Mutant and mislocalized cell surface nucleophosmin 1 as diagnostic and therapeutic targets for human disease

Abstract

Provided herein are antibodies specific for nucleophosmin 1 (NPM1) that are capable of binding to wild-type and / or mutant nucleophosmin 1 located on the surface of cells. These antibodies, as well as antibody conjugates that include these antibodies, are useful for the detection and treatment of cancers in which NPM1 is expressed on the surface of cancer cells.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to Greek Application No. 20220100348, entitled "MUTANT AND MISLOCALIZED CELL SURFACE NUCLEOPHOSMIN 1 AS A DIAGNOSTIC AND THERAPEUTIC TARGET OF HUMAN DISEASE," filed on 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 (C123370224WO00-SEQ-VLJ.xml; size: 47,034 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 targeting cytotoxic payloads to cells with cell surface expression of WT and / or mutant NPM1. Accordingly, some aspects of the disclosure relate to antibodies that bind to NPM1.

[0005] 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.

[0006] 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.

[0007] 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.

[0008] 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. In some embodiments, the antibody is a human or humanized antibody.

[0009] 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. 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.

[0010] 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.

[0011] 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. Another aspect of the disclosure relates to a composition comprising an antibody that binds to NPM1 described herein. In some embodiments, the composition further comprises a pharmacologically acceptable excipient.

[0012] Another aspect of the present disclosure relates to a nucleic acid or a nucleic acid set that encodes an antibody that binds to NPM1 as described herein.In some embodiments, the nucleic acid or the nucleic acid set comprises a vector or a vector set.In some embodiments, the vector or the vector set is an expression vector or a vector set. Another aspect of the present disclosure pertains to a cell comprising a nucleic acid or set of nucleic acids encoding an antibody that binds NPM1 as described herein.

[0013] Another aspect of the present disclosure relates to a method of producing an antibody that binds to NPM1 described herein, the method comprising culturing cells containing a nucleic acid or set of nucleic acids encoding an antibody that binds to NPM1 described herein, harvesting the cultured cells and / or culture medium, and isolating the antibody from the cultured cells and / or culture medium. Another aspect of the disclosure relates to a conjugate comprising an antibody that binds NPM1 as described herein conjugated to an agent.

[0014] In some embodiments, the agent is a drug. In some embodiments, the drug is one of the following: 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, apoptolidin, L-asparaginase, azacytidine, bafilomycin A1, buffycin, bafilom ... thromycin 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), Glucopyericidin A, Gramicidin A, Helboxidien, 9-Hydroxyellipticine, Hydroxyurea, Hygrolysine, Hypothemycin, Idarubicin, Irimaquinone, Isatropolone A, Isofistularin-3, Iki Sabepilone, JW55, 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,The drug is selected from the group consisting of ortataxel, 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.

[0015] 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.

[0016] 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. 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.

[0017] In some embodiments, the agent is a radioisotope, hi some embodiments, the radioisotope is selected from the group consisting of iodine-131, rhenium-188, yttrium-90, bismuth-213, and actinium-225. In some embodiments, the agent is an imaging agent. In some embodiments, the imaging agent is a luminescent imaging agent or a fluorescent imaging agent. In some embodiments, the imaging agent is an agent detectable by magnetic resonance imaging (MRI). In some embodiments, the imaging agent is gadolinium-diethylenetriamine (Gd-DTPA).

[0018] Another aspect of the present disclosure relates to a method of treating nucleophosmin 1 (NPM1)-expressing cancer, the method comprising administering to a subject in need thereof an effective amount of an antibody or conjugate described herein.

[0019] In some embodiments, the NPM1 expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells. 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.

[0020] In some embodiments, the antibody or conjugate is administered systemically or locally. In some embodiments, the antibody or conjugate is administered via injection. In some embodiments, the injection is intravenous, subcutaneous, intraperitoneal, or intratumoral. In some embodiments, the antibody or conjugate is administered orally. In some embodiments, administration occurs more than once, hi some embodiments, administration occurs between once per day and once per six months.

[0021] In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. 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.

[0022] Another aspect of the present disclosure relates to a method for assessing the presence of a nucleophosmin 1 (NPM1)-expressing cancer, the method comprising administering to a subject in need thereof an effective amount of a conjugate described herein, imaging the conjugate in the subject, and determining the presence of an NPM1-expressing cancer in the subject based on the level and location of the antibody conjugate imaged in the subject.

[0023] In some embodiments, the NPM1 expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells. 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.

[0024] In some embodiments, the imaging is luminescence imaging or fluorescence imaging. In some embodiments, the imaging is magnetic resonance imaging (MRI). In some embodiments, the method further comprises administering to the subject an effective amount of an antibody or conjugate described herein if an NPM1-expressing cancer is determined to be present in the subject.

[0025] Another aspect of the present disclosure relates to a method for assessing the presence of a nucleophosmin 1 (NPM1)-expressing cancer, the method comprising collecting a biological sample from a subject in need thereof, contacting the biological sample with a conjugate described herein, analyzing binding between the conjugate and NPM1-expressing cancer cells in the biological sample, and determining the presence of an NPM1-expressing cancer in the subject based on the level of binding between the conjugate and the NPM1-expressing cancer cells analyzed in the sample.

[0026] In some embodiments, the NPM1-expressing cancer is a cancer in which NPM1 is expressed on the surface of cancer cells. 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.

[0027] In some embodiments, the biological sample collected from the subject is a blood sample, a serum sample, or a plasma sample. In some embodiments, the analysis is luminescence or fluorescence analysis. In some embodiments, the analysis is performed via flow cytometry. In some embodiments, the method further comprises administering to the subject an effective amount of an antibody or conjugate described herein if an NPM1-expressing cancer is determined to be present in the subject. [Brief description of the drawings]

[0028] 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 figure. In the drawing:

[0029] [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 cytosol and membrane fractions collected from various human leukemia cell lines. [Figure 1-2] FIG. 1B shows FACS analysis of NPM1 surface expression in human leukemia cell lines. [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 IE shows FACS analysis demonstrating 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).

[0030] [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.

[0031] [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 an antibody that binds to WT NPM1 against OCI-AML3 cells. Streptavidin-saporin was conjugated to either biotinylated anti-WT NPM1 or anti-mouse IgG, after which the complexes were added to OCI-AML3 cells and incubated for 24 hours. After incubation, cells were washed and cell killing was analyzed by FACS. [Figure 3-2] FIG. 3B shows the same as FIG. 3A with 48 hours of incubation.

[0032] [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 the human and rhesus monkey NPM1 amino acid sequences (SEQ ID NOs:39 and 41).

[0033] [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, whereas 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 murine 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 murine MLL-AF4 / FLT3ltd / +, MLL-AF9 / FLT3ltd / +, MLL-ENL / FLT3ltd / +, and Npm1c / FLT3ltd / + AML cells.

[0034] [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 as separate panels, together with quantification of the percentage of cells positive for binding 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. Applicable cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type 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. Applicable cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type 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 intracellular WT and mutant NPM1. Applicable cells expressed empty TY1 lentiviral vector, TY1-tagged NPM1 wild-type lentiviral vector, or TY1-tagged NPM1c lentiviral vector. [Figure 6-6] Figure 6E shows the binding between anti-NPM1 antibodies (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 6-7] Figure 6F shows immunofluorescence illustrating the binding between anti-NPM1 antibodies (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.

[0035] [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.

[0036] [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 the 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] FIG. 8B shows binding between isolated WT and mutant anti-NPM1 antibodies and NPM1 localized on the surface of PDX-2 (NPM1c) cells. [Figure 8-3] 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 8-4] 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.

[0037] [Figure 9] Figures 9A-9B show intracellular and cell surface staining of isotype and NPM1 from the cell line OCI-AML3 (Figure 9A), as well as denaturing protein gels and western blotting (WB) of biochemical fractions (cytosolic and membrane fractions) from the four cell lines (Figure 9B).

[0038] [Figure 10-1] Figures 10A-10B show results from live cell staining of a human suspension cell line (Figure 10A) and a primary mouse cell line (Figure 10B) cultured with anti-NPM1 antibody (AF647 signal). [Figure 10-2] Figures 10A-10B show results from live cell staining of a human suspension cell line (Figure 10A) and a primary mouse cell line (Figure 10B) cultured with anti-NPM1 antibody (AF647 signal). [Figure 10-3] Figures 10A-10B show results from live cell staining of a human suspension cell line (Figure 10A) and a primary mouse cell line (Figure 10B) cultured with anti-NPM1 antibody (AF647 signal).

[0039] [Figure 11] Figures 11A-11B show Western blots of cells after cell surface biotinylation with a cell-impermeant biotinylation reagent. The Western blot detects NPM1 in the membrane fraction of these cells, and NPM1 IP can enrich NPM1 more robustly from membrane lysates (Figure 11A). Examination of the biotin signal from these fractions (cell surface exposed proteins) demonstrates isolation of a single band in the NPM1 IP (Figure 11B), indicating that full-length NPM1 is exposed to the surface of live cells.

[0040] [Figure 12-1] Figures 12A-12B are diffraction-limited (DL) and super-resolution (SR) reconstructions of anti-NPM1 staining the cell surface of both HL-60 and OCI-AML3 (Figure 12A) and the adherent cell line PANC1 (Figure 12B). Cell surface NPM1 appears as distinct clusters. [Figure 12-2] Figures 12A-12B are diffraction-limited (DL) and super-resolution (SR) reconstructions of anti-NPM1 staining the cell surface of both HL-60 and OCI-AML3 (Figure 12A) and the adherent cell line PANC1 (Figure 12B). Cell surface NPM1 appears as distinct clusters.

[0041] [Figure 13]FIG. 13 shows fluorescent images of live (top) or fixed and permeabilized (bottom) OCI-AML3 cells stained with either Ab2.2 or the commercial NPM1 antibody.

[0042] [Figure 14-1] 14A-14B show bone marrow samples from three healthy donors stained with Ab2.2 and sorted for various markers of the hematopoietic system. Ab2.2 binds in part to CD33+ cells, however, there is no observable binding to CD34+ (HSC) cells. [Figure 14-2] Figure 14B shows the binding of a commercial anti-NPM1 antibody and Ab2.2 to protein lysate samples by Western blotting. The banding patterns are identical.

[0043] [Figure 15] FIG. 15 is a table listing 12 AML patients.

[0044] [Figure 16-1] 16A-16B show flow cytometry analysis of 12 AML cases. [Figure 16-2] 16A-16B show flow cytometry analysis of 12 AML cases. [Figure 16-3] 16A-16B show flow cytometry analysis of 12 AML cases. [Figure 16-4] 16A-16B show flow cytometry analysis of 12 AML cases. [Figure 16-5] 16A-16B show flow cytometry analysis of 12 AML cases. [Figure 16-6] 16A-16B show flow cytometry analysis of 12 AML cases.

[0045] [Figure 17] FIG. 17 is a table listing samples collected from 15 AML patients.

[0046] [Figure 18-1] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-2] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-3] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-4] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-5] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-6] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-7] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-8] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-9] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-10] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-11] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-12] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-13] 18A-18E show flow cytometry analysis of 15 AML patients. [Figure 18-14] 18A-18E show flow cytometry analysis of 15 AML patients.

[0047] [Figure 19-1]Figures 19A-19B show how the toxicity of Ab2.2 was assessed in vivo. Figure 19A is a schematic of the experimental protocol. Figure 19B shows the weight results and results from blood sample analysis of WBC, PLT, and HGB. [Figure 19-2] FIG. 19B shows the weight results and results from blood sample analysis of WBC, PLT, and HGB. [Figure 19-3] FIG. 19B shows the weight results and results from blood sample analysis of WBC, PLT, and HGB.

[0048] [Figure 20-1] Figures 20A-20G show how the efficacy of Ab2.2 treatment was assessed in vivo. Figure 20A is a schematic of the experimental design. Figure 20B shows antibody binding to this AML model. [Figure 20-2] Figure 20C shows the WBC counts, and Figure 20D shows the results from qPCR analysis of AML alleles. [Figure 20-3] Figure 20E shows the spleens of IgG and Ab2.2 treated mice and the spleen weights. Figure 20F shows the results of HGB and HCT analysis of blood samples. [Figure 20-4] FIG. 20G shows the results from the survival assay.

[0049] [Figure 21-1] Figures 21A-21C show how mice were subjected to sublethal irradiation and transplantation of primary murine AML cells to assess efficacy (efficacy model 2). Figure 21A is a schematic of the experimental design. [Figure 21-2] Figure 21B shows antibody binding, and Figure 21C shows the results from a viability assay.

[0050] [Figure 22-1]Figures 22A-22I show that treatment with Ab2.2 reduces tumor burden in a transplantation model. Figure 22A is a schematic diagram of the experimental design to test the effect of secondary transplantation after Ab2.2 treatment. Figure 22B shows the weights of spleen, lung, and liver in mice receiving transplants from IgG or Ab2.2 treated mice. [Figure 22-2] FIG. 22C shows the results from the survival assay. [Figure 22-3] 22D-22E show the results of flow cytometry. [Figure 22-4] Figures 22D-22E show the results of flow cytometry, and Figure 22F shows the WBC count and PLT levels from a blood sample. [Figure 22-5] Figure 22G shows WBC over time. Figure 22H shows spleen images and a graph of spleen weight in isotype or Ab2.2 treated mice. [Figure 22-6] FIG. 22I shows AML% in BM and PB.

[0051] [Figure 23] Figures 23A-23B show the immune dependence of the tumor killing activity of Ab2.2 versus an intact immune system. Figure 23A shows the experimental design. Figure 23B shows the survival curves of mice treated with either Ab2.2 or IgG.

[0052] [Figure 24-1] Figures 24A-24C show the solid tumor activity of Ab2.2. Figure 24A shows the experimental design. Figure 24B shows the tumor volumes on days 10 and 13. [Figure 24-2] FIG. 24C shows tumor volume over time from days 7 to 13.

[0053] [Figure 25-1] Figures 25A-25B show that wild-type healthy mice treated with sublethally irradiation and Ab2.2 exhibited no observable effect of Ab2.2 treatment. Figure 25A shows the experimental design. [Figure 25-2] FIG. 25B shows the weight, and WBC count, HGB, and PLT analysis from blood samples. [Figure 25-3] FIG. 25B shows the weight, and WBC count, HGB, and PLT analysis from blood samples.

[0054] [Figure 26-1] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-2] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-3] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-4] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-5] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-6] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-7] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-8]Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-9] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors. [Figure 26-10] Figures 26A-26O show in vitro models of various cancer cell types that were analyzed using flow cytometry to assess the ability of Ab2.2 to bind to human or mouse tumors.

[0055] [Figure 27] FIG. 27 shows high surface detection of Npm1c on Dmnt3a R882H single mutant cells (pre-leukemic cells).

[0056] [Figure 28] Figure 28 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.

[0057] [Figure 29-1] Figures 29A-29C show that Ab2.2 extends life span in a transplant model of a human AML cell line. Figure 29A is a schematic of the experimental design. [Figure 29-2] FIG. 29B shows antibody binding using flow cytometry. [Figure 29-3] FIG. 29C is a graph of the results of the viability assay.

[0058] [Figure 30-1]Figures 30A-30C show that Ab2.2 extends life span in a transplant model of human AML-PDX cell line. Figure 30A is a schematic of the experimental design. Figure 30B shows antibody binding using flow cytometry. [Figure 30-2] FIG. 30C is a graph of the results of the viability assay. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] 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, radioisotopes) to certain cell types (e.g., cancer cells) in a subject. Without wishing to be bound by theory, NPM1 is a multifunctional protein that 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 may occur in any type of cancer, it is particularly common in leukemias (e.g., acute myeloid leukemia (AML)) due to mutations occurring 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 transported to the cell membrane and exposed to the extracellular environment. It is also known that NPM1c occurs only in cancer cells, so that 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, even without conjugation to a cytotoxic payload, an NPM1-specific antibody may be sufficient by itself to stimulate antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) by binding to cancer cells. However, although 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 currently there are no monoclonal antibodies available that bind to NPM1c. Therefore, new antibodies were 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 in 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.

[0060] Antibody that binds to NPM1 The present disclosure provides antibodies that bind to NPM1, e.g., wild-type (WT) NPM1 or mutant NPM1. Such antibodies may have a higher affinity for WT NPM1 than for mutant NPM1, or may have a higher affinity for mutant NPM1 than for WT NPM1.

[0061] 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 CH Each light chain is composed of a light chain variable region (referred to herein as V L The light chain constant region consists of one domain, C L It consists of: V H 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 L consists 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 can 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 can be a polyclonal or monoclonal antibody.

[0062] In some embodiments, antibodies are heterotetrameric glycoproteins consisting 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, and secreted IgA antibodies can polymerize to form multivalent assemblies containing 2-5 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, and 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 in each of the α and γ chains (C H ), and four C H Each L chain has a variable domain (VL ), followed by a constant domain (C L ) V L is V H Align with C L aligns 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 L The pairing of these together forms a single antigen-binding site. For the structures and properties of non-limiting examples of different 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, which is incorporated herein by reference. In some embodiments, the antibody is an IgG.

[0063] 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 different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. 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. By way of example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0064] The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed across the entire 110 amino acid span 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 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs that adopt a predominantly β-sheet configuration and are connected by three hypervariable regions. These 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, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991), incorporated herein by reference). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0065] In some embodiments, the antibody is a monoclonal antibody. A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies constituting the population are identical with the exception of possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that 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 without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256:495 (1975), or can 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 can 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.

[0066] The monoclonal antibodies described herein encompass "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences on antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences on antibodies from another species or belonging to another antibody class or subclass, so long as they exhibit the desired biological activity (see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). 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 monkeys, apes, etc.) and human constant region sequences.

[0067] In some embodiments, the antibody is a polyclonal antibody. A "polyclonal antibody" is a mixture of different antibody molecules that react with more than one immunogenic determinant of an antigen. Polyclonal antibodies can be isolated or purified from mammalian blood, secretions, or other fluids, or from eggs. Polyclonal antibodies can also be recombinant. Recombinant polyclonal antibodies are polyclonal antibodies that are produced by the use of recombinant technology. Recombinantly produced polyclonal antibodies usually contain a high concentration of different antibody molecules. All or most of these (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 that is composed of more than one epitope.

[0068] In some embodiments, the antibody is "humanized" for use in humans (e.g., as a therapeutic). "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, that has the desired antibody specificity, affinity, and quality. In some cases, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further improve antibody performance. In general, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least some portion of an immunoglobulin constant region (Fc), typically that 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).

[0069] 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 to a particular antigen that occurs more frequently, at a higher rate, for a greater duration, and / or with greater affinity 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. That is, 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 contains one or more amino acid insertions, deletions, or substitutions (i.e., a mutant NPM1), and a variant of NPM1 that contains one or more amino acid insertions, deletions, or substitutions (i.e., a mutant NPM1) may or may not bind to WT NPM1.

[0070] 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 generally accepted reference sequence for intact, fully functional NPM1. For example, WT NPM1 can be an NPM1 isoform (for example, 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 can 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 can have reduced activity and / or altered subcellular localization compared to WT NPM1. The WT NPM1 or mutant NPM1 described herein can 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.

[0071] In some embodiments, the antibody that binds to NPM1 is a full-length antibody. The full-length antibody described herein can be any antibody class based on the amino acid sequence of its heavy chain constant region. For example, the full-length antibody that binds to NPM1 can be Immunoglobulin G (IgG), Immunoglobulin A (IgA), Immunoglobulin D (IgD), Immunoglobulin E (IgE), and Immunoglobulin M (IgM), or subclasses thereof (e.g., IgG1, IgG2, IgG3, IgG4). In some embodiments, the antibody described herein is an antigen-binding fragment. The antigen-binding fragment can be in the 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 can be of mouse, rat, human, or any other origin. In some embodiments, the antibody described herein is a human antibody or a humanized antibody.

[0072] 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 can be determined using any numbering scheme commonly known in the art (e.g., Kabat, Chothia, Contact, IGMT).

[0073] 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).

[0074] 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).

[0075] Table 1 provides the amino acid sequences of the heavy and light chain CDRs of exemplary antibodies ("Ab1" and "Ab2") that are 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.

[0076] Table 1: CDR sequences of anti-NPM1 antibodies [Table 1]

[0077] 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).

[0078] 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.

[0079] Table 2: Heavy and light chain sequences of anti-NPM1 antibodies [Table 2-1]

[0080] [Table 2-2]

[0081] [Table 2-3]

[0082] In some embodiments, the antibody that binds to NPM1 has the CDRs, VDRs, VV ... H , V L , CH , C L In some embodiments, an antibody that binds to NPM1 may include 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 include 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 ... H , V L , C H , C L , heavy chain, or 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 CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H17, CDR-H18, CDR-H20, CDR-H21, CDR-H22, CDR-H23, CDR-H24, CDR-H25, CDR-H26, CDR-H27, CDR-H28, CDR-H29, CDR-H30, CDR-H31, CDR-H32, CDR-H33, CDR-H40, CDR-H41, CDR-H42, CDR-H43, CDR-H44, CDR-H45, CDR-H46, CDR-H47, CDR-H48, CDR-H49, CDR-H50, CDR-H51, CDR-H52, CDR-H61, CDR-H62, CDR-H63, CDR-H64, CDR-H65, CDR-H66, CDR-H67, CDR-H68, CDR-H69, CDR-H70, CDR-H71, CDR-H72, CDR-H73, CDR-H74, CDR-H75, CDR-H76, CDR-H77, CDR-H78, CDR-H79, CDR-H80, CDR-H81, CDR-H82,

[0083] 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).

[0084] 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).

[0085] 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).

[0086] 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).

[0087] 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).

[0088] 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 compared to a VH that comprises 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 compared to a VL that comprises the amino acid sequence of SEQ ID NO: 29.

[0089] 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 compared to a VH comprising the amino acid sequence of SEQ ID NO: 30. 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 compared to a VL comprising the amino acid sequence of SEQ ID NO: 31.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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, for example, 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 fusing agent (e.g., polyethylene glycol or electric current) to produce an immortalized cell line capable of producing and secreting the desired antibody. Hybridomas can be cultured in a culture medium under conditions known in the art.

[0102] Alternatively, the nucleic acids encoding the heavy and light chains of the anti-NPM1 antibodies 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 particular cell type. The one or more expression vectors contain one or more promoters for driving expression of the operably linked genes (typically 3' of the promoter sequence). Each of the nucleotide sequences encoding the heavy and light chains can be operably linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operably linked to the same promoter (e.g., within 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. When necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences to enhance recombinant expression. An expression vector encoding an anti-NPM1 antibody can be inserted into a cell for expression using any technique known in the art (e.g., chemical transfection, viral transfection). The cell can be a cell that can be cultured using techniques known in the art. The cell can be any cell type capable of expressing a heterologous antibody sequence, such as 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 the 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.

[0103] 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.

[0104] In some embodiments, the conjugate described herein comprises an anti-NPM1 antibody linked to a drug. A conjugate comprising an anti-NPM1 antibody conjugated to a drug may be referred to as an "ADC". In some embodiments, the drug is a small molecule. The term "small molecule" refers to a molecule 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, and heterocycles, etc.). In some embodiments, the molecular weight of a 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 a small molecule is at least about 100 g / mol, at least about 200 g / mol, at least about 300 g / mol, at least about 400 g / mol, at least about 500 g / mol, at least about 600 g / mol, at least about 700 g / mol, at least about 800 g / mol, or at least about 900 g / mol, or at least about 1,000 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and up to about 500 g / mol) are also possible.

[0105] 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, ansatrienins, ansamitocin P-3, aphidicolin, apo- putolysin, 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, cyclopa 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, helvoxacin Sidiene, 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, taltobrine, 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 include another drug (e.g., a chemotherapy drug) known in the art to be cytotoxic or cytostatic to a particular cell type (e.g., a cancer cell).

[0106] In some embodiments, the conjugate described herein comprises an anti-NPM1 antibody linked to an imaging agent. The imaging agent may be a small molecule. The imaging agent has one or more properties by which the presence of the agent can be detected. For example, the imaging agent described herein may be a luminescent imaging agent (e.g., luciferase) or a fluorescent imaging agent (e.g., sodium fluorescein, methylene blue, indocyanine green (ICG)). It can be detected by measuring the emission at one or more specific wavelengths using techniques commonly known in the art. Alternatively, the imaging agent may be an agent detectable using magnetic resonance imaging (MRI), such as, but not limited to, gadolinium-diethylenetriamine (Gd-DTPA).

[0107] In some embodiments, the anti-NPM1 antibodies described herein are conjugated to more than one molecule of an agent (e.g., a drug, an imaging agent). That is, the antibody and the agent are conjugated together in a ratio greater than 1:1. In some embodiments, the ratio between the antibody and the agent is between 1:1 and 1:10. In some embodiments, the ratio between the antibody and the agent is 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0108] 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 a therapeutic or diagnostic benefit of the association between the two entities is realized.

[0109] 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, incorporated herein by reference.

[0110] In some embodiments, the linker is non-cleavable. In some embodiments, the non-cleavable linker is a linker that is not cleaved in the subject's systemic circulation. 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.

[0111] Any of the conjugates described herein can be synthesized using methods known in the art. See, for example, Yao et al., Int J Mol Sci. 2016 Feb 2;17(2).

[0112] ADCs comprising anti-NPM1 antibodies conjugated to drugs are advantageous for therapeutic use, in part because the drugs (e.g., chemotherapy drugs) are toxic and can be targeted to specific cell types expressing 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%, at least 99% compared to the drug in its free form. Conjugates comprising anti-NPM1 antibodies conjugated to imaging agents are advantageous, as they are specific to specific cells and tissues expressing cell surface NPM1 (e.g., NPM1-expressing cancer cells), allowing greater specificity of the imaging agent to cells or tissues than can be achieved by the free imaging agent. Such conjugates are useful for detecting the presence and abundance of cells and tissues expressing cell surface NPM1 (e.g., NPM1-expressing cancer cells) and can be administered, for example, prior to, during, or after any course of treatment.

[0113] 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 treating cancer, such as a radioisotope that is useful for treating 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 treating 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 can be reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% compared to the radioisotope in its free form.

[0114] 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 compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio. A "pharma-ceutically 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 agent 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 components of the formulation and not toxic to the patient's tissues (e.g., physiologically compatible, sterile, physiological pH, etc.). The term "carrier" denotes a natural or synthetic organic or inorganic ingredient with which the active ingredient is combined to facilitate 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) cellulose acetate, cellulose esters, cellulose esters, cellulose acetate ... ) polyols, such as glycerin, 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 substances 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.

[0115] Pharmaceutical compositions can be conveniently presented in unit dosage form and can be prepared by any method well known in the art of pharmacy.The term "unit dose" refers to a physically discrete unit suitable as a unitary dosage for administration to a subject when referring to the pharmaceutical compositions of the present disclosure, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect, in association with a required diluent; i.e., carrier or base.

[0116] 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, which may be formulated according to known art using suitable dispersing or wetting agents and suspending agents. Injectable preparations may also be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, for example as solutions in 1,3 propanediol or 1,3 butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution USP, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as solvents or suspending media. For this purpose, any non-irritating, fixed oil may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. 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.

[0117] 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.

[0118] In some embodiments, the compositions provided herein (e.g., pharmaceutical compositions administered to a subject) must be sterile. Sterility is readily achieved 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. A variety of preservatives are well known, including, for example, phenol and ascorbic acid. The pharmaceutical composition will typically 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 6-8, although in some instances, higher or lower pH values ​​may also be appropriate.

[0119] Administration of anti-NPM1 conjugates The present 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. In some embodiments, a method is provided for assessing the presence of 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.

[0120] 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).

[0121] 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 thereof (e.g., pharmaceutical composition) described herein in or to 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 a composition thereof (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 encompass administering an agent (e.g., antibody, conjugate) or a composition thereof (e.g., pharmaceutical composition) described herein to a subject with the intent of curing, reversing, mitigating, alleviating, modulating, resolving, ameliorating, improving or affecting a disease, a symptom of a disease, or a predisposition to a disease in the subject. Methods for treating a disease may encompass prophylaxis, in which an agent is administered to a subject for the purpose of preventing the onset of the disease, e.g., in a subject not known to have the disease but who may develop or be at risk of developing the disease in the future.

[0122] 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) that is sufficient to induce a desired biological response in a subject, for example, to alleviate one or more symptoms of a disease (e.g., cancer). A therapeutically effective amount can be an amount administered to a subject, either alone or in combination with one or more other agents. As will be recognized by those skilled in the art, the effective amount will vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the administered agent, 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 allied factors within the knowledge and opinion of the medical practitioner to determine. These factors are well known to those skilled in the art and can be addressed with a degree of routine experimentation. It is generally preferred that the maximum dose of an individual agent (e.g., an 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 a medical professional may select a lower dose (e.g., a minimum effective dose) to mitigate any potential risks of the treatment, such as side effects of the treatment.

[0123] In some embodiments, for a normal weight adult subject, a dose of an agent (e.g., an antibody or conjugate described herein) may be administered that ranges from about 0.01 to 1000 mg / kg. In some embodiments, the dose is between 1 and 200 mg. The specific dosage regimen, i.e., dose, timing, and repetition, will depend on the particular subject and the subject's medical history, as well as the properties of the agent (such as the pharmacokinetics of the agent) and other considerations that are well known in the art.

[0124] 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 suspend, hinder, slow down, delay, stabilize, and / or postpone the progression of the disease in a subject. Delaying the progression of a disease may include delaying or preventing the spread of the disease occurring in a subject, such as delaying or preventing the metastasis of the cancer occurring in a subject to one or more organs or tissues that have not yet been affected by the cancer. This delay may be 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 the disease in a given time frame and / or reduces the degree of symptoms in a given time frame, compared to the absence of such a method. The comparison is typically based on a clinical study using a sufficient number of subjects to give a statistically significant result.

[0125] "Onset" or "progression" of a disease (e.g., cancer) refers to the initial manifestation and / or subsequent progression of the disease in a subject. 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 a disease. As used herein, "onset" or "onset" of a disease includes the initial onset of a disease and the recurrence of a disease (i.e., in a subject who previously had the disease).

[0126] 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 a minor) 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 or 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 animal or an adult animal. The non-human animal may be a transgenic or genetically engineered animal.

[0127] In some embodiments, the subject is a companion animal (e.g., a pet or service animal). As used herein, "companion animal" refers to pets and other domestic animals. 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 is a research animal. Non-limiting examples of research animals include rodents (e.g., rats, mice, guinea pigs, and hamsters), rabbits, or non-human primates.

[0128] 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 compared to non-cancerous cells), which may further be characterized by changes in 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 chemotherapy drug). In some embodiments, NPM1-expressing cancer is a solid (tissue) or liquid (body 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 can be cancer such as leukemia, carcinoma, or lymphoma resulting from previous treatment with chemotherapy or radioisotope. In some embodiments, the NPM1-expressing cancer is therapy-related AML (t-AML), or a secondary malignancy of non-Hodgkin's lymphoma.

[0129] In some embodiments, the subject has been previously treated for NPM1-expressing cancer. In some embodiments, the subject has NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapy drugs). NPM1-expressing cancer is said to be resistant to a treatment (e.g., treatment with one or more chemotherapy drugs) when the treatment cannot effectively kill and / or inactivate the cancer cells, especially when the treatment was previously effective for killing and / or inactivating the cancer cells, for example, due to genetic and / or epigenetic changes occurring in the cancer that cause 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. In some embodiments, NPM1-expressing cancer that is resistant to one or more treatments (e.g., treatment with one or more chemotherapy drugs) expresses NPM1 on the surface of cancer cells as a result of a previous treatment (e.g., previous treatment with one or more chemotherapy drugs) to which the cancer is resistant or has become resistant. 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 previous treatment (e.g., previous treatment with one or more chemotherapeutic drugs) to which the cancer is resistant or has become resistant.

[0130] Depending on the type of disease (e.g., cancer) or the site of the disease (e.g., cancer) to be treated, 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. The antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) can be administered systemically (i.e., throughout the body) or locally (i.e., to one or more specific organs, tissues, or locations of the body). The antibody, conjugate, or composition thereof (e.g., pharmaceutical composition) can be administered via any conventional route. For example, it can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, bucally, vaginally, intraperitoneally, or via an implanted reservoir. As used herein, the term "parenteral" 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 the subject via an injectable depot administration route, e.g., using 1, 3, or 6 month depot injection 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.

[0131] 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.

[0132] In some embodiments, the administration results in a reduced growth of the NPM1-expressing cancer cells of the subject, compared to the growth of the NPM1-expressing cancer cells of the subject in the absence of the administration. 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, the administration results in a reduced growth of the NPM1-expressing cancer cells of the subject by at most 5%, at most 10%, at most 15%, at most 20%, at most 25%, at most 30%, at most 35%, at most 40%, at most 45%, at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, at most 100%, at most 2-fold, at most 3-fold, at most 4-fold, at most 5-fold, at most 6-fold, at most 7-fold, at most 8-fold, at most 9-fold, or at most 10-fold, compared to the growth of the NPM1-expressing cancer cells in the absence of the administration.

[0133] In some embodiments, the administration results in increased cell death of the NPM1-expressing cancer cells of the subject, compared to the cell death of the NPM1-expressing cancer cells of the subject in the absence of the administration.As used herein, "cell death" refers to the increased rate of cell death that occurs in the NPM1-expressing cancer cells of the subject, through any pathway by which the cell ceases to survive, such as, but not limited to, apoptosis, autophagy, necrosis, and entosis. In some embodiments, administration results in increased cell death of NPM1-expressing cancer cells of the subject by up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 60%, up to 70%, up to 80%, up to 90%, up to 100%, up to 2-fold, up to 3-fold, up to 4-fold, up to 5-fold, up to 6-fold, up to 7-fold, up to 8-fold, up to 9-fold, or up to 10-fold compared to cell death of NPM1-expressing cancer cells of the subject in the absence of administration.

[0134] In some aspects, the disclosure further provides a method for assessing the presence of (detecting) 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 imaging agent described herein, or a composition thereof (e.g., a pharmaceutical composition).

[0135] In some embodiments, such methods include administering an effective dose of an anti-NPM1 antibody described herein, a conjugate between an anti-NPM1 antibody described herein and an imaging agent, or a composition thereof (e.g., a pharmaceutical composition) to a subject in need thereof, imaging the antibody or conjugate, and determining the presence of an NPM1-expressing cancer in the subject based on the level and location of the antibody or conjugate imaged in the subject. As recognized by those skilled in the art, the effective amount will vary depending on the specific disease to be detected, the severity of the disease, individual subject parameters including age, physical condition, size, sex, and weight, the duration of detection, the specific administration route, and allied factors within the knowledge and opinion of medical professionals. In some embodiments, the NPM1-expressing cancer is a cancer in which WT NPM1 and / or mutant NPM1 is expressed on the surface of cancer cells. In some embodiments, the NPM1-expressing cancer is a solid (tissue) or liquid (body fluid, e.g., blood) cancer. In some embodiments, the NPM1 expressing cancer is selected from blood cancer, lung cancer, breast cancer, brain cancer, digestive 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. In some embodiments, the NPM1 expressing cancer is therapy-related AML (t-AML) or secondary malignancy of non-Hodgkin's lymphoma.

[0136] In some embodiments, the imaging carried out is luminescence imaging or fluorescence imaging.The imaging method can be selected based on the type of imaging agent administered to subject, for example, the imaging agent that is conjugated or bound to the anti-NPM1 antibody administered to subject.In some embodiments, the imaging is magnetic resonance imaging (MRI).

[0137] In some embodiments, the method of evaluating the presence of NPM1-expressing cancer in a subject (detecting NPM1-expressing cancer) further comprises administering to the subject an effective amount of an anti-NPM1 antibody, a conjugate comprising an anti-NPM1 antibody and an agent (e.g., drug, radioisotope) described herein, or a composition thereof (e.g., pharmaceutical composition), for example, when it is determined that an NPM1-expressing cancer exists in the subject. The effective amount of an anti-NPM1 antibody, a conjugate comprising an anti-NPM1 antibody and an agent (e.g., drug, radioisotope), or a composition thereof (e.g., pharmaceutical composition) to be administered to the subject may depend on a variety of factors. These are within the knowledge and opinion of a medical professional to determine, including the amount and location of the NPM1-expressing cancer that is determined to exist in the subject.

[0138] In further aspect, the present disclosure provides an ex vivo method for assessing the presence of NPM1-expressing cancer in a subject (detecting NPM1-expressing cancer).For example, some aspects of the present disclosure relate to a method for assessing the presence of NPM1-expressing cancer in a subject (detecting NPM1-expressing cancer), which comprises: collecting a biological sample from a subject in need thereof (or providing a biological sample previously obtained from such a subject); contacting the biological sample with an anti-NPM1 antibody or a conjugate between an anti-NPM1 antibody and an imaging agent as described herein; analyzing the binding between the antibody or conjugate and NPM1-expressing cancer cells in the biological sample; and determining the presence of NPM1-expressing cancer in the subject based on the level of binding between the conjugate and NPM1-expressing cancer cells analyzed in the sample.

[0139] In some embodiments, the NPM1 expressing cancer is a cancer in which WT NPM1 and / or mutant NPM1 are expressed on the surface of cancer cells. In some embodiments, the NPM1 expressing cancer is a solid (tissue) or liquid (body 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, 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 a metastatic cancer. In some embodiments, the NPM1 expressing cancer is a therapy-related cancer or a secondary malignancy. In some embodiments, the NPM1-expressing cancer is therapy-related AML (t-AML), or a secondary malignancy of non-Hodgkin's lymphoma.

[0140] In some embodiments, the biological sample collected from the subject comprises a tissue biopsy, a blood sample, a serum sample, a plasma sample, a saliva sample, a sputum sample, a urine sample, a fecal sample, a lymphatic fluid sample, a synovial fluid sample, a cerebrospinal fluid sample, or an interstitial fluid sample.In some embodiments, the tissue biopsy is a tumor biopsy.In some embodiments, the biological sample collected from the subject is a blood sample, a serum sample, or a plasma sample.

[0141] In some embodiments, the analysis performed is luminescence analysis or fluorescence analysis. The method of analysis can be selected based on the type of imaging agent conjugated to the anti-NPM1 antibody described herein. For example, in some embodiments, the anti-NPM1 antibody is conjugated to a luminescence imaging agent or a fluorescence imaging agent. In some embodiments, the analysis is performed via flow cytometry. Various techniques for assessing the binding between a labeled antibody (e.g., an anti-NPM1 antibody conjugated to a luminescence imaging agent or a fluorescence imaging agent) and cells in a sample (e.g., NPM1-expressing cancer cells) using flow cytometry are well known in the art (see, for example, McKinnon, KM, “Flow Cytometry: An Overview.” Curr Protoc Immunol. 2018; 120:5.1.1-5.1.11, the contents of which are incorporated herein by reference).

[0142] Alternative approaches for analyzing binding by antibodies or antibody conjugates and cells in a sample are also well known in the art and are further contemplated herein. For example, the binding between the antibodies or conjugates described herein and NPM1-expressing cancer cells in a sample can be analyzed by enzyme-linked immunosorbent assay (ELISA) instead. In some embodiments, the binding between the antibodies or conjugates described herein and NPM1-expressing cancer cells in a sample is analyzed by cell-based ELISA using techniques well known in the art (see, for example, Molnar E., "Cell-Based Enzyme-Linked Immunosorbent Assay (Cell-ELISA) Analysis of Native and Recombinant Glutamate Receptors." Methods Mol Biol. 2019; 1941:47-54, the contents of which are incorporated herein by reference). In some embodiments, the total level of NPM1 (WT or mutant NPM1) expressed by cells (e.g., cancer cells) in a sample and / or the level of NPM1 (WT or mutant NPM1) localized on the surface of cells (e.g., cancer cells) in a sample can be further analyzed using techniques well known in the art, such as cell membrane fractionation followed by immunoblotting for NPM1, e.g., by using one or more anti-NPM1 antibodies described herein.

[0143] In some embodiments, the method of assessing the presence of NPM1-expressing cancer in a subject (detecting NPM1-expressing cancer) further comprises administering to the subject an effective amount of an anti-NPM1 antibody, a conjugate comprising an anti-NPM1 antibody and an agent (e.g., a drug, a radioisotope) described herein, or a composition thereof (e.g., a pharmaceutical composition), for example, when it is determined that an NPM1-expressing cancer exists in the subject. The effective amount of an anti-NPM1 antibody, a conjugate comprising an anti-NPM1 antibody and an agent (e.g., a drug, a radioisotope), or a composition thereof (e.g., a pharmaceutical composition) to be administered to the subject may depend on a variety of factors. These are within the knowledge and expertise of a medical professional to determine, including the amount and location of the NPM1-expressing cancer that is determined to exist in the subject.

[0144] Those skilled in the art will recognize that the treatment and detection methods described herein may also be suitable for treating or detecting 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.

[0145] 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 to the cytosol of each cell type, significant quantities 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 various levels of NPM1 on the surface of each cell type assayed.

[0146] These results suggest that antibodies specific for WT and / or mutant NPM1 can be used to deliver cytotoxic payloads to NPM1-expressing cancers, for example in patients 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, particularly on the surface of cells from which the cancer originally originated. 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.

[0147] 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).

[0148] 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 of 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 a biotinylated anti-mouse isotype control (Strep-ZAP-IgG) (Thermo Scientific # 31800). After binding, Strep-ZAP-IgG complexes were added to the cells and the cells were left to grow at 37° C. for either 24 or 48 hours. After each time point, the cells were harvested, washed with PBS, and stained with Annexin V to detect apoptosis and with DAPI to detect whether the cells remained viable. After staining, the 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.

[0149] Although these studies were performed in human cell lines, it should be noted that due to the high conservation of NPM1, NPM1 antibodies can be used to target endogenous NPM1 on the surface of non-human cell types. 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 conservation also suggests that results obtained by testing the effects of anti-NPM1 antibodies or ADCs, for example, in mouse cells or mice, would be expected to extend to humans.

[0150] Example 3 - Antibodies detect WT and mutant NPM1 on the surface of human 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, live cells were assayed to observe cell surface-localized NPM1. In other cases, fixed cells were assayed to observe intracellular and cell surface levels of NPM1. Significant levels of NPM1 were observed on the surface of live OCI-AML3 cells. However, NPM1 was not robustly observed in significant quantities 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 of 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). Using live and fixed cell flow cytometry, TY1 signals were found to be present on the surface of cells expressing either WT-NPM1-TY1 or NPM1c-TY1, confirming that NPM1c protein can also be presented on the cell surface (Figure 6A-6E). An orthogonal immunofluorescence detection strategy confirms the surface distribution of TY1-tagged NPM1 molecules (Figure 6F).

[0151] Considering 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. As determined by FACS analysis, the isolated Ab1 anti-NPM1 (Figure 2A) and Ab2 anti-NPM1 (Figure 2B) antibodies were able to bind to surface-localized NPM1 on primary mouse MLL-rearranged AML cells (Figure 7A) and primary mouse NPM1c AML cells (Figure 7B), respectively. Furthermore, the antibodies bound to NPM1 on human patient-derived xenograft (PDX) cells transplanted into a mouse model without binding to host mouse bone marrow cells (Figures 8A-8D). These results, in combination 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 non-cancerous tissues. These antibodies can also be used in other applications. For example, antibodies may alternatively be conjugated with imaging agents to include diagnostic agents, which in turn may be used to locate and measure the relative abundance of cancer cells in a patient.

[0152] 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 comparison, 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. NPM1 inside the cells is in the nucleolar form, while NPM1 on the cell surface forms clusters (Figure 9A). To examine whether the biochemical fractionation is specific, other RNA-binding proteins (RBPs) were examined, including heterogeneous nuclear ribonucleoprotein U (HNRNPU), nucleolar RNA helicase 2 (DDX21), dolichol diphosphooligosaccharide protein glycosyltransferase subunit 1 (RPN1), and RIO kinase 1 (RIOK1). RBPs were measured in the cytosol and membrane compartments of various cell lines, including 293, A549, K562, and AML3. Results from biochemical fractionation and Western blot (WB) indicate that some, but not all, highly abundant cytosolic proteins, RBPs, are found in the membrane fractions of various cells (Figure 9B).

[0153] In addition to assessing cell surface expression of NPM1 on human cells, primary mouse 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 mouse AML cells (Figures 10A-10B).

[0154] We also examined cell surface localization of NPM1 using Western blot. Anti-NPM1 captures full-length NPM1 from the cell membrane fraction (Fig. 6A). Cell surface NHS-biotinylation (surface protein only) followed by anti-NPM1 IP from the membrane fraction selectively isolates a biotinylated band at the molecular weight of NPM1 (Fig. 6B).

[0155] 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 nonrandom pattern of NPM1 markers suggests a highly regulated cell biology process (Figures 12A-12B). Super-resolution microscopy performed on PANC1 cell line (pancreatic cancer) also demonstrated that NPM1 forms nanoclusters on the surface of cancer cells (Figure 12B).

[0156] 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 on the cell surface, NPM1 forms nanoclusters on various human cancer cell lines, including acute myeloid leukemia and pancreatic cancer.

[0157] Example 6: The Ab2.2 antibody targets NPM1. An antibody having a heavy chain (Ab2.2) (SEQ ID NO: 44) and a light chain (Ab2.2) (SEQ ID NO: 45), hereafter "Ab2.2", was generated and used to target NPM1 on the surface of human and non-human cell types.

[0158] The ability of Ab2.2 to bind to NPM1 on the cell surface of cancer cells (OCI-AML3 cells) was tested using both live cell microscopy and 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 Ab2.2 stain surface puncta. Thus, Ab2.2 performs similarly to the commercially available NPM1 antibody on the cell surface (Figure 13). Fixed and permeabilized cell microscopy shows that the commercially available NPM1 antibody results in nucleolar and cytoplasmic staining, while the 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 commercially available NPM1 antibody (Figure 13).

[0159] 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), bone marrow 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 14A).

[0160] 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 14B).

[0161] 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 were used. Cells were isolated from bone marrow of 12 acute myeloid leukemia (AML) patients (Figure 15) and 15 AML patients (Figure 17) and analyzed using flow cytometry (Figures 16A-16B and Figures 18A-18C). Results show that Ab2.2 binds strongly to AML bone marrow blasts (Figures 16A-16B, patient 5) and NPM1c AML bone marrow (Figures 18D-18E). Furthermore, NPM1c blasts are low CD34, 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 18D-18E). Results from 12 AML patients in Figure 15 indicate that Ab2.2 strongly stains blasts agnostic to mutation status, disease status, or prior treatment. Results from 15 AML patients in Figure 17 show that Ab2.2 strongly stains blasts. Ab2.2 also stains strongly in NPM1c patients whose LSCs are CD34-, thus Ab2.2 will target leukemia-initiating cells.

[0162] 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 received weekly treatments of Ab2.2. There were five mice per group. Target groups received Ab2.2 administered by IP injection at doses of 2.5 mg / kg, 5 mg / kg, or 10 mg / kg. The control group received 5 mg / kg IgG (Figure 19A). One dose was delivered per week, for a total of four doses. Weekly blood draws for sample collection were performed on days 1, 7, 14, 20, and 27 (D) of treatment. 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 19B).

[0163] Example 9: Ab2.2 treatment improves survival in a mouse model of AML without affecting 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 with 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, as well as molecular phenotyping (Figure 20A). The primary mouse cells used were NPM1c / Flt3-ITD AML, and a syngenic 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 20B-20D). 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 20G). The survival study was terminated at day 100 due to protocol-related causes, not toxicity or AML relapse.

[0164] 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 treatments of Ab2.2 at a dose of 5 mg / kg. Administration occurred by IP injection. Weekly blood sampling was performed and overall survival and molecular phenotyping of mice was performed (Figure 21A). 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 (Figure 21B). The mean survival of the control group was about 20 days compared to about 55 days in the target group (Figure 21C). 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 lead to a survival benefit with Ab2.2 treatment.

[0165] Spleen, lung, and liver samples collected 3 days after the first dose of Ab2.2 treatment in efficacy model 2 were compared between the control (IgG) and Ab2.2-treated groups. The spleens, lungs, and livers of Ab2.2-treated mice all weighed less than control mice (Figure 22B). These results indicate that a single dose of Ab2.2 results in a robust reduction in organ weights, indicative of tumor clearance.

[0166] Efficacy model 2 was used to assess LSC targeting of Ab2.2 in secondary recipients as well (Figure 22A). 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, graft examination, and LSC function measurements were performed. 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 22C). The extended survival in 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 22D) and the MLL-AF9 model had low expression of NPM1 on the surface (Figure 22E). WBC counts were reduced in Ab2.2-treated mice and PLTs were increased (Figure 22F). Further analysis of WBC over time demonstrated that WBC remained lower in Ab2.2-treated mice compared to IgG treatment over time (Figure 22G). Spleens of Ab2.2-treated secondary recipients weighed less than IgG-treated mice (Figure 22H). 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 BM and PB, there was a reduction in AML% in the Ab2.2-treated group compared to the IgG control (Figure 22I). These results indicate that Ab2.2 targets the tumor after only one dose.

[0167] 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 weekly via IP injection, 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. Routine blood cell counts, animal phenotyping, and measurement of adverse / toxic effects were performed (Figure 25A). The results show that no significant differences were observed between the weights, WBC counts, HGB levels, or PLT levels of IgG- and Ab2.2-treated mice between day 1 (D) and D27 (Figure 25B). Thus, WT mice do not exhibit observable effects of Ab2.2 treatment.

[0168] Example 10: The effect of Ab2.2 on survival is dependent on the immune system. 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 23A). The transplanted primary mouse cells were MLL-AF9 / Flt3-ITD AML. The mouse model used was an immunodeficient 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 mean survival of the control and Ab2.2-treated groups was approximately 30 days (Figure 23B). Thus, Ab2.2 does not provide a survival benefit without an intact immune system.

[0169] 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 24A). Implanted cells were from MC38 mouse colon 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. Results of tumor burden calculations demonstrate that Ab2.2 reduces tumor volume on days 10 and 13 (Figures 24B-24C).

[0170] 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 that there is high surface detection of Npm1c even on Dmnt3a R882H single mutant cells (Figure 27).

[0171] Example 13: Ab2.2 binds to human tumors in vitro. To assess the ability of Ab2.2 to bind to human tumors, various types of in vitro models were examined 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 26A-26O). The results show that Ab2.2 binds to a diverse set of human tumor models in vitro.

[0172] Example 14: Ab2.2-saporin ADCs exhibit in vitro activity. 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. 28). Thus, NPM1 may serve as an ADC target in at least some models.

[0173] Example 15: Ab2.2 treatment extends the life span of mice engrafted with a human AML cell line. The ability of Ab2.2 to extend life span in a mouse model xenografted with a human AML cell line was assessed. SCID CB17 mice received transplants of OCI-AML3 cells (human AML). The engrafted mice then received weekly treatments of 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 4 doses. Mice were assessed for overall survival and molecular phenotyping (Figure 29A).

[0174] Antibody binding was assessed using flow cytometry (Figure 29B). Results of survival assays demonstrate that Ab2.2-treated grafted mice survived significantly longer than IgG-treated mice, surviving for more than 40 days after transplantation (p=0.0015) (Figure 29C). Thus, extended life span is observed in Ab2.2-treated natural killer (NK) cells and complement, as well as in mice grafted with human AML cell lines.

[0175] Example 16: Ab2.2 treatment extends the life span of mice engrafted with human AML-PDX cell lines. The ability of Ab2.2 to extend life span in a mouse model xenografted with human AML-PDX was assessed. SCID CB17 mice received transplantation of PDX (MLL-R) cells (human AML-PDX). The engrafted mice then received weekly treatments of 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 4 doses. Mice were assessed for overall survival and molecular phenotype analysis (Figure 30A).

[0176] Antibody binding was assessed using flow cytometry (Figure 30B). Survival assay results demonstrated that Ab2.2-treated grafted mice survived for more than 60 days after transplantation, significantly longer than IgG-treated mice (p=0.0023) (Figure 30C). Thus, extended life span is observed in Ab2.2-treated natural killer (NK) cells and complement, as well as mice grafted with human AML-PDX cell lines.

[0177] 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.

[0178] 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 should be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.

[0179] 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 methods 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.

[0180] 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.

[0181] 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 be able to take any particular value within the claimed range in some embodiments, 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 be able to take any subrange within the given range, 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.

[0182] 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.

[0183] 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 expressly described herein.

Claims

1. Antibodies, next: (1) Heavy chain (HC) complementarity-determining region (CDR) 1 containing the amino acid sequence NIFVH (SEQ ID NO: 1), HC CDR2 containing the amino acid sequence KIDPANDNTKFAPNFQG (SEQ ID NO: 2), and HC CDR3 containing the amino acid sequence DSSGYDAVDY (SEQ ID NO: 3); and Light chain variable region containing 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); or (2) Heavy chain (HC) complementarity determining region (CDR) 1 containing the amino acid sequence SYAMS (SEQ ID NO: 15), HC CDR2 containing the amino acid sequence AISGSGGSTYYADSVKG (SEQ ID NO: 16), and HC CDR3 containing the amino acid sequence WRNNAFDY (SEQ ID NO: 17); and Light chain variable region containing 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, which includes the antibody.

2. (1) The heavy chain variable region contains the amino acid sequence described in SEQ ID NO: 28, and / or the light chain variable region contains the amino acid sequence described in SEQ ID NO: 29; or (2) The heavy chain variable region contains the amino acid sequence described in SEQ ID NO: 30, and / or the light chain variable region contains the amino acid sequence described in SEQ ID NO:

31. The antibody according to claim 1.

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

4. The antibody according to claim 3, 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).

5. The antibody according to claim 3, 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.

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

7. The antibody according to claim 1, further comprising a heavy chain constant region, wherein the heavy chain constant region comprises an amino acid sequence described in SEQ ID NO: 32 or SEQ ID NO:

46.

8. The antibody according to claim 1, further comprising a light chain constant region, wherein the light chain constant region comprises an amino acid sequence described in SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO:

47.

9. Antibodies, (1) A heavy chain containing the amino acid sequence of SEQ ID NO: 35 and / or a light chain containing the amino acid sequence of SEQ ID NO: 37; (2) A heavy chain containing the amino acid sequence of SEQ ID NO: 36 and / or a light chain containing the amino acid sequence of SEQ ID NO: 38; or (3) A heavy chain containing the amino acid sequence of SEQ ID NO: 44 and / or a light chain containing the amino acid sequence of SEQ ID NO: 45 The antibody according to claim 1, comprising:

10. A composition comprising the antibody described in claim 1 and a pharmacologically acceptable excipient.

11. A nucleic acid or set of nucleic acids encoding the antibody described in claim 1.

12. A cell comprising the nucleic acid or nucleic acid set described in claim 11.

13. A method for producing antibodies, wherein the method is as follows: (i) Culturing the cells described in claim 12 in a culture medium under conditions sufficient for antibody expression; (ii) Collecting cultured cells and / or culture media; and (iii) Isolating antibodies from cultured cells and / or culture media. The method, including the method described above.

14. A conjugate comprising the antibody described in claim 1, conjugated to a drug.

15. The conjugate according to claim 14, wherein the drug is a pharmacokinetic.

16. Drugs, (1) 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, Ixabepyrone, JW55, Lactacystine, Ruisol A, Maytancinol Meltansine (DM1), Maytansine DM3, Labtansine (DM4), Maytansinoid AP-3, Mekelcarmycin A, Menthalcin, Methotrexate, 6-Mercaptopurine, Microcholine B, Microcystine LR, Mitoxantrone, Muscotoxin A, Myosevelin, Mytoxin B, Nelarabine, Nemorubicin, Noquarin 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, tubulicin A, tubulicin M, tubulicin IM-1, tubulicin IM-2, tubulicin IM-3, venetoclax, and vincristine; or, (2) Saporin, daunorubicin, venetoclax, or azacitidine The conjugate according to claim 15.

17. The conjugate according to claim 15, wherein the antibody-to-drug ratio is between 1:1 and 1:

10.

18. The conjugate according to claim 14, wherein the drug is a radioactive isotope.

19. The conjugate according to claim 18, wherein the radioactive isotope is selected from the group consisting of iodine-131, rhenium-188, yttrium-90, bismuth-213, and actinium-225.

20. The conjugate according to claim 18, wherein the drug is a contrast agent.

21. The contrast agent (1) Luminescent contrast agent or fluorescent contrast agent; (2) A drug detectable by magnetic resonance imaging (MRI); or (3) Gadolinium-diethylenetriamine (Gd-DTPA), The conjugate according to claim 20.

22. Use of an antibody according to any one of claims 1 to 9 in the treatment of nucleophosphatine 1 (NPM1) expressing cancer.

23. 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) Select 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 use described in claim 22.

24. The use according to claim 22, wherein the antibody or conjugate is administered systemically or locally.

25. The use according to claim 22, wherein the subject is a mammal or a human.

26. Use of the antibody according to any one of claims 1 to 9 in the assessment of the presence of nucleophosphatine 1 (NPM1) expressing cancer.

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