). Engrafted mice were then treated weekly with Ab2.2 (target group) or IgG (control group). The target group received a 10 mg / kg dose of Ab2.2. Mice received a total of four doses. Mice were assessed for overall survival and molecular phenotyping (Figure 30A).

[0181] Antibody binding was assessed using flow cytometry (Figure 30B). Survival assay results demonstrate that survival of engrafted mice treated with Ab2.2 was significantly longer than IgG-treated mice, surviving for more than 40 days after transplantation (p=0.0015) (Figure 30C). Thus, extended survival is observed in mice engrafted with human AML cell lines and natural killer (NK) cells and complement treated with Ab2.2.

[0182] Example 16: Ab2.2 treatment extends survival of mice engrafted with human AML-PDX cell lines. The ability of Ab2.2 to extend survival in a mouse model xenografted with human AML-PDX was assessed. SCID CB17 mice were transplanted with PDX (MLL-R) cells (human AML-PDX). Engrafted mice were then treated weekly with Ab2.2 (target group) or IgG (control group). The target group received a 10 mg / kg dose of Ab2.2. Mice received a total of four doses. Mice were assessed for overall survival and molecular phenotyping (Figure 31A).

[0183] Antibody binding was assessed using flow cytometry (Figure 31B). Survival assay results demonstrated that engrafted mice treated with Ab2.2 survived for more than 60 days after transplantation, significantly longer than IgG-treated mice (p=0.0023) (Figure 31C). Thus, prolonged survival is observed in mice engrafted with human AML-PDX cell lines and natural killer (NK) cells and complement treated with Ab2.2.

[0184] Example 17: Low dose chemo induces cell surface NPM1. To determine whether low doses of the chemotherapy drugs daunorubicin and venetoclax induce cell surface NPM1, OCI-AML3 cells were exposed to control or low dose chemotherapy treatment. OCI-AML3 cells are resistant to BLC2i. Daunorubicin was administered at 10 nanomolar (nm) and venetoclax was administered at 40 nm. Administration of daunorubicin increased NPM1 on the cell surface at 48 hours and maintained higher expression for at least 9 days. Administration of venetoclax and 5-aza individually increased NPM1 at 48 hours and maintained higher expression for at least 7 days (Figure 32). These results indicate that low doses of chemotherapy drugs induce cell surface NPM1 on AML cells and that Ab2.2 enhances the safety or efficacy of approved drugs.

[0185] The ability of low dose chemotherapy drugs daunorubicin and venetoclax to induce cell surface NPM1 on non-transformed cells was tested. Normal hematopoietic progenitor cell line HPC7 and normal myeloid progenitor line HOXB8 were exposed to either control or low dose chemotherapy treatment. Daunorubicin and venetoclax doses were administered at 10 nanomolar (nM) and 40 nM, respectively. Antibody binding results measured by flow cytometry show no change in low dose chemotherapy drugs compared to control (Figure 33). Thus, low dose chemotherapy drugs do not induce expression of NPM1 on normal non-transformed cells.

[0186] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to 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.

[0187] Articles such as "a", "an" and "the" can mean one or more than one, unless indicated to the contrary or otherwise clear from the context. Unless indicated to the contrary or otherwise clear from the context, a claim or description that includes "or" between two or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present. The disclosure of a group that includes "or" between two or more group members provides for embodiments in which exactly one member of the group is present, embodiments in which more than one member of the group is present, and embodiments in which all of the group members are present. For the sake of brevity, these embodiments are not exhaustively written out individually herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0188] 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 of the claims or from one or more of the relevant parts of the specification are introduced into another claim. For example, a claim that is dependent on another claim may be modified to include one or more of the limitations found in any other claim that is dependent on the same original claim. Moreover, where a claim describes a composition, it should be understood that the method of making or using the composition according to any of the methods of making or using disclosed herein, or according to any method known in the art, as appropriate, is included, unless otherwise indicated or it is obvious to one skilled in the art that a contradiction or inconsistency would arise.

[0189] Where elements are presented as lists, for example in Markush group format, it should be understood that any possible subgroups of the elements are also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term "comprising" is intended to be open-ended, permitting the inclusion of additional elements or steps. In general, where an embodiment, product, or method is referred to as comprising a particular element, feature, or step, it should be understood that an embodiment, product, or method consisting of or consisting essentially of such element, feature, or step is also provided. For the sake of brevity, these embodiments are not exhaustively described herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0190] Where ranges are given, the endpoints are included. Moreover, unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to mean that in some embodiments, any particular value within the claimed range can be taken to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For brevity, the values ​​in each range are not written out individually and exhaustively herein, but it will be understood that each of these values ​​is provided herein and can be specifically claimed or disclaimed. Also, unless otherwise indicated or otherwise evident from the context and / or the understanding of one of ordinary skill in the art, values ​​expressed as ranges should be understood to mean that any subrange within the given range can be taken, where the endpoint of the subrange is expressed to the same degree of precision as the tenth of the unit of the lower limit of the range.

[0191] Where websites are provided, the URL addresses are provided as non-browser executable code and the periods in each web address are within parentheses. The actual web addresses do not contain parentheses.

[0192] In addition, it should be understood that any particular aspect of the present disclosure may be expressly excluded from any one or more of the claims. Where ranges are given, any value within the range may be expressly excluded from any one or more of the claims. Any aspect, element, feature, application, or aspect of the compositions and / or methods of the present disclosure may be excluded from any one or more of the claims. For the sake of brevity, all of the aspects in which one or more elements, features, purposes, or aspects are excluded are not explicitly described herein.

Claims

1. An antibody that binds to nucleophosmin 1 (NPM1), for use in treating cancers that express NPM1 in subjects requiring such treatment.

2. Antibodies, next: Heavy chain variable regions including heavy chain (HC) complementarity-determining region (CDR) 1 containing the amino acid sequence NIFVH (SEQ ID NO: 1), HC CDR 2 containing the amino acid sequence KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and HC CDR 3 containing the amino acid sequence DSSGYDAVDY (SEQ ID NO: 3); and Light chain variable region including light chain (LC) CDR1 containing amino acid sequence RASESVYTYLA (SEQ ID NO: 9), LC CDR2 containing amino acid sequence NAKTLTE (SEQ ID NO: 10), and LC CDR3 containing amino acid sequence QHHYGTPYT (SEQ ID NO: 11). The antibody according to claim 1, comprising:

3. The antibody according to claim 2, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 28, and / or the light chain variable region includes the amino acid sequence described in SEQ ID NO:

29.

4. Antibodies, next: Heavy chain variable regions including heavy chain (HC) complementarity-determining region (CDR) 1 containing the amino acid sequence SYAMS (SEQ ID NO: 15), HC CDR 2 containing the amino acid sequence AISGSGGGSTYYADSVKG (SEQ ID NO: 16), and HC CDR 3 containing the amino acid sequence WRNNAFDY (SEQ ID NO: 17); and Light chain variable region including light chain (LC) CDR1 containing amino acid sequence QGDSLRSYYAS (SEQ ID NO: 22), LC CDR2 containing amino acid sequence GKNNRPS (SEQ ID NO: 23), and LC CDR3 containing amino acid sequence NSSPRLKHRVV (SEQ ID NO: 24). The antibody according to claim 1, comprising:

5. The antibody according to claim 4, wherein the heavy chain variable region includes the amino acid sequence described in SEQ ID NO: 30, and / or the light chain variable region includes the amino acid sequence described in SEQ ID NO:

31.

6. The antibody according to claim 1, wherein the antibody is a full-length antibody or an antigen-binding fragment thereof.

7. The method according to claim 6, wherein the antibody is a full-length antibody selected from immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), and immunoglobulin M (IgM).

8. The antibody according to claim 1, wherein the antibody is an antigen-binding fragment selected from Fab fragment, F(ab')2 fragment, Ig monomer, Fd fragment, scFv, scAb, dAb, Fv, affibody, diabody, single-domain heavy chain antibody, and single-domain light chain antibody.

9. The antibody according to claim 1, wherein the antibody is a human antibody or a humanized antibody.

10. The antibody according to claim 1, further comprising a heavy chain constant region.

11. The antibody according to claim 10, wherein the heavy chain constant region comprises the amino acid sequence described in SEQ ID NO:

32.

12. The antibody according to claim 1, further comprising a light chain constant region.

13. The antibody according to claim 12, wherein the light chain constant region comprises the amino acid sequence described in SEQ ID NO: 33 or SEQ ID NO:

34.

14. Antibodies, (i) comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 35 and a light chain containing the amino acid sequence of SEQ ID NO: 37; (ii) comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 36 and a light chain containing the amino acid sequence of SEQ ID NO: 38; or (iii) A heavy chain containing the amino acid sequence of SEQ ID NO: 44 and a light chain containing the amino acid sequence of SEQ ID NO: 45 The antibody according to claim 1.

15. The antibody according to claims 1 to 14, wherein the antibody is conjugated to a drug.

16. The antibody according to claim 15, wherein the drug is a drug.

17. Drugs, (i) 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, Aeropricinin, Aldoxorubicin, Aglochelin, Ansatrienin, Ansamitosin P-3, Aphydicolin, Apoptolysin, L-Asparaginase, Azacitidine, Bafilomycin A1, Bafilomycin B1, Bafilomycin B2, Buff Iromycin C1, Bafilomycin C2, Bafilomycin D, Bafilomycin E, Kaliceamicin, Campatecin, Ketocin, Ketoglobosin, Chlamydosin, Synervin B, Cladribine, Colchicine, Combretastatin A1, Combretastatin A4, Cordycepin, Cryptophycin, Cucurbitacin B, Cucurbitacin E, Culburin, Cyclopamine, Cyclophosphamide, Cytarabine, Dactinomycin, Daunorubicin, Decitabine, Dexamethasone, Dorastatin 10, Dorastatin 15, Duocalmycin SA, Duocalmycin Syn™, Duocalmycin MA, Duocalmycin DM, Doxorubicin, Englelin A, Epotilon A, Epotilon B, Epotilon C, Etoposide, Fludarabine, Fumagiline, Geldanamycin, Tanespimycin (17-AAG), Glucopyricidine A, Gramicidin A, Herboxydiene, 9-Hydroxyellipticin, Hydroxyurea, Hygloridine, Hypotemycin, Idarubicin, Ilimaquinone, Isatropolone A, Isophystraline-3, Isabepyrone, JW55, Lactacystine, Ruisol A, Meitansinol Meltansine (DM1), Maytansine DM3, Rabtansine (DM4), Maytansinoid AP-3, Mekelcalmycin A, Menthalcin, Methotrexate, 6-Mercaptopurine, Microcholine B, Microcystine LR, Mitoxantrone, Muscotoxin A, Myosevelin, Mytoxin B, Nelarabine, Nemorubicin, Noquorin A, Ocyclomycin, Oligomycin A, Oligomycin B, Paclitaxel, Larotaxel, Mirataxel, Ortataxel, Tesetaxel, Phalacidine, Phalloidin, Phytosphingosine,Piericidine A, pyronetin, podophyllotoxin, polyketomycin, prednisone, pseudohydramycin B, shulotin A, pwinaficin F, pyrrolobenzodiazepine, quinaldopeptin, rakelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinalin, saporin, synefungin, tartobrin, telomestatin, 6-thioguanine, thiocolchicine, tritoxin, triporin A, triptolid, tubastatin A, tubulicin A, tubulicin M, tubulicin IM-1, tubulicin IM-2, tubulicin IM-3, venetoclax, and vincristine; or, (ii) Saporin, daunorubicin, venetoclax, and azacitidine, The antibody according to claim 16, selected from the group consisting of the following.

18. The antibody according to claim 16, wherein the antibody and the drug are conjugated via a linker.

19. Linker, (i) It is a linker that can be cut; (ii) A linker that is pH-sensitive, glutathione-sensitive, or protease-cleavable; (iii) A cleavable linker selected from the group consisting of N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 3-(2-pyridyldithio)butanoate (SPDB), sulfo-SPDB, valine-citrulline (Val-cit), acetylbutyrate, CL2A, maleimidocaproyl (MC), and Mal-EBE-Mal; (iv) an inseparable linker; or (v) An uncleavable linker selected from the group consisting of N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and maleimidomethylcyclohexane-1-carboxylate (MCC), MC-VC-PAB, The antibody according to claim 16.

20. The antibody according to claim 16, wherein the antibody-to-drug ratio is between 1:1 and 1:

10.

21. The antibody according to claim 15, wherein the drug is a radioactive isotope.

22. The antibody according to claim 21, wherein the radioactive isotope is selected from the group consisting of iodine-131, rhenium-188, yttrium-90, bismuth-213, and actinium-225.

23. (i) NPM1-expressing cancer is cancer in which NPM1 is expressed on the surface of cancer cells; (ii) NPM1-expressing cancer is cancer in which NPM1 is expressed on the surface of cancer cells as a result of the administration of or treatment with a chemotherapeutic drug; or (iii) NPM1-expressing cancer is a cancer in which wild-type NPM1 and / or mutant NPM1 are expressed on the surface of cancer cells. The antibody according to claim 1.

24. Cancer, (i) A solid or humoral cancer selected from the group consisting of blood 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 or neck cancer, or skin cancer; (ii) Cancer 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); (iii) It is metastatic cancer; (iv) Treatment-related cancer or secondary malignancy; or (v) Treatment-related AML (t-AML), or a secondary malignancy of non-Hodgkin lymphoma, The antibody according to claim 1.

25. The antibody according to claim 1, wherein the subject is currently receiving or has previously received chemotherapy.

26. Chemotherapy drugs, (i) 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, Aeropricinin, Aldoxorubicin, Aglochelin, Ansatrienin, Ansamitosin P-3, Aphydicolin, Apoptolysin, L-Asparaginase, Azacitidine, Bafilomycin A1, Bafilomycin B1, Bafilomycin B2, Buff Iromycin C1, Bafilomycin C2, Bafilomycin D, Bafilomycin E, Kaliceamicin, Campatecin, Ketocin, Ketoglobosin, Chlamydosin, Synervin B, Cladribine, Colchicine, Combretastatin A1, Combretastatin A4, Cordycepin, Cryptophycin, Cucurbitacin B, Cucurbitacin E, Culburin, Cyclopamine, Cyclophosphamide, Cytarabine, Dactinomycin, Daunorubicin, Decitabine, Dexamethasone, Dorastatin 10, Dorastatin 15, Duocalmycin SA, Duocalmycin Syn™, Duocalmycin MA, Duocalmycin DM, Doxorubicin, Englelin A, Epotilon A, Epotilon B, Epotilon C, Etoposide, Fludarabine, Fumagiline, Geldanamycin, Tanespimycin (17-AAG), Glucopyricidine A, Gramicidin A, Herboxydiene, 9-Hydroxyellipticin, Hydroxyurea, Hygloridine, Hypotemycin, Idarubicin, Ilimaquinone, Isatropolone A, Isophystraline-3, Isabepyrone, JW55, Lactacystine, Ruisol A, Meitansinol Meltansine (DM1), Maytansine DM3, Rabtansine (DM4), Maytansinoid AP-3, Mekelcalmycin A, Menthalcin, Methotrexate, 6-Mercaptopurine, Microcholine B, Microcystine LR, Mitoxantrone, Muscotoxin A, Myosevelin, Mytoxin B, Nelarabine, Nemorubicin, Noquorin A, Ocyclomycin, Oligomycin A, Oligomycin B, Paclitaxel, Larotaxel, Mirataxel, Ortataxel, Tesetaxel, Phalacidine, Phalloidin, Phytosphingosine,Selected from the group consisting of piericidine A, pyronetin, podophyllotoxin, polyketomycin, prednisone, pseudophosphate B, shulotin A, pwinaficin F, pyrrolobenzodiazepine, quinaldopeptin, rakelmycin, rebeccamycin, Ro 5-3335, safracin B, sandramycin, sanguinalin, saporin, synefungin, tartobrin, telomestatin, 6-thioguanine, thiocolchicine, tritoxin, triporin A, triptolid, tubastatin A, tubulisin A, tubulisin M, tubulisin IM-1, tubulisin IM-2, tubulisin IM-3, venetoclax, and vincristine; (ii) Saporin, daunorubicin, venetoclax, or azacitidine; (iii) A chemotherapy drug that cancer has developed resistance to, The antibody according to claim 25.

27. The administration is (i) occurring systemically or locally; (ii) Caused orally or by injection; (iii) Caused by intravenous injection, subcutaneous injection, intraperitoneal injection, or intratumoral injection; or (iv) Occurring between once a day and once every six months, The antibody according to claim 1.

28. The antibody according to claim 1, wherein the target is a mammal or a human.

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