Antibody specific to CD123 and antibody-drug conjugate, and use of the same

Abstract

To provide an antibody specific to CD123 and an antibody-drug conjugate, and use thereof.SOLUTION: The present invention provides an antibody that specifically binds to CD123. The present invention further relates to an immune conjugate (e.g., antibody drug conjugate or ADC) including the antibody, nucleic acid encoding the antibody, and a method for obtaining the antibody. The present invention further relates to a therapeutic method regarding use of the antibody and ADC for treating symptoms related to a cell expressing CD123 (e.g., cancer or autoimmune disease).SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] A reference to an array list This application has been filed electronically via EFS-Web and contains an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing entitled "PC72333_SEQ_LIST_ST25.txt" created on October 27, 2017, and having a size of 78KB. The sequence listing contained in this .txt file is a part of the present specification, which is hereby incorporated by reference in its entirety.

[0002] The present invention relates to antibodies, e.g., full-length antibodies, that specifically bind to CD 123. The present invention further relates to conjugates (e.g., antibody-drug conjugates or "ADCs") comprising CD 123 antibodies, compositions comprising CD 123 antibodies or conjugates thereof, and methods of using CD 123 antibodies or conjugates thereof to treat conditions associated with cells expressing CD 123, e.g., cancer. [Background technology]

[0003] CD123 is the alpha chain of the interleukin-3 receptor (IL-3Rα or IL3RAa), which forms a heterodimer with the common beta chain CD131 to help transmit the IL-3 signal. The biological role of IL-3 is to stimulate the survival and proliferation of pluripotent cells. CD123 is believed to be involved in stimulating the proliferation of acute myeloid leukemia (AML) cells and has a direct function in tumor biology. CD123 is expressed at high frequencies on leukemia stem cells (LSCs), a cell population associated with relapse in patients. In normal human tissues, CD123 expression is largely restricted to hematopoietic cells, especially plasmacytoid dendritic cells (pDCs), which constitute less than 0.4% of peripheral blood mononuclear cells in humans. As components of the innate immune system, pDCs produce large amounts of type 1 interferons (IFN-α / β) in response to viral and bacterial stimuli. Importantly, CD123 is not expressed on hematopoietic stem cells.

[0004] New cases of leukemia, lymphoma and myeloma are expected to account for 10.2% of the estimated 1,688,780 new cancer cases diagnosed in the United States in 2017. CD123 is expressed on cancer cells in a variety of hematological malignancies, including acute myeloid leukemia (AML), where its expression rate is greater than 80%. Examples of hematological cancer cells that express CD123 include blasts and leukemic stem cells. Diseases associated with CD123 expression include AML, myelodysplastic syndromes (MDS; low and high risk), acute lymphoblastic leukemia (ALL, all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myelogenous leukemia (CML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0005] Currently, treatments for these diseases include more than 50 individual drugs, and others are under investigation and in clinical trials. Radiation therapy is also commonly used to treat blood cancers, sometimes administered together with drug therapy. Immunotherapy, gene therapy, and personalized medicine are also used. However, these therapies can have significant side effects and adverse reactions. Thus, there is a need for new and improved treatments for blood cancers that express CD123 (IL-3Rα). Summary of the Invention

[0006] The invention disclosed herein is directed to antibodies that bind to CD123 and conjugates comprising antibodies that bind to CD123, such as antibody-drug conjugates (ADCs), and methods of preparing and using such antibodies and ADCs to treat disorders.

[0007] Provided herein are antibodies that specifically bind to CD123. In one embodiment, the present invention provides an isolated antibody that specifically binds to CD123, wherein the antibody comprises a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) of VH comprising the amino acid sequence of SEQ ID NO:6, 24, 32, 44, 51, or 64, and a light chain variable region (VL) comprising three CDRs of VL comprising the amino acid sequence of SEQ ID NO:17, 28, 39, 48, 57, or 71. In one embodiment, the VH can comprise (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7, 33, 52, or 65; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:8, 25, 34, 45, 53, or 66; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9, 35, 46, or 67. In some embodiments, the VL region can comprise (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18, 40, 58, or 72; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19, 42, 60, or 74; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 20, 42, 60, or 74. In some embodiments, the VH can comprise (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 25; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 20, 42, 60, or 74. In some embodiments, the VL can comprise (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH can comprise a variant thereof having one or several conservative amino acid substitutions at residues not within the sequence or CDRs depicted in SEQ ID NO:24, and / or the VL can comprise a variant thereof having one or several amino acid substitutions at amino acids not within the amino acid sequence or CDRs depicted in SEQ ID NO:28. In some embodiments, the antibody can comprise a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the VH can comprise a variant thereof having one or several conservative amino acid substitutions at residues not within the sequence or CDRs depicted in SEQ ID NO:24, and / or the VL can comprise a variant thereof having one or several amino acid substitutions at amino acids not within the amino acid sequence or CDRs depicted in SEQ ID NO:28. The light chain may comprise the sequence shown in SEQ ID NO:30 and the heavy chain may comprise the sequence shown in SEQ ID NO:27.

[0008] Also provided is an isolated antibody that specifically binds CD123 and comprises a heavy chain variable region produced by an expression vector having ATCC Accession No. PTA-124283 and a light chain variable region produced by an expression vector having ATCC Accession No. PTA-124284.

[0009] Also provided is an isolated antibody that specifically binds to CD123 and competes for binding to CD123 with an antibody comprising a heavy chain variable region (VH) comprising three CDRs, the VH comprising the amino acid sequence of SEQ ID NO:6, 24, 32, 44, 51 or 64; and a VL comprising three CDRs, the VL comprising the amino acid sequence of SEQ ID NO:17, 28, 39, 48, 57 or 71.

[0010] VH comprising the amino acid sequences of SEQ ID NOs:7, 8 and 9, and An isolated CD123 antibody comprising a VL comprising the amino acid sequence of NO:18, 19 and 20 is also provided.

[0011] Also provided is an isolated CD123 antibody comprising a VH comprising the amino acid sequence of SEQ ID NOs:7, 25, and 9 and a VL comprising the amino acid sequence of SEQ ID NOs:18, 19, and 20.

[0012] VH comprising the amino acid sequences of SEQ ID NOs: 33, 34 and 35, and SEQ Isolated CD123 antibodies comprising a VL comprising the amino acid sequence of ID NO:40, 41 and 42 are also provided.

[0013] VH comprising the amino acid sequences of SEQ ID NOs: 33, 45 and 46, and SEQ Isolated CD123 antibodies comprising a VL comprising the amino acid sequence of ID NO:40, 41 and 42 are also provided.

[0014] VH comprising the amino acid sequences of SEQ ID NOs: 52, 53 and 54, and SEQ Isolated CD123 antibodies comprising a VL comprising the amino acid sequence of ID NOs:58, 59 and 60 are also provided.

[0015] VH comprising the amino acid sequences of SEQ ID NOs: 65, 66 and 67, and SEQ Isolated CD123 antibodies comprising a VL comprising the amino acid sequence of ID NOs:72, 73 and 74 are also provided.

[0016] In certain embodiments, the CD123 antibodies described herein can contain an engineered acyl donor glutamine containing tag at a specific site. In an embodiment, the tag is Q, LQG, LLQGG (SEQ ID NO:77), LLQG (SEQ ID NO:78), LSLSQG (SEQ ID NO:79), GGGLLQGG (SEQ ID NO:80), GLLQG (SEQ ID NO:81), LLQ, GSPLAQSHGG (SEQ ID NO:82), GLLQGGG (SEQ ID NO:83), GLLQGG (SEQ ID NO:84), GLLQ (SEQ ID NO:85), LLQLLQGA (SEQ ID NO:86), LLQGA (SEQ ID NO:87), LLQYQGA (SEQ ID NO:88), LLQGSG (SEQ ID NO:89), LLQYQG (SEQ ID NO:90), LLQLLQG (SEQ ID NO:91), SLLQG (SEQ ID NO:92), NO:92), LLQLQ(SEQ ID NO:93), LLQLLQ(SEQ ID NO:94), LLQGR(SEQ ID NO:95), LLQGPP(SEQ ID NO:96), LLQGPA(SEQ ID NO:97), GGLLQGPP(SEQ ID NO:98), GGLLQGA(SEQ ID NO:99), LLQGPGK(SEQ ID NO:100), LLQGPG(SEQ ID NO:101), LLQGP(SEQ ID NO:102), LLQP(SEQ ID NO:103), LLQPGK(SEQ ID NO:104), LLQAPGK(SEQ ID NO:105), LLQGAPG(SEQ ID In some embodiments, the glutamine-containing tag can include an amino acid sequence selected from the group consisting of LLQGAP (SEQ ID NO:107) and LLQLQG (SEQ ID NO:108). In some embodiments, the glutamine-containing tag is LLQG (SEQ ID NO:78).

[0017] In some embodiments, the CD123 antibodies described herein can include an amino acid modification at position 222, 340, or 370. In some embodiments, the amino acid modification can be a lysine to arginine substitution. In some embodiments, the amino acid modification can be K222R.

[0018] In some embodiments, the CD123 antibody described herein may include a linker. In some embodiments, the linker is cleavable. In some embodiments, the linker is Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val-Cit- PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine. In some embodiments, the linker can be Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl).

[0019] In some embodiments, the antibody described herein comprises a constant region. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is an antibody of human IgG1, IgG2 or IgG2Δa, IgG3 or IgG4 subclass. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody comprises an N60G mutation.

[0020] Also provided is a conjugate comprising the CD123 antibody described herein conjugated to a drug. In some embodiments, the drug can be selected from the group consisting of cytotoxic drugs, immunomodulatory agents, imaging agents, therapeutic proteins, biopolymers and oligonucleotides. In some embodiments, the drug can be a cytotoxic drug. In some embodiments, the cytotoxic drug can be selected from the group consisting of anthracyclines, auristatins, camptothecins, combretastatins, CBI dimers, cyclopropylpyrroloindoline (CPI) dimers, CTI dimers, dolastatins, duocarmycins, enediynes, geldanamycins, indolinobenzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterins, spliceostatins, pladienolides, and their stereoisomers, isosteres, analogs or derivatives. In some embodiments, the cytotoxic agent can be a CPI dimer. In some embodiments, the CPI dimer is CPI-8314. In some embodiments, the CPI dimer can have the following structure:

[0021] [ka]

[0022] . In some embodiments, the cytotoxic agent can have the following IUPAC name: (8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-5-hydroxy-8-methyl-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl dihydrogen phosphate. In some embodiments, the cytotoxic agent can be C31H31Cl2N4O7P or a pharma- ceutically acceptable salt or solvate. In some embodiments, the cytotoxic agent can be in the trifluoroacetic acid (TFA) salt form: C31H31Cl2N4O7P·C2HF3O2.

[0023] In other embodiments, the cytotoxic agent is MMAD (monomethyl auristatin D), 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4- {(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), 0131 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), 2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide hyl-L-valinamide) or 0121 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide).

[0024] In some embodiments, the conjugate can comprise the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic agent). In some embodiments, the acyl donor glutamine-containing tag can comprise the amino acid sequence LLQG (SEQ ID NO:78), and the linker can comprise acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl. In some embodiments, the acyl donor glutamine-containing tag can be inserted into the antibody at positions E294-N297. In some embodiments, the conjugate can further comprise a lysine to arginine amino acid substitution (K222R) at position 222 of the antibody according to the EU index of Kabat numbering.

[0025] Also provided is a pharmaceutical composition comprising a therapeutically effective amount of a CD123 antibody described herein, or a CD123 ADC described herein, and a pharma- ceutically acceptable carrier. Also provided is an isolated polynucleotide comprising a nucleotide sequence encoding the CD123 antibody described herein. Also provided is a vector comprising such a polynucleotide.

[0026] Also provided are isolated host cells that recombinantly produce any of the CD123 antibodies described herein. Also provided are methods of producing antibodies, including culturing such host cells under conditions that result in the production of the antibody and isolating the antibody from the host cells or culture.

[0027] Also provided is a method of treating a condition associated with cells expressing CD123 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the CD123 antibodies or a conjugate of any of the antibodies described herein and a pharma- ceutically acceptable carrier. In some embodiments, the condition is cancer.In some embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), hairy cell leukemia, B cell non-Hodgkin's lymphoma (NHL), multiple myeloma, malignant plasma cell neoplasms, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, macroglobulienemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma lymphoma), Waldenström macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (lower limb type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma with inflammation, intravascular large B-cell lymphoma The cancer may be selected from the group consisting of AML, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma occurring in HHV8-associated multicentric Castleman disease, unclassified B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with intermediate characteristics between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-related lymphomas. In some embodiments, the cancer is AML.

[0028] Also provided is a method of inhibiting tumor growth or progression in a subject having malignant cells that express CD123, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the CD123 antibodies provided herein or a conjugate of any of the antibodies and a pharma- ceutically acceptable carrier.

[0029] Also provided is a method of inhibiting metastasis of malignant cells expressing CD123 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the CD123 antibodies provided herein or a conjugate of any of the antibodies and a pharma- ceutically acceptable carrier.

[0030] Also provided is a method of inducing tumor regression in a subject having malignant cells that express CD123, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any of the CD123 antibodies provided herein or a conjugate of any of the antibodies and a pharma- ceutically acceptable carrier.

[0031] In another aspect, the present invention provides an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein for treating a condition associated with CD123 expression (e.g., cancer or an autoimmune disorder) in a subject in need thereof. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprises a CD123 antibody or CD123 ADC described herein for inhibiting tumor growth or progression in a subject having malignant cells expressing CD123. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprises a CD123 antibody or CD123 ADC described herein for inhibiting metastasis of malignant cells expressing CD123 in a subject in need thereof is provided. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprises a CD123 antibody or CD123 ADC described herein for inducing tumor regression in a subject having malignant cells expressing CD123.

[0032] In another aspect, the present invention provides a CD123 antibody or CD123 ADC described herein for use in treating a condition associated with CD123 expression (e.g., cancer or autoimmune disorder) in a subject in need thereof. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inhibiting tumor growth or progression in a subject having malignant cells expressing CD123. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inhibiting metastasis of malignant cells expressing CD123 in a subject in need thereof. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inducing tumor regression in a subject having malignant cells expressing CD123.

[0033] In another aspect, the present invention provides the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for treating a condition associated with CD123 expression (e.g., cancer or an autoimmune disorder). In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inhibiting tumor growth or progression is provided. In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing CD123 is provided. In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inducing tumor regression is provided.

[0034] Also provided herein is a method for conjugating an antibody to AcLysValCitPABC-DMAE-CO_CPI-000638314 (AcLysPABC-CPI-8314). In some embodiments, the method includes preparing a composition comprising an antibody and AcLysPABC-CPI-8314 in a buffer comprising 30-100 mM KPO4 and 150-200 mM NaCl; adding bacterial transglutaminase to the composition; and incubating the composition to allow conjugation of the antibody to AcLysPABC-CPI-8314. In some embodiments, the pH of the composition is 7. In some embodiments, the composition comprises 0.5-2 units (U) of bacterial transglutaminase per mg of antibody. In some embodiments, the composition comprises 1 U of bacterial transglutaminase per mg of antibody. In some embodiments, AcLysPABC-CPI-8314 is present in a 10-fold molar excess over the antibody. In some embodiments, the composition is incubated overnight at 25° C. with continuous mixing. In some embodiments, the composition further comprises 7.5% (v / v) dimethylsulfoxide (DMSO). In some embodiments, the buffer comprises 30 mM KPO4 and 150 mM NaCl. In some embodiments, the buffer comprises 100 mM KPO4 and 200 mM NaCl. In some embodiments, the antibody is an anti-tumor antibody. In some embodiments, the anti-tumor antibody is a CD123 antibody. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows Western blot results from analysis of cells treated with IL-3 plus antibody ("Ab"). 7G3 is the benchmark antibody, 3D1, 18G3 and 16D6 are CD123 antibodies; 8.8 is a negative control antibody that does not bind to CD123. Levels of STAT5, phosphorylated STAT5 and actin were analyzed. [Diagram 2]FIG. 2 shows representative flow cytometry analyses demonstrating the percentage of tumor cells remaining in peripheral blood (left graph) and bone marrow (right graph) in animal models following treatment with either vehicle or CD123-ADC (CD123-18G3-CPI). [Diagram 3] FIG. 3 shows a graph summarizing the length of survival (in days) of animals treated with either vehicle or CD123-ADC at the indicated doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The invention disclosed herein provides antibodies and antibody-drug conjugates (ADCs) that specifically bind to CD123 (e.g., human CD123). The invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods of making and using these antibodies. The invention also provides methods for treating disorders associated with CD123 expression in a subject, such as cancer or autoimmune disease.

[0037] general technique The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of those in the art. Such techniques are explained fully in the literature, for example in Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989). Cold SpringHarbor Press; Oligonucleotide Synthesis (MJ Gait, editor, 1984); Met hods inMolecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Gellis, editor, 1998) Academic Press; Animal Cell Culture (RI. Freshney, editor, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 19 98) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, editors, 1993 - 1998) J. Wiley and Sons; Methods in Enzy mology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir an d C.C. Blackwell, editors); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, editors, 1987); Current Protocols in Molecular Biology (F.M. Ausub el et al., editors, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., editors , 1994); Current Protocols in Immunology (J.E. Coligan et al., editors, 1991); Shor t Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Jan eway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach(D. Catty., editor, IRL Press, 1988 - 1989); Monoclonal antibodies: a prac tical approach(P. Shepherd and C. Dean, editors, Oxford University Press, 2000); U singantibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor LaboratoryPress), 1999); The Antibodies (M. Zanetti and J.D. Capra, editors Harwood AcademicPublishers, 1995).

[0038] definition An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only complete polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain antibodies (ScFv) and single domain antibodies (including, e.g., shark and camel antibodies) as well as fusion proteins comprising antibodies and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or subclasses thereof), and an antibody need not be of a particular class. Depending on the antibody amino acid sequence of the constant region of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0039] The term "antigen-binding fragment" or "antigen-binding portion" of an antibody, as used herein, refers to one or more fragments of an intact antibody that retain the ability to specifically bind to a given antigen (e.g., CD123). The antigen-binding function of an antibody may be performed by fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody are Fab; Fab'; F(ab')2; an Fd fragment consisting of the heavy chain variable region (VH) and CH1 domain; an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a single domain antibody (dAb) fragment (Wardet et al., 2002). al., Nature 341:544-546, 1989) and isolated complementarity determining regions (CDRs). .

[0040] An antibody, ADC, or polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., CD123 protein) is a term well understood in the art, and methods for determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for greater duration, and / or with greater affinity than it reacts or associates with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and / or for greater duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to a CD123 epitope is an antibody that binds to this epitope with greater affinity, avidity, more readily, and / or for greater duration than it binds to other CD123 epitopes or non-CD123 epitopes. It is also understood by reading this definition that, for example, an antibody (or moiety or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it can include exclusive binding). In general, reference to binding means preferential binding, but does not necessarily mean preferential binding.

[0041] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Maryland) and (2) an approach based on the variability of sequences between different species (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Maryland). (Bethesda, Florida); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273:927-948). , the CDRs can refer to CDRs defined by either approach, or by a combination of both approaches.

[0042] The "CDRs" of a variable domain are the amino acid residues within a variable region identified according to the Kabat, Chothia definition, both the Kabat and Chothia pool, AbM, contact, and / or conformational definitions, or any method of CDR determination known in the art. The CDRs of an antibody can be identified as hypervariable regions as originally defined by Kabat et al. See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., NIH Public Health Service, Washington, DC. The locations of CDRs can also be identified as structural loop structures as originally described by Chothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other approaches to CDR identification include, but are not limited to, the following: The approach is a compromise between Kabat and Chothia and includes the "AbM definition" obtained using Oxford Molecular's AbM antibody modeling software (now Accelrys®), or the "contact definition" of CDRs based on observed antigen contacts as described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. In another approach, referred to herein as the "conformational definition" of CDRs, the positions of CDRs can be identified as residues that make an enthalpic contribution to antigen binding. See, e.g., Makabeet al., Journal of Biological Chemistry, 283:1156-1166, 2008. See. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will still overlap with at least a portion of the Kabat CDRs, but they may be shorter or longer, taking into account predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly affect antigen binding. As used herein, CDRs can refer to CDRs defined by any approach known in the art, including combinations of approaches. The methods used herein can utilize CDRs defined according to any of these approaches. For any given embodiment that contains more than one CDR, the CDRs can be defined according to any of the Kabat, Chothia, extended, AbM, contact and / or conformational definitions.

[0043] The terms "polypeptide," "oligopeptide," "peptide," and "protein" are used interchangeably herein to refer to a chain of amino acids of any length, preferably relatively short (e.g., 10-100 amino acids). The chain can be linear or branched, it can include modified amino acids, and / or it can be interrupted by non-amino acids. The term also embraces amino acid chains that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides containing, for example, one or more analogs of an amino acid, including, for example, unnatural amino acids, as well as other modifications known in the art. It is understood that the polypeptides can exist as single chains or associated chains.

[0044] The antibodies of the present invention can be generated using techniques well known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, or a combination of such techniques or other techniques readily known in the art (see, e.g., Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA, et al, J. MoI. Biol.,373(4):924-40, 2007). (see ).

[0045] As known in the art, "polynucleotide" or "nucleic acid", as used interchangeably herein, refers to a chain of nucleotides of any length, including DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide can include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the chain. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a label component. Other types of modifications include, for example, "caps", substitution of one or more analogs of naturally occurring nucleotides, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), modifications containing alkylating agents, modifications with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide(s). Additionally, any of the hydroxyl groups normally present in the sugar can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide for additional linkage to additional nucleotides, or conjugated to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1-20 carbon atoms.Other hydroxyls can also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, alpha or beta anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), where each R or R' is independently H, or a substituted or unsubstituted alkyl (1-20C) optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0046] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

[0047] A "host cell" includes an individual cell or cell culture that can be or has been a recipient for a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell, which progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with a polynucleotide(s) of the invention.

[0048] As known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of an immunoglobulin heavy chain Fc region can vary, the human IgG heavy chain Fc region is usually defined to extend from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus thereof. The numbering of residues in the Fc region is that of the EU guidelines, as in Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., National Institutes of Health, Public Health Service, Bethesda, MD, 1991). The Fc region of an immunoglobulin generally comprises two constant regions, CH2 and CH3.

[0049] The term "compete" as used herein with respect to antibodies means that a first antibody or antigen-binding fragment (or portion) thereof binds to an epitope in a manner sufficiently similar to that of a second antibody or antigen-binding portion thereof such that the result of binding of the first antibody to its cognate epitope is detectably reduced in the presence of the second antibody compared to binding of the first antibody in the absence of the second antibody. However, the alternative that binding of the second antibody to its epitope is also detectably reduced in the presence of the first antibody may not necessarily be true. That is, a first antibody can inhibit binding of a second antibody to its epitope without the second antibody inhibiting binding of the first antibody to its respective epitope. However, if each antibody detectably inhibits the binding of the other antibody to its cognate epitope or ligand, whether to the same extent, a greater extent, or a lesser extent, the antibodies are said to "cross-compete" with each other for binding their respective epitope(s). Both competing and cross-competing antibodies are encompassed by the present invention. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), one of skill in the art will understand, based on the teachings provided herein, that such competing and / or cross-competing antibodies are encompassed by and can be useful for the methods disclosed herein.

[0050] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. A "variant Fc region" comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by at least one amino acid modification but retains at least one effector function of the native sequence Fc region. In certain embodiments, a variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about 1 to about 10 amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide, preferably from about 1 to about 5 amino acid substitutions. A variant Fc region herein will preferably have at least about 80% sequence identity with a native sequence Fc region and / or with the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

[0051] As used herein, the term "CD123" refers to all forms of CD123 and variants thereof that retain at least some of the activity of CD123. Unless otherwise indicated, such as by specific reference to human CD123, CD123 includes native sequence CD123 of all mammalian species, e.g., human, canine, feline, equine, and bovine. Exemplary CD123 sequences are shown in Table 1.

[0052] [Table 1-1]

[0053] [Table 1-2]

[0054] As used herein, "CD123 antibody" refers to an antibody that specifically binds to CD123 and modulates the biological activity and / or downstream event(s) mediated by CD123. In some embodiments, the CD123 antibody is an antagonist antibody. CD123 antagonist antibodies include antibodies that block, antagonize, inhibit or reduce (to any extent, including significantly) the biological activity of CD123, including downstream events mediated by CD123, such as IL-3 binding and / or downstream signaling, phosphorylation of STAT5 and survival of pluripotent cells. Examples of CD123 antibodies and CD123 antibody-drug conjugates ("CD123 ADCs") are provided herein.

[0055] As used herein, "treatment" is an approach to obtain beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reduction in proliferation (or destruction) of neoplastic or cancerous cells, inhibition of metastasis of neoplastic cells, amelioration of a disease associated with CD123 (e.g., cancer or autoimmune disease), reduction in symptoms resulting from a disease associated with CD123 (e.g., cancer or autoimmune disease), increasing the quality of life of a person suffering from a disease associated with CD123 (e.g., cancer or autoimmune disease), reducing the dosage of other medications required to treat a disease associated with CD123 (e.g., cancer or autoimmune disease), slowing the progression of a disease associated with CD123 (e.g., cancer or autoimmune disease), curing a disease associated with CD123 (e.g., cancer or autoimmune disease) and / or prolonging the survival of a subject with a disease associated with CD123 (e.g., cancer or autoimmune disease).

[0056] "Ameliorating" refers to a decrease or improvement in one or more symptoms compared to when the CD123 antibody or CD123 ADC is not administered. "Ameliorating" also includes a shortening or reduction in the duration of a symptom.

[0057] As used herein, an "effective dosage" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve any one or more beneficial or desired therapeutic results. For prophylactic use, beneficial or desired results include eliminating or reducing the risk of, reducing the severity of, or delaying the onset of, a disease, including the biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that exist during the manifestation of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing or ameliorating the incidence of one or more symptoms of various CD123-associated diseases or conditions (e.g., but not limited to, cancer), reducing the dose of other medications required to treat the disease, enhancing the effect of another medication, and / or delaying the progression of a CD123-associated disease in a subject. An effective dosage can be administered in one or more administrations. For purposes of this invention, an effective dosage of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish (either directly or indirectly) a prophylactic or therapeutic treatment. As understood in a clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if a desired result can or is achieved in conjunction with one or more other agents.

[0058] An "individual" or "subject" is a mammal, more preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats.

[0059] As used herein, "vector" refers to a construct capable of delivering, and preferably expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0060] As used herein, "expression control sequence" refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, e.g., a constitutive or inducible promoter, or an enhancer. An expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[0061] As used herein, a "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system. Examples include, but are not limited to, all of the standard pharmaceutical carriers, such as phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, and various types of wetting agents. A preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, Vol. 18). Edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and Remington, The (See Science and Practice of Pharmacy 21st ed. Mack Publishing, 2005).

[0062] The term "acyl donor glutamine-containing tag" or "glutamine tag" as used herein refers to a polypeptide or protein that contains one or more Gln residue(s) that serve as an amine acceptor for transglutaminase. See, e.g., WO2012059882 and WO2015015448, which are incorporated by reference in their entireties.

[0063] The term “k” on " as used herein refers to the rate constant for the association of an antibody to an antigen. Specifically, the rate constant (k on and k off ) and the equilibrium dissociation constant are measured using IgG and CD123 protein (e.g., CD123-Fc fusion protein).

[0064] The term “k off " as used herein refers to the rate constant for dissociation of an antibody from the antibody / antigen complex. The term “K D " as used herein refers to the equilibrium dissociation constant of an antibody-antigen interaction.

[0065] Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter per se. For example, a reference to "about X" includes the description of "X." Numeric ranges include the numbers defining the range.

[0066] As used herein, CPI refers to 1,2,8,8,8a-tetrahydrocyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one or a substituted or derivatized form thereof. CPI can also refer to the seco form of CPI, also known as 8-(chloromethyl)-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-ol, or seco-CPI or a substituted or derivatized form(s) thereof.

[0067] Wherever embodiments are described herein with the language "comprising," it is understood that otherwise similar embodiments are also provided that are described with the terms "consisting of" and / or "consisting essentially of."

[0068] When aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the invention includes not only the entire group recited as a whole, but also each member of the group individually, and all possible subgroups of the main group, as well as the main group lacking one or more of the group members. The invention also envisions the explicit exclusion of any one or more of the group members in the claimed invention.

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Throughout this specification and claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but in no way to imply the exclusion of other integers or groups of integers. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0070] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative and not intended to be limiting.

[0071] CD123 antibodies and antibody-drug conjugates The present invention provides antibodies that bind to CD123 (e.g., human CD123 (e.g., SEQ ID NO:1)) and conjugates (e.g., antibody-drug conjugates or ADCs) comprising anti-CD123 antibodies, which are characterized by any one or more of the following features: (a) treat, prevent, ameliorate one or more symptoms of a condition associated with malignant cells that express CD123 in a subject (e.g., cancer, including but not limited to, AML, B-ALL, HCL, etc.); (b) inhibit tumor growth or progression in a subject (having a malignant tumor that expresses CD123); (c) inhibit metastasis of cancer (malignant) cells that express CD123 in a subject (having one or more malignant cells that express CD123); (d) induce regression (e.g., long-term regression) of tumors that express CD123; and (e) exert cytotoxic activity on malignant cells that express CD123.

[0072] The antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), variants thereof, fusion proteins containing antibody portions (e.g., domain antibodies), humanized antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies can be of murine, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the CD123 antibodies described herein are monoclonal antibodies. For example, the CD123 antibodies can be humanized monoclonal antibodies, human antibodies, or chimeric monoclonal antibodies.

[0073] In some embodiments, the antibody can comprise an altered constant region, such as, for example, but not limited to, a constant region with increased potential to elicit an immune response. For example, the constant region can be altered to have increased affinity for an Fc gamma receptor, such as, for example, FcγRI, FcγRIIA, or FcγIII. In some embodiments, the antibody can comprise a constant region that is immunologically inert, i.e., has reduced potential to elicit an immune response. In some embodiments ... The Fc is modified as described in patent application No. 98099518. The Fc can be human IgG1, human IgG2, human IgG3 or human IgG4. The Fc can be human IgG2 (IgG2Δa) containing the mutation A330P331→S330S331, where the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 29:2613-2624, 1999. In one embodiment, the antibody comprises the following: Mutation: (Armouret al., Molecular Immunology 40 585-593, 2003): E233F234L In yet another embodiment, the Fc comprises a constant region of IgG4 comprising the deletion G236 at position 235→P233V234A235 (IgG4Δc), where the numbering refers to wild type IgG4. In yet another embodiment, the Fc is human IgG4 E233F234L235→P233V234A235 (IgG4Δb). In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing the hinge stabilizing mutation S228→P228 (Aalbersee et al., Immunology 105, 9-19, 2002). In another embodiment, the Fc comprises a constant region of IgG4 comprising the deletion G236 at position 235→P233V234A235 (IgG4Δc), where the numbering refers to wild type IgG4. In yet another embodiment, the Fc comprises human IgG4 E233F234L235→P233V234A235 (IgG4Δb). In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing the hinge stabilizing mutation S228→P228 (Aalbersee et al., Immunology 105, 9-19, 2002). , the Fc can be an aglycosylated Fc.

[0074] In some embodiments, the constant region can be deglycosylated by mutating the oligosaccharide attachment residue (e.g., Asn297) and / or adjacent residues that are part of a glycosylation recognition sequence in the constant region. In some embodiments, the constant region is enzymatically deglycosylated for N-linked glycosylation. The constant region can be deglycosylated enzymatically for N-linked glycosylation or by expression in a glycosylation-deficient host cell.

[0075] One method of determining the binding affinity of an antibody to CD123 is by measuring the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, an antibody (e.g., an IgG) can be cleaved with papain or recombinantly expressed. The affinity of the CD123 Fab fragment of the antibody was determined by measuring the binding affinity of the monofunctional Fab fragment in running buffer (0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v / v Surfactant P20) with pre-immobilized streptavidin sensor chips (SA) or surface plasmon resonance (Biacore™ 3000™ Surface Plasmon Resonance (SPR) system, Biacore™, INC, Piscataway, NJ). Biotinylated or Fc-fused human CD123 can be diluted to a concentration of less than 0.5 μg / mL in HBS-EP buffer and injected across individual chip channels with variable contact times to achieve two ranges of antigen densities (either 50-200 response units (RU) for detailed kinetic studies or 800-1,000 RU for screening assays). Regeneration studies show that 25 mM NaOH in 25% v / v ethanol effectively removes bound Fab while maintaining the activity of CD123 on the chip for over 200 injections. Typically, serial dilutions (0.1-10× estimated K ) of purified Fab samples are injected over individual chip channels. DFab proteins (ranging from 0.1 to 1.0 ... on ) and dissociation rate (k off ) fits the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-1 10) using the BIAevaluation program. D ) is the value of k off / k on The binding affinity (K ) of an antibody to any CD123, including human CD123, another mammalian CD123 (e.g., mouse CD123, rat CD123, or primate CD123), as well as different forms of CD123 (e.g., glycosylated CD123). Antibody binding affinity is typically measured at 25° C., but can also be measured at 37° C. The binding affinity (K ) of a CD123 antibody as described herein to CD123 (e.g., human CD123 (e.g., (SEQ ID NO:1)) is calculated as DIn some embodiments, the binding affinity is about 6500 nm, 6000 nm, 5986 nm, 5567 nm, 5500 nm, 4500 nm, 4000 nm, 3500 nm, 3000 nm, 2500 nm, 2134 nm, 2000 nm, 1500 nm, 1000 nm, 750 nm, 500 nm, 400 nm, 300 nm, 250 nm, 200 nM, 193 nM, 100 nM, 90nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 19nM, 18nM, 17nM, 16nM, 15nM, 10nM, 8nM, 7.5nM, 7nM, 6.5nM, 6nM, 5.5nM, 5nM, 4nM, 3nM, 2nM, 1nM, 0.5nM, 0.3nM, 0.1nM, 0.01nM or 0.002nM. In certain embodiments, the binding affinity is less than about any of 6500 nm, 6000 nm, 5500 nm, 5000 nm, 4000 nm, 3000 nm, 2000 nm, 1000 nm, 900 nm, 800 nm, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM or 0.5 nM.

[0076] The CD123 antibody described herein can be produced by any method known in the art.For the generation of hybridoma cell lines, the route and schedule of immunization of host animals generally conforms to the established conventional techniques for stimulating and producing antibodies as further described herein.The general techniques for producing human and mouse antibodies are known in the art and / or described herein.

[0077] In certain embodiments, the CD123 antibody can comprise a heavy chain variable region (VH) comprising three CDRs of the VH comprising the amino acid sequence of SEQ ID NO:6, 24, 32, 44, 51 or 64. In certain aspects of the invention, the antibody comprises a light chain variable region (VL) comprising three CDRs of the VL comprising the amino acid sequence of SEQ ID NO:17, 28, 39, 48, 57 or 71. In certain aspects of the invention, the antibody comprises a VH comprising three CDRs of the VH comprising the amino acid sequence of SEQ ID NO:6, 24, 32, 44, 51 or 64 and a VL comprising three CDRs of the VL comprising the amino acid sequence of SEQ ID NO:17, 28, 39, 48, 57 or 71. In other embodiments, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO:7, 8 and 9 and a VL comprising the amino acid sequence of SEQ ID NO:18, 19 and 20. In other embodiments, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NOs:7, 25, and 9, and a VL comprising the amino acid sequence of SEQ ID NOs:18, 19, and 20. In other embodiments, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NOs:33, 34, and 35, and a VL comprising the amino acid sequence of SEQ ID NOs:40, 41, and 42. In other embodiments, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NOs:33, 45, and 46, and a VL comprising the amino acid sequence of SEQ ID NOs:40, 41, and 42. In another embodiment, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO:52, 53, and 54, and a VL comprising the amino acid sequence of SEQ ID NO:58, 59, and 60. In another embodiment, the CD123 antibody can comprise a VH comprising the amino acid sequence of SEQ ID NO:65, 66, and 67, and a VL comprising the amino acid sequence of SEQ ID NO:72, 73, and 74.

[0078] Representative CD123 antibody heavy and light chain variable regions can respectively comprise the amino acid sequences of SEQ ID NOs: 6, 24, 32, 44, 51 and 64, and SEQ ID NOs: 17, 28, 39, 48, 57 and 71. Representative CD123 antibody heavy and light amino chains can respectively comprise the amino acid sequences of SEQ ID NOs: 15, 27, 37, 47, 55 and 69, and SEQ ID NOs: 23, 30, 43, 49, 62 and 76. Sequences of representative CD123 antibodies are shown in Table 2.0.

[0079] [Table 2]

[0080] Exemplary CD123 antibodies provided herein include 18G3, humanized 18G3 (h18G3), 16D6, humanized 16D6 (h16D6), 3D1 and 20D7, as shown in Table 2.1. The sequences shown in Table 2.1 are amino acid sequences unless otherwise indicated.

[0081] [Table 3-1]

[0082] [Table 3-2]

[0083] [Table 3-3]

[0084] [Table 3-4]

[0085] [Table 3-5]

[0086] [Table 3-6]

[0087] [Table 3-7]

[0088] Substitutional variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, although framework changes are also contemplated. Conservative substitutions are shown in Table 3 under the heading of "conservative substitutions". If such substitutions result in a change in biological activity, more substantial changes, such as those referred to in Table 3 as "exemplary substitutions" or as further described below with reference to classes of amino acids, can be introduced and the products screened.

[0089] [Table 4]

[0090] Substantial alterations in the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the polypeptide backbone in the region of the substitution, e.g., beta-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Naturally occurring residues are divided into groups based on common side chain properties: (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile; (2) Uncharged polar: Cys, Ser, Thr, Asn, Gln; (3) Acidic (negatively charged): Asp, Glu; (4) Basic (positively charged): Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe, His.

[0091] Non-conservative substitutions are made by exchanging a member of one of these classes for a member of another class. One type of substitution that can be made is to change one or more cysteines in an antibody, which may be, for example, chemically reactive, to another residue, such as (without limitation) alanine or serine. For example, a non-canonical cysteine ​​may be substituted. The substitution may be made in the CDR or framework regions of the variable domain or in the constant region of the antibody. In some embodiments, the cysteine ​​is canonical. Any cysteine ​​residue that is not involved in maintaining the proper conformation of the antibody may also be substituted with serine, generally to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to the antibody to improve its stability, especially when the antibody is an antibody fragment such as an Fv fragment.

[0092] The present invention also provides conjugates of the CD123 antibodies described herein, or of antigen-binding fragments thereof, in which the antibodies are conjugated, either directly or indirectly via a linker, to agents (e.g., cytotoxic agents) for targeted immunotherapy (e.g., antibody-drug conjugates or ADCs). For example, a cytotoxic agent can be linked or conjugated to the CD123 antibodies as described herein for targeted, localized delivery of the cytotoxic agent moiety to tumors (e.g., tumors expressing CD123).

[0093] Methods for conjugating cytotoxic drugs or other therapeutic agents to antibodies have been described in various publications. For example, chemical modifications can be made to antibodies, either through lysine side chain amines or through cysteine ​​sulfhydryl groups activated by reducing interchain disulfide bonds, for the conjugation reaction to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, 2005 and Gentle et al., 2006, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2016, 2015, 2016, 2017 ... See, e.g., et al., Bioconjugate Chem. 15:658-663, 2004. Engineered reactive cysteine ​​residues at specific sites on antibodies for conjugation of drugs have also been described. See, e.g., Junutula et al., Nature Biotechnology, 26:925-932, 2008. Transglutaminase and amines (e.g., containing reactive amines) can be used to treat or treat inflammatory conditions. Conjugation using endogenous glutamine engineered to be reactive (i.e., capable of forming a covalent bond as an acyl donor) in the presence of an acyl donor glutamine-containing tag or polypeptide in the presence of a cytotoxic agent attached to a reactive amine (i.e., a cytotoxic agent attached to a reactive amine) is also described in WO 2012 / 059882 and WO 2015015448, which are incorporated herein by reference in their entirety. Transglutaminases are protein-glutamine γ-glutamyltransferases (EC 2.3.2.13), which typically catalyze the pH-dependent transamidation of glutamine residues with lysine residues. Transglutaminases used in the inventions described herein can be obtained or produced from a variety of sources, or can be engineered to catalyze the transamidation of one or more endogenous glutamine residues with one or more lysine residues or amine donor agents containing one or more reactive amines. Methods for preparing ADCs using transglutaminase are described, for example, in U.S. Patent Application Publication No. 20170043033, which is incorporated by reference in its entirety.

[0094] ADCs comprise an antibody component conjugated to an agent, typically through the use of a linker. In some embodiments, the ADCs disclosed herein comprise an antibody site-specifically conjugated via an engineered glutamine-containing tag, an endogenous glutamine (i.e., a native glutamine without engineering, e.g., a native glutamine in a variable domain, CDR, etc.) and / or a reactive endogenous glutamine to an amine donor agent (e.g., a small molecule attached to a linker bearing an amine donor unit).

[0095] Endogenous glutamine can be made reactive (i.e., capable of forming a covalent bond as an acyl donor in the presence of an amine and a transglutaminase) by modifying (e.g., amino acid deletion, insertion, substitution or mutation) one or more amino acid(s) in the antibody by enzymatic deglycosylation or by reaction with an engineered transglutaminase. Thus, in one aspect, an antibody having the formula: antibody-(T-(XYZ a ) b ) cwherein T is 1) an engineered glutamine-containing tag at a specific site, 2) an endogenous glutamine, and / or 3) an endogenous glutamine made reactive by antibody engineering or engineered transglutaminase; X is an amine donor unit; Y is a linker; and Z is a drug moiety; XYZ is an amine donor drug site-specifically conjugated to the glutamine-containing tag, the endogenous glutamine, and / or the reactive endogenous glutamine; a is an integer from 1 to 6; b is an integer from 1 to 6; c is an integer from 1 to 20; and wherein the product of a, b, and c (drug antibody ratio) is at least about 1. Both the glutamine-containing tag, the endogenous glutamine and / or the reactive glutamine on the antibody and the amine donor drug (XYZ) described herein are substrates for transglutaminase, and the linkage between the glutamine-containing tag and / or the endogenous / reactive glutamine and the amine donor drug is of the formula CH2-CH2-CO-NH-, where NH- is connected to the linker and to the drug moiety.

[0096] In certain embodiments, the CD123 antibody or conjugate described herein comprises an acyl donor glutamine-containing tag engineered into a specific site of the antibody (e.g., the carboxyl terminus, the amino terminus, or another site in the CD123 antibody). In certain embodiments, the tag comprises the amino acid glutamine (Q) or the amino acid sequence LQG, LLQGG (SEQ ID NO: 1). NO:77), LLQG(SEQ ID NO:78), LSLSQG(SEQ ID NO:79), GGGLLQGG(SEQ ID NO:80), GLLQG(SEQ ID NO:81), LLQ, GSPLAQSHGG(SEQ ID NO:82), GLLQGGG(SEQ ID NO:83), GLLQGG(SEQ ID NO:84), GLLQ(SEQ ID NO:85), LLQLLQGA(SEQ ID NO:86), LLQGA(SEQ ID NO:87), LLQYQGA(SEQ ID NO:88), LLQGSG(SEQ ID NO:89), LLQYQG(SEQ ID NO:90), LLQLLQG(SEQ ID NO:91), SLLQG(SEQ LLQGPP (SEQ ID NO: 103), LLQPGK (SEQ ID NO: 104), LLQAPGK (SEQ ID NO: 105), LLQGAPG (SEQ ID NO: 106), LLQGAP (SEQ ID NO: 107) and LLQLQG (SEQ ID NO: 108).

[0097] In some embodiments, the acyl donor glutamine-containing tag comprises, for example, LLQG (SEQ ID NO:78), which replaces amino acid residues E294 to N297 in an antibody heavy chain. Also provided is an isolated antibody comprising an acyl donor glutamine-containing tag and an amino acid modification at position 222, 340 or 370 (EU numbering scheme) of the antibody, wherein the modification is an amino acid deletion, insertion, substitution, mutation or any combination thereof. In some embodiments, the amino acid modification is a lysine to arginine substitution (e.g., K222R, K340R or K370R).

[0098] Agents that can be conjugated to the CD123 antibodies or antigen-binding fragments of the invention include, but are not limited to, a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, or an oligonucleotide.

[0099] Examples of cytotoxic drugs include, but are not limited to, anthracyclines, auristatins, dolastatins, combretastatins, duocarmycins, pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, enediynes, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, camptothecins, tubulysins, hemiasterins, spliceostatins, pladienolides, and their stereoisomers, isosteres, analogs, or derivatives.

[0100] In some aspects, the drug / agent is cyclopropylpyrroloindoline (CPI) dimer, CTI dimer or CBI dimer.CPI dimer induces interstrand DNA crosslinking and strong cytotoxicity.PCT International Publication No. WO2015 / 110935, which is incorporated herein by reference in its entirety, discloses the CPI and CBI dimers that are useful in the CD123 ADC of the present invention, and provides the method of producing CPI dimers and CBI dimers. For example, the drug (8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-5-hydroxy-8-methyl-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl dihydrogen phosphate (also known as “CPI-8314 dimer”) has the structure:

[0101] [ka]

[0102] and has the formula C31H31Cl2N4O7P. Anthracyclines are derived from the bacteria Streptomyces and have been used to treat a wide range of cancers, such as leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunorubicin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0103] Dolastatins, and their peptidic analogs and derivatives, the auristatins, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, e.g., U.S. Patent No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998. Exemplary dolastatins and auristatins are The auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethylauristatin D or monomethyldolastatin 10), MMAF (monomethylauristatin F or N-methylvaline-valine-dolaisoleucine-dolaproine-phenylalanine), MMAE (monomethylauristatin E or N-methylvaline-valine-dolaisoleucine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (e.g., the auristatins described in U.S. Patent Application Publication No. 2013 / 0129753). In some embodiments, the auristatin is 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), having the following structure:

[0104] [ka]

[0105] . In some embodiments, the auristatin is 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), which has the following structure:

[0106] [ka]

[0107] . In some embodiments, the auristatin is OMe (N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), having the following structure:

[0108] [ka]

[0109] . In other embodiments, the auristatin is 0131 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide y-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

[0110] [ka]

[0111] . In other embodiments, the auristatin is 0121(2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide -N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) having the following structure:

[0112] [ka]

[0113] . Camptothecins are cytotoxic quinoline alkaloids that inhibit the enzyme topoisomerase I. Examples of camptothecins and their derivatives include, but are not limited to, topotecan and irinotecan, and their metabolites, such as SN-38.

[0114] Combretastatins are natural phenols that have vascular disrupting properties in tumors. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

[0115] Duocarmycins and CC-1065 are DNA alkylating agents with cytotoxic potency. See Bogerand Johnson, PNAS92:3642-3649 (1995). Exemplary duocarmycins and CC-1065s include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, (+)-CC-1065, and compounds as disclosed in International Application PCT / IB2015 / 050280, including, but not limited to, the following compounds: structure:

[0116] [ka]

[0117] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(1S)-1-(chloromethyl)-3-[(5-{[(1S)-1-(chloromethyl)-5-(phosphonooxy)-1,2-dihydro-3H-benzo[e]indol-3-yl]carbonyl}thiophen-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide, structure:

[0118] [ka]

[0119] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide, structure:

[0120] [ka]

[0121] N-2-acetyl-L-lysyl-L-valyl-N-5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(4-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}pentacyclo[4.2.0.0-2,5.0-3,8.0-4,7]oct-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide.

[0122] Enediynes are a class of antitumor bacterial products characterized by either the presence of 9- and 10-membered rings or conjugated triple-double-triple bond ring systems. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and their derivatives.

[0123] Geldanamycins are benzoquinone ansamycin antibiotics that bind to Hsp90 (heat shock protein 90) and have been used as antitumor drugs. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0124] Hemiasterlin and its analogs (eg, HTI-286) bind to tubulin, disrupting normal microtubule dynamics and depolymerizing microtubules in stoichiometric amounts. Maytansines or their derivatives, maytansinoids, inhibit cell proliferation by blocking the formation of microtubules during mitosis through inhibition of tubulin polymerization. See Remillard et al., Science 189:1002-1005, 1975. Exemplary maytansines and maytansinoids, including, but not limited to, mertansine (DM1) and its derivatives and ansamitocins.

[0125] Pyrrolobenzodiazepine dimers (PBDs) and indolino-benzodiazepine dimers (IGNs) are antitumor agents or their equivalents that contain one or more imine functional groups that bind to double-stranded DNA. PBD and IGN molecules are based on the natural product athramycin and interact with DNA in a sequence-selective manner that favors purine-guanine-purine sequences. Exemplary PBDs and their analogs include, but are not limited to, SJG-136.

[0126] Spliceostatins and pladienolides are antitumor compounds that inhibit splicing and interact with the splicesome, SF3b. ​​Examples of spliceostatins include spliceostatin A, FR901464, and

[0127] [ka]

[0128] and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate having the structure:

[0129] Examples of pladienolides include, but are not limited to, pladienolide B, pladienolide D, or E7107. Taxanes are diterpenes that act as antitubulin or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g., TAXOL®) and docetaxel (TAXOTERE®).

[0130] Tubulysins are natural products isolated from strains of myxobacteria that have been shown to depolymerize microtubules and induce mitotic arrest. Exemplary tubulysins include, but are not limited to, tubulysin A, tubulysin B, and tubulysin D.

[0131] Vinca alkaloids are also antitubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0132] In certain embodiments, the cytotoxic agent is selected from the group consisting of the following compounds: MMAD (monomethyl auristatin D), 0101 (2-methylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-2-methyl-3-oxo-3-{[(1S)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino}propyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 3377 (N,2-dimethylalanyl- N-{(1S,2R)-4-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxyl-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-2-methoxy-1-[(1S)-1-methylpropyl]-4-oxobutyl}-N-methyl-L-valinamide), 0131 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl 2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide ethyl-L-valinamide), 0131-OMe (N,2-dimethylalanyl-N-[(3R,4S,5S)-3-methoxy-1-{(2S)-2-[(1R,2R)-1-methoxy-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide), 0121 (2-methyl-L-prolyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2-methyl-L-proly-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-{[(2S)-1-methoxy-1-oxo-3-phenylpropan-2-yl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide rrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamide) and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazinyl-2-oxoethyl)-4-hydroxy-1,6-dioxaspiro[2.5]oct-5-yl]-3-methylpenta-2,4-dien-1-yl}-2,5-dimethyltetrahydro-2H-pyran-3-yl]amino}-5-oxopent-3-en-2-yl acetate.

[0133] In some embodiments, the agent is an immunomodulatory agent. Examples of immunomodulatory agents include ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporin, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, and the like. mofetil), methotrexate, glucocorticoids and analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21), colony stimulating factors (e.g., granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)), interferons (e.g., interferon-α, -β, and -γ), stem cell growth factors referred to as "S1 factors," erythropoietin, and thrombopoietin, or combinations thereof.

[0134] In some embodiments, the drug moiety is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rhodamine, lanthanide fluorophores and their derivatives or radioisotopes bound to a chelator. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101). Examples of chelating agents include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

[0135] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein amidite (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor® (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethylrhodamine (TAMRA) (e.g., 5-TAMRA), tetramethylrhodamine (TMR), and sulforhodamine (SR) (e.g., SR101).

[0136] In certain embodiments, therapeutic or diagnostic radioisotopes or other labels (e.g., PET or SPECT labels) can be incorporated into the agent for conjugation to an anti-CD123 antibody or antigen-binding fragment as described herein. Examples of radioisotopes or other labels include: 3 H, 11 C. 13 N, 14 C. 15 N, 15 O. 35 S, 18 F, 32 P, 33 P, 47 Sc, 51 Cr, 57 Co, 58 Co, 59 Fe, 62 Cu, 64 Cu, 67 Cu, 67 Ga, 68 Ga, 75 Se, 76 Br, 77 Br, 86 Y, 89 Zr, 90 Y, 94 Tc, 95 Ru, 97 Ru, 99 Tc, 103 Ru, 105 Rh, 105 Ru, 107 Hg, 109 Pd, 111 Ag, 111 In, 113 In, 121 Te, 122 Te, 123 I, 124 I, 125 I, 125 Te, 126 I, 131 I, 131 In, 133 I, 142 Pr, 143 Pr, 153 Pb, 153 Sm, 161 Tb, 165 Tm, 166 Dy,166 H, 167 Tm, 168 Tm, 169 Yb, 177 Lu, 186 Re, 188 Re, 189 Re, 197 Pt, 198 Au, 199 Au, 201 Tl, 203 Hg, 211 At, 212 Bi, 212 Pb, 213 Bi, 223 Ra, 224 Ac and 225 Including, but not limited to, Ac.

[0137] In certain embodiments, the agent is a therapeutic protein, including, but not limited to, toxins, hormones, enzymes, and growth factors. Examples of toxin proteins (or polypeptides) include diptheria (e.g., diptheria A chain), Pseudomonas exotoxins and endotoxins, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii proteins, dianthin proteins, ribonuclease (RNase), DNase I, Staphylococcus enterotoxin A, pokeweed antiviral protein, gelonin, diphtherin toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, Saponaria officinalis toxin, and the like. officinalis inhibitors, mitogellin, restrictocin, phenomycin, enomycin, trichothecenes, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxins), and conotoxins (e.g., KIIIA or SmIIIa).

[0138] In some embodiments, the agent is a biocompatible polymer. The CD123 antibody or antigen-binding fragment described herein can be conjugated to a biocompatible polymer to increase serum half-life and bioactivity and / or extend half-life in vivo. Examples of biocompatible polymers include water-soluble polymers, such as polyethylene glycol (PEG) or its derivatives, and zwitterion-containing biocompatible polymers (e.g., phosphorylcholine-containing polymers).

[0139] In certain embodiments, the agent is an oligonucleotide, such as an antisense oligonucleotide. In another aspect, the invention provides a conjugate of an antibody described herein, wherein the conjugate comprises the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic agent), wherein the acyl donor glutamine-containing tag is engineered at a specific site of the antibody (e.g., at the carboxyl terminus of the heavy or light chain, after residue T135 in the antibody heavy chain, or at another site), wherein the tag is conjugated to a linker (e.g., a linker containing one or more reactive amines (e.g., primary amine NH2)), and wherein the linker is conjugated to a cytotoxic agent (e.g., MMAD or other auristatins, e.g., 0101, 0131, or 3377).

[0140] Examples of linkers containing one or more reactive amines are Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2 (or amino PEG6-propionyl), Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine or Ac-Lys-putrescine.

[0141] In some embodiments, the (linker)-(cytotoxic agent) is N 2 -Acetyl-L-lysyl-L-valyl-N 5-carbamoyl-N-[4-({[(2-{[({(8S)-8-(chloromethyl)-6-[(3-{[(1S)-1-(chloromethyl)-8-methyl-5-(phosphonooxy)-1,6-dihydropyrrolo[3,2-e]indol-3(2H)-yl]carbonyl}bicyclo[1.1.1]pent-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithineamide, molecular formula CHNOP and structure:

[0142] [ka]

[0143] has. In some embodiments, the (linker)-(cytotoxic agent) is the trifluoroacetate salt form of C63H82Cl2N13O15P, which has the following structure:

[0144] [ka]

[0145] has. The formula for the trifluoroacetate salt form above is C63H82Cl2N13O15P·C2HF3O2.

[0146] In some embodiments, the ADC is 1) antibody-LLQG (SEQ ID NO: 78)-Ac-Lys-Val-Cit-PABC-CPI-8314. In some embodiments, the acyl donor glutamine-containing tag comprises LLQG (SEQ ID NO: 78), e.g., replacing amino acid residues E294-N297 in the antibody heavy chain. In some embodiments, the ADC comprises an amino acid modification at position K222 (in the hinge). In some embodiments, the modification is K222R. Examples of antibodies include, but are not limited to, h18G3, 18G3, h18G3, 16D6, h16D6, 3D1, and 20D7.

[0147] In some embodiments, the invention encompasses compositions comprising an antibody or ADC as described herein, or a pharmaceutical composition comprising an antibody or ADC produced by a method as described herein and having the characteristics as described herein. As used herein, a composition comprises one or more antibodies or ADCs that bind to CD123. These compositions can further comprise suitable excipients, such as pharma- ceutically acceptable excipients including buffers, as are well known in the art.

[0148] Methods of Using CD123 Antibodies and ADCs Thereof The antibodies and ADCs of the invention are useful in a variety of applications, including, but not limited to, therapeutic and diagnostic treatments.

[0149] In one aspect, the present invention provides a method for treating a condition associated with CD123 expression in a subject. In one embodiment, the method for treating a condition associated with CD123 expression in a subject comprises administering to a subject in need thereof an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein. Conditions associated with CD123 expression include, but are not limited to, aberrant CD123 expression, altered or ectopic CD123 expression, malignant cells expressing CD123 and proliferative disorders (e.g., cancer) or autoimmune disorders.

[0150] Thus, in certain embodiments, methods of treating cancer in a subject are provided, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC described herein. As used herein, cancer includes, for example, but not limited to, multiple myeloma, malignant plasma cell neoplasms, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia (HCL), B-cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL, B-ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma. , marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), myeloid leukemia, Waldenstrom macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma lymphoma), Waldenström macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (lower limb type), EBV in the elderly The cancer can be ALK positive large B cell lymphoma, diffuse large B cell lymphoma with inflammation, intravascular large B cell lymphoma, ALK positive large B cell lymphoma, plasmablastic lymphoma, large B cell lymphoma occurring in HHV8-associated multicentric Castleman disease, unclassified B cell lymphoma with intermediate characteristics between diffuse large B cell lymphoma and Burkitt lymphoma, unclassified B cell lymphoma with intermediate characteristics between diffuse large B cell lymphoma and classical Hodgkin lymphoma and other B cell-related lymphoma.In some embodiments, the cancer is AML, B-ALL, BPDCN, NHL or HCL.

[0151] In one embodiment, a method is provided for inhibiting tumor growth or progression in a subject having malignant cells expressing CD123, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC as described herein. In another embodiment, a method is provided for inhibiting metastasis of cells expressing CD123 in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC as described herein. In another embodiment, a method is provided for inducing tumor regression in malignant cells in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC as described herein.

[0152] In certain embodiments, a method of treating an autoimmune disorder in a subject is provided comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC described herein.

[0153] As used herein, an autoimmune disorder includes systemic lupus erythematosus, rheumatoid arthritis, diabetes mellitus (type I), multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, Hashimoto's encephalopathy, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren's syndrome, acute disseminated encephalomyelitis, agammaglobulinemia, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, antisynthetase syndrome, atopic allergy, atopic dermatitis, autoimmune enteropathy, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune peripheral neuropathy, autoimmune pancreatitis, autoimmune polyendocrine syndrome, and autoimmune inflammatory bowel disease. syndrome, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Behcet's disease, Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barre syndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, narcolepsy, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, relapsing polychrondritis, rheumatic fever, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis.

[0154] In another aspect, the present invention provides an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein for treating a condition associated with CD123 expression (e.g., cancer or an autoimmune disorder) in a subject in need thereof. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein for inhibiting tumor growth or progression in a subject having malignant cells expressing CD123 is provided. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein for inhibiting metastasis of malignant cells expressing CD123 in a subject in need thereof is provided. In one embodiment, an effective amount of a composition (e.g., a pharmaceutical composition) comprising a CD123 antibody or CD123 ADC described herein for inducing tumor regression in a subject having malignant cells expressing CD123 is provided.

[0155] In another aspect, the present invention provides a CD123 antibody or CD123 ADC described herein for use in treating a condition associated with CD123 expression (e.g., cancer or autoimmune disorder) in a subject in need thereof. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inhibiting tumor growth or progression in a subject having malignant cells expressing CD123. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inhibiting metastasis of malignant cells expressing CD123 in a subject in need thereof. In one embodiment, a CD123 antibody or CD123 ADC described herein is provided for inducing tumor regression in a subject having malignant cells expressing CD123.

[0156] In another aspect, the present invention provides the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for treating a condition associated with CD123 expression (e.g., cancer or an autoimmune disorder). In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inhibiting tumor growth or progression is provided. In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inhibiting metastasis of malignant cells expressing CD123 is provided. In one embodiment, the use of a CD123 antibody or CD123 ADC described herein in the manufacture of a medicament for inducing tumor regression is provided.

[0157] In another aspect, methods for detecting, diagnosing and / or monitoring the condition associated with CD123 expression are provided.For example, the CD123 antibody described herein can be labeled with detectable moieties, such as imaging agents and enzyme-substrate labels.The antibody described herein can also be used for in vivo diagnostic assays, such as in vivo imaging (e.g. PET or SPECT), or staining reagents.

[0158] In some embodiments, the methods described herein further comprise treating the subject with an additional form of therapy. In some embodiments, the additional form of therapy is an additional anti-cancer therapy, including but not limited to chemotherapy, radiation, surgery, hormonal therapy, and / or additional immunotherapy.

[0159] In certain embodiments, the additional form of therapy includes administering one or more therapeutic agents in addition to a CD123 antibody or CD123 ADC described herein. The one or more therapeutic agents can be, for example, a second antibody (e.g., an anti-VEGF (vascular endothelial growth factor) antibody (e.g., AVASTIN®), an anti-HER2 antibody (e.g., HERCEPTIN®), an anti-CD25 antibody, an anti-CD33 antibody, an anti-CD20 antibody (e.g., RITUXAN®), an anti-mucin-like glycoprotein antibody, an anti-TNF antibody, an anti-PD-1 antibody or PD-L1 antibody, and / or an epidermal growth factor receptor (EGFR) antibody (e.g., ERBITUX®), an angiogenesis inhibitor, a cytotoxic agent (e.g., anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin, barbicin, and mitoxantrone), taxanes (e.g., paclitaxel and docetaxel), dolastatins, duocarmycins, enediynes, geldanamycins, or the like). sine, maytansine, puromycin, vinca alkaloids (e.g. vincristine), topoisomerase inhibitors (e.g. etoposide), tubulysin, pyrimidine analogues (e.g. fluorouracil), platinum-containing drugs (e.g. cisplatin, carboplatin and oxaliplatin), alkylating agents (e.g. melphalan, cyclophosphamide or carmustine) and hemiasterin), immunomodulators (e.g. prednisone), anti-inflammatory agents (e.g. dexamethasone), aromatase inhibitors (e.g. anastrozole, exemestane, letrozole, vorozole, formestane or testolactone), proteasome inhibitors (e.g. bortezomib, e.g. VELCADE® ([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl -2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid)) as well as other drugs such as tamoxifen.

[0160] For example, in one embodiment, a method of treating AML is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC described herein and one other therapeutic agent, such as a chemotherapeutic agent or thalidomide or a derivative thereof (e.g., lenalidomide). In one embodiment, the one other therapeutic agent is selected from the group consisting of bortezomib (e.g., VELCADE®), melphalan, prednisone, doxorubicin, lenalidomide, thalidomide, prednisone, carmustine, etoposide, cisplatin, cyclophosphamide, and vincristine. In one embodiment, the other therapeutic agent is bortezomib (e.g., VELCADE®), melphalan, or prednisone. In one embodiment, the subject is relapsed or refractory to a previous AML therapy.

[0161] CD123 antibodies or CD123 ADCs can be administered to an individual via any suitable route. Those skilled in the art should understand that the examples described herein are not intended to be limiting, but are intended to illustrate available techniques. Thus, in some embodiments, CD123 antibodies or CD123 ADCs are administered according to known methods, for example, intravenously, for example, as a bolus or by continuous infusion over a period of time, intramuscularly, intraperitoneally, intracerebrospinal, intracranially, transdermally, subcutaneously, intraarticularly, sublingually, intrasynovially, via insufflation, intrathecally, orally, by inhalation or topical route. Administration can be systemic, for example, intravenously, or topical. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the CD123 antibody or CD123 ADC can be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[0162] In one embodiment, CD123 antibody or CD123 ADC is administered by site-specific or targeted local delivery technique.Examples of site-specific or targeted local delivery technique include various implanted depot sources of CD123 antibody or CD123 ADC, or local delivery catheters, such as injection catheters, indwelling catheters, or needle catheters, artificial grafts, adventitial wraps, shunts and stents or other implanted devices, site-specific carriers, direct injection, or direct application.See, for example, PCT Publication WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0163] Various formulations of CD123 antibody or CD123 ADC can be used for administration. In some embodiments, CD123 antibody or CD123 ADC can be administered neat. In some embodiments, CD123 antibody (or CD123 ADC) and pharma- ceutically acceptable excipients can be in various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate administration of pharmacologically active substances. For example, excipients can provide form or consistency or act as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts for varying osmolality, encapsulating agents, buffers, and skin permeation enhancers. Excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington, The Science and Practice of Pharmacy, 21st Edition, Mack Publishing, 2005.

[0164] In certain embodiments, these agents are formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Thus, these agents can be combined with a pharma- ceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, etc. The particular dosing regimen, i.e., dosage, timing and repetition, will depend on the particular individual and that individual's medical history.

[0165] The CD123 antibodies or CD123 ADCs described herein can be administered using any suitable method, including administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). The CD123 antibodies or CD123 ADCs can also be administered by inhalation, as described herein. In general, for administration of CD123 antibodies or CD123 ADCs, the initial candidate dosage can be about 2 mg / kg. For purposes of the present invention, a typical daily dosage can range anywhere from about 3 μg / kg to 30 μg / kg to 300 μg / kg to 3 mg / kg, up to 30 mg / kg, up to 100 mg / kg or more, depending on the factors mentioned above. For example, dosages of about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 10 mg / kg, and about 25 mg / kg can be used. For repeated administration over several days or longer periods, depending on the symptoms, treatment is maintained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved, for example to inhibit or slow tumor growth / progression or metastasis of cancer cells. One typical dosing regimen includes administering an initial dose of about 2 mg / kg of CD123 antibody or CD123 ADC, followed by a weekly maintenance dose of about 1 mg / kg, or followed by a biweekly maintenance dose of about 1 mg / kg. Another typical dosing regimen includes administering an initial dose of about 0.21, about 0.5, or about 0.8 mg / kg every week or every three weeks. Other typical dosing regimens include administering increasing doses (e.g., an initial dose of 1 mg / kg and gradually increasing to one or more higher doses every week or at longer time intervals). Other dosing regimens may also be useful depending on the pattern of pharmacokinetic decay the practitioner wishes to achieve. For example, in some embodiments, administration 1-4 times per week is contemplated. In other embodiments, administration once a month or once every two or three months is contemplated. The progress of this therapy is easily monitored by conventional techniques and assays. The dosage regimen, including the CD123 antibody or CD123 ADC used, can be varied over time.

[0166] For the purposes of the present invention, the appropriate dosage of CD123 antibody or CD123 ADC will depend on the CD123 antibody or CD123 ADC (or composition thereof) used, the type and severity of the condition to be treated, whether the agent is administered for therapeutic purposes, previous therapy, the subject's medical history and response to the agent, the subject's clearance rate for the agent administered, and the discretion of the attending physician. Typically, the clinician will administer CD123 antibody or CD123 ADC until a dosage is reached that achieves the desired result. Dosage and / or frequency can vary over the course of treatment. Empirical considerations, such as half-life, will generally contribute to the determination of dosage. For example, antibodies that are compatible with the human immune system, such as humanized or fully human antibodies, can be used to extend the half-life of the antibody and prevent the antibody from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of therapy and is generally, but not necessarily, based on treating and / or suppressing and / or ameliorating and / or slowing symptoms, such as suppressing or slowing tumor growth. Alternatively, sustained continuous release formulations of CD123 antibodies or CD123 ADCs may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0167] In one embodiment, dosages for CD123 antibodies or CD123 ADCs can be empirically determined in individuals who have been given one or more doses (singular or multiple) of the CD123 antibody or CD123 ADC. Individuals are given increasing doses of the CD123 antibody or CD123 antagonist. Disease indicators can be followed to assess efficacy.

[0168] Administration of a CD123 antibody or CD123 ADC in the methods of the invention can be continuous or intermittent depending, for example, on the physiological disorder of the recipient, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to those of skill in the art. Administration of the CD123 antibody or CD123 ADC can be essentially continuous over a preselected period of time, or can be in a series of spaced doses.

[0169] In some embodiments, more than one CD123 antibody or CD123 ADC can be present. At least one, at least two, at least three, at least four, at least five different or more CD123 antibodies or CD123 ADCs can be present. In general, the CD123 antibodies or CD123 ADCs can have complementary activities that do not adversely affect each other. For example, one or more of the following CD123 antibodies can be used: a first CD123 antibody directed against an epitope on CD123 and a second CD123 antibody directed against a different epitope on CD123.

[0170] Therapeutic formulations of CD123 antibodies or CD123 ADCs used in accordance with the present invention may comprise the antibody having the desired degree of purity in any pharma- ceutically acceptable carrier, excipient, or stabilizer (see Remington, The Science and Practice of Pharmacy, 21st ed., Mack Publishing). , 2005) in the form of a lyophilized formulation or aqueous solution prepared for storage. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include buffers, such as phosphates, citrates, and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. , such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0171] Liposomes containing a CD123 antibody or a CD123 ADC can be formulated using methods known in the art, e.g., the methods described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688, 1985; ng, et al., Proc. Natl Acad. Sci. USA 77:4030, 1980; and U.S. Pat. No. 4,485,336. Liposomes having enhanced circulation time are prepared by the methods described in U.S. Patent Nos. 5,013,556 and 4,544,545. Particularly useful liposomes can be generated by the reverse phase evaporation method using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to generate liposomes with the desired diameter.

[0172] The active ingredient can also be entrapped in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, for example by coacervation techniques or in microcapsules prepared by interfacial polymerization (e.g. hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively). Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 21st Edition, Mack Publishing, 2005.

[0173] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose isobutyl acetate, and poly-D-(−)-3-hydroxybutyric acid.

[0174] Formulations to be used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic CD123 antibody or CD123 ADC compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0175] Compositions of the invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

[0176] To prepare solid compositions such as tablets, the primary active ingredient is mixed with pharmaceutical carriers, such as conventional tableting ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of extended action. For example, the tablets or pills can include an inner dosage component and an outer dosage component, the latter being in the form of an outer membrane covering the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.

[0177] Suitable surfactants include in particular non-ionic agents such as polyoxyethylene sorbitans (e.g. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions containing a surfactant will conveniently contain 0.05 to 5% surfactant, which may be 0.1 to 2.5%. It will be appreciated that other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, may be added as required.

[0178] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition or it can be dissolved in oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion is formed upon mixing with phospholipids (e.g. egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be understood that other ingredients such as glycerol or glucose can be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example 5-20%. The fat emulsion can contain fat droplets of 0.1-1.0 μm, especially 0.1-0.5 μm, and can have a pH in the range of 5.5-8.0.

[0179] The emulsion composition can be an emulsion composition prepared by mixing a CD123 antibody or a CD123 ADC with Intralipid™ or its components (soybean oil, egg phospholipids, glycerol and water).

[0180] Compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions can contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal respiratory route for local or systemic action. Compositions, preferably in sterile pharma- ceutically acceptable solvents, can be nebulized by the use of gases. Nebulized solutions can be breathed directly from the nebulizing device, or the nebulizing device can be attached to a face mask, tent, or intermittent positive airway pressure simulator. Solution, suspension, or powder compositions can be administered from a device that delivers the formulation in an appropriate manner, preferably orally or nasally.

[0181] composition The composition used in the method of the present invention comprises an effective amount of the CD123 antibody or CD123 ADC described herein. Examples of such compositions and how to formulate them are also described in the previous section and below. In some embodiments, the composition comprises one or more CD123 antibodies or CD123 ADCs. For example, the CD123 antibody recognizes human CD123. In some embodiments, the CD123 antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the CD123 antibody comprises a constant region that can induce a desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the CD123 antibody comprises a constant region that does not induce an unwanted or undesired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the CD123 antibody comprises one or more CDR(s) of the antibody (e.g., one, two, three, four, five, or in some embodiments, all six CDRs).

[0182] It is understood that a composition can include more than one CD123 antibody or CD123 ADC (e.g., a mixture of CD123 antibodies that recognize different epitopes of CD123). Other exemplary compositions include more than one CD123 antibody or CD123 ADC that recognize the same epitope(s) or CD123 antibodies or CD123 ADCs of different species that bind to different epitopes of CD123 (e.g., human CD123).

[0183] In certain embodiments, the CD123 antibody can be administered in combination with the administration of one or more additional therapeutic agents. These include chemotherapeutic agents, vaccines, CAR-T cell-based therapies, radiation therapy, cytokine therapy, vaccines, CD123 bispecific antibodies, inhibitors of other immunosuppressive pathways, inhibitors of angiogenesis, T cell activators, inhibitors of metabolic pathways, mTOR inhibitors, inhibitors of the adenosine pathway, tyrosine kinase inhibitors including but not limited to inlighters, ALK inhibitors and sunitinib, BRAF inhibitors, epigenetic modifiers, IDO1 inhibitors, inhibitors or depletors of Treg cells and / or myeloid-derived suppressor cells, JAK inhibitors, STAT inhibitors, cyclin-dependent kinase inhibitors, biotherapeutics (VEGF inhibitors, ... , VEGFR, EGFR, Her2 / neu, other growth factor receptors, CD20, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, CD123, PD-L1, TIGIT and ICOS), immunogenic agents (e.g., attenuated cancerous cells, tumor antigens, antigen presenting cells, such as dendritic cells pulsed with tumor-derived antigens or nucleic acids, immunostimulatory cytokines (e.g., IL-2, IFNα2, GM-CSF) and cells transfected with genes encoding immunostimulatory cytokines, such as, but not limited to, GM-CSF).Examples of chemotherapeutic agents include the following agents: alkylating agents, such as thiotepa and cyclophosphamide; alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins, particularly bullatacin and bullatacinone; camptothecin, including the synthetic analog topotecan; bryostatin; kallistatin; CC-1065, including its adozelesin, carzelesin, and bizelesin synthetic analogs; cryptophycins, particularly cryptophycin 1 and cryptophycin 2. tophicin 8); dolastatins; duocarmycins (including their synthetic analogs KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictiin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichiin, phenesterine, prednimustine, trophosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (such as calicheamicins, especially calicheamicin gamma 1I and calicheamicin phi I1, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemicin (d. ynemicins) (including dynemicin A); bisphosphonates, e.g., clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, ausramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin , dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine. , thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diazicon;Eflornithine;Elliptinium acetate;Epothilone;Etoglucide;Gallium nitrate;Hydroxyurea;Lentinan;Lonidamine;Maytansinoids, e.g. maytansine and ansamitocins;Mitoguazone;Mitoxantrone;Mopidamol;Nitracrine;Pentostatin;Phenamet;Pirarubicin;Losoxantrone;Podophyllic acid;2-Ethylhydrazide;Procarbazine;Razoxane;Rhizoxin;Sizofiran;Spirogermanium;Tenuazonic acid;Triaziquone;2,2',2''-Trichlorotriethylamine;Tricothecenes (especially T-2 toxin, veracrine A, roridin A and anguidine);Urethane;Vindesine;Dacarbazine;Mannomustine;Mitobronitol;Mitolactol;Pipobroman;Gashi gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitors RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharma- ceutically acceptable salts, acids, or derivatives of any of the above. antihormonal agents that act to control or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droxifene, 4-hydroxytamoxifen, trioxyfene, keoxifene, LY117018, onapristone, and toremifene (Fareston); aromatase inhibitors that inhibit the enzyme aromatase, which controls estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole, and anastrozole;and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharma- ceutically acceptable salts, acids or derivatives of any of the above.

[0184] In some embodiments, the CD123 antibody is administered in combination with one or more other therapeutic agents targeting immune checkpoint regulators, such as (but not limited to) CD123, PD-1, PD-L1, CTLA-4, LAG-3, B7-H3, B7-H4, B7-DC (PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD 86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1), OX40, OX40L, GITR, GITRL, CD70, CD27, 4-1BB, 4-BBL, DR3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM(SLAMF1, CD150 ), SLAMF2(CD48), SLAMF3(CD229), SLAMF4(2B4, CD244), SLAMF5(CD84), SLAMF6(NTB-A), SLAMCF7(CS1), SLAMF 8(BLAME), SLAMF9(CD2F), CD28, CEACAM1(CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CE It is used in combination with agents that target ACAM1-3AS, CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, the CD39-CD73-adenosine pathway (A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, the VEGF pathway, CSF-1, or innate immune response regulators.In some embodiments, the CD123 antibody is, for example, an anti-PD-L1 antagonist antibody, such as BMS-936559 (MDX-1105); an CD123 antibody, such as nivolumab, pembrolizumab, and pidilizumab; an anti-CTLA-4 antagonist antibody, such as ipilimumab; an anti-LAG-3 antagonist antibody, such as BMS-986016 and IMP701; an anti-TIM-3 antagonist antibody; Anti-B7-H3 antagonistic antibody, such as MGA271; anti-VISTA antagonistic antibody; anti-TIGIT antagonistic antibody; anti-CD28 antagonistic antibody; anti-CD80 antibody; anti-CD86 antibody; anti-B7-H4 antagonistic antibody; anti-ICOS agonist antibody; anti-CD28 agonist antibody; natural immune response regulator (such as TLRs, KIR, NKG2A) and IDO inhibitor are used together. In some embodiments, CD123 antibody is used together with 4-1BB (CD137) agonist, such as PF-05082566 or BMS-663513. In some embodiments, CD123 antibody is used together with OX40 agonist, such as anti-OX-40 agonist antibody. In some embodiments, CD123 antibodies are used in combination with GITR agonists, such as anti-GITR agonist antibodies, such as (but not limited to) TRX518. In some embodiments, CD123 antibodies are used in combination with IDO inhibitors. In some embodiments, CD123 antibodies are used in combination with cytokine therapy, such as (but not limited to) IL-15, CSF-1, MCSF-1, etc.

[0185] In certain embodiments, a CD123 antibody is used in combination with one or more other therapeutic antibodies, including but not limited to antibodies targeting CD19, CD22, CD40, CD52, or CCR4.

[0186] In some embodiments, the CD123 antibody therapy can precede the other agent treatment by an interval ranging from minutes to weeks, or can follow the other agent treatment by an interval ranging from minutes to weeks. In embodiments in which the other agent and / or protein or polynucleotide is administered separately, one will generally ensure that no significant period of time passes between each delivery, so that the agents and compositions of the present invention will still be able to exert their advantageous combined effect on the subject. In such cases, it is contemplated that both modalities can be administered within about 12 to 24 hours of each other, more preferably within about 6 to 12 hours of each other. However, in some situations, where several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) pass between the respective administrations, it may be desirable to extend the period for administration considerably.

[0187] In certain embodiments, the CD123 antibody composition comprises a second agent selected from crizotinib, palbociclib, gemcitabine, cyclophosphamide, fluorouracil, FOLFOX, folinic acid, oxaliplatin, axitinib, sunitinib malate, tofacitinib, bevacizumab, rituximab, and traztuzumab.

[0188] In certain embodiments, the CD123 antibody composition is combined with a treatment regimen that further comprises traditional therapies selected from the group including surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, anti-angiogenic therapy, and palliative care.

[0189] The compositions used in the present invention may be in the form of lyophilized formulations or aqueous solutions and may further include pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington: The Science and Practice of Pharmacy, 21st Edition, 2005, Lippincott Williams and W ilkins, editor KE Hoover). An acceptable carrier, excipient, or stabilizer is These agents are non-toxic to the recipient in the dosages and concentrations and include buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins. The excipient may include immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0190] Polynucleotides, Vectors and Host Cells The present invention also provides isolated polynucleotides encoding the antibodies of the present invention, as well as vectors and host cells comprising the polynucleotides. In another aspect, the present invention provides compositions (e.g., pharmaceutical compositions) comprising any of the polynucleotides of the present invention. In some embodiments, the compositions comprise an expression vector comprising a polynucleotide encoding any of the antibodies described herein. In another aspect, the present invention provides methods of making any of the polynucleotides described herein.

[0191] Polynucleotides complementary to any such sequences are also encompassed by the present invention. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond in a one-to-one manner to DNA molecules, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences can, but need not, be present within the polynucleotides of the present invention, and polynucleotides can, but need not be linked to other molecules and / or support materials.

[0192] The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. A variant of a polynucleotide contains one or more substitutions, additions, deletions and / or insertions such that the immunoreactivity of the encoded polypeptide is not diminished compared to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide may generally be assessed as described herein. The variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity to the polynucleotide sequence encoding a natural antibody or a portion thereof.

[0193] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences are the same when aligned for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20, usually about 30 to about 75 or about 40 to about 50, contiguous positions in which a sequence can be compared against a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[0194] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc., Madison, Wis.) with default parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins- Matrices for detecting distant relationships. In Dayhoff, MO (Editor) Atlas of Protein Sequence and Structure, National Biomedical Research Fo undation, Washington, DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Appro ach toAlignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Acad emic Press,Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1 989, CABIOS5:151-153; Myers, EW and Muller W., 1988, CABIOS 4:11-17; Robinson , ED, 1971,Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4 :406-425;Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principle s and Practice of Numerical Taxonomy, Freeman Press, San Diego, CA Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730. Preferably, a "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, where the portion of the polynucleotide or polypeptide sequence in the comparison window can contain up to 20 percent, usually 5-15 percent or 10-12 percent, additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0195] Variants can also (or alternatively) be substantially homologous to a native gene or a portion or complementary sequence thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[0196] Suitable "moderately stringent conditions" include prewashing in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing overnight in 5x SSC at 50°C to 65°C; followed by two washes for 20 minutes each in 2x, 0.5x and 0.2x SSC containing 0.1% SDS at 65°C.

[0197] As used herein, "highly stringent conditions" or "high stringency conditions" refers to (1) the use of low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50° C.; (2) the use of a denaturing agent during hybridization, e.g., formamide, e.g., 50% (v / v) formamide, with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer (pH 6.5), 750 mM sodium chloride, 75 mM sodium citrate, at 42° C.; or (3) 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42° C., washed in 0.2× SSC (sodium chloride / sodium citrate) at 42° C., washed in 50% formamide at 55° C., followed by a high stringency wash consisting of 0.1× SSC containing EDTA at 55° C. One of skill in the art will know how to adjust temperature, ionic strength, etc. as necessary to accommodate factors such as probe length, etc.

[0198] Those skilled in the art will understand that there are many nucleotide sequences that code for the polypeptides described herein as a result of the degeneracy of the genetic code. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. Nevertheless, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes that include the polynucleotide sequences provided herein are within the scope of the present invention. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as deletion, addition, and / or substitution of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using standard techniques (e.g., hybridization, amplification, and / or database sequence comparison).

[0199] The polynucleotide of the present invention can be obtained by chemical synthesis, recombinant method or PCR.The method of chemical polynucleotide synthesis is well known in the art and does not need to be described in detail here.Those skilled in the art can use the sequences provided herein and commercial DNA synthesis equipment to generate desired DNA sequences.

[0200] To prepare a polynucleotide using recombinant methods, a polynucleotide containing a desired sequence can be inserted into a suitable vector, which in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein. The polynucleotide can be inserted into the host cell by any means known in the art. The cell is transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained in the cell as a non-integrated vector (e.g., a plasmid) or can be integrated into the host cell genome. The polynucleotide thus amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., J. Molecular Biology 1999, 143:1311-1323, 1999. See et al., 1989.

[0201] Alternatively, PCR allows the duplication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202 and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0202] RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA is then expressed as, for example, as described in Sambrook et al., 1989 (supra). The peptides can be isolated using methods well known to those skilled in the art.

[0203] A suitable cloning vector can be constructed according to standard techniques or can be selected from the many cloning vectors available in the art. Although the cloning vector selected can vary depending on the host cell intended to be used, a useful cloning vector will generally be capable of autonomous replication, can have a single target for a particular restriction endonuclease, and / or can have a gene for a marker that can be used in the selection of clones containing the vector. Suitable examples include plasmids and bacterial viruses, such as pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, such as pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Strategene, and Invitrogen.

[0204] An expression vector is generally a replicable polynucleotide construct that contains the polynucleotide of the present invention. It is implied that an expression vector must be replicable in a host cell, either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, adenoviruses, adeno-associated viruses, retroviruses, viral vectors including cosmids, and the expression vector(s) disclosed in PCT Publication WO 87 / 04462. Vector components generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; appropriate transcriptional regulatory elements (e.g., promoters, enhancers and terminators). For expression (i.e., translation), one or more translational regulatory elements are usually also required, such as a ribosome binding site, a translation initiation site, and a stop codon.

[0205] The vector containing the polynucleotide of interest can be introduced into the host cell by any of several suitable means, including electroporation, transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (e.g., when the vector is an infectious agent such as vaccinia virus).The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0206] The present invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating the gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS cells, HeLa cells and CHO cells. See also PCT Publication WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., E. coli or B. subtillis) and yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe; or K. lactis). Preferably, the host cell expresses the cDNA at a level about 5-fold higher, more preferably about 10-fold higher, and even more preferably about 20-fold higher than the level of the corresponding endogenous antibody or protein of interest (if present) in the host cell. Screening of the host cells for specific binding to CD123 is accomplished by immunoassay or FACS. Cells overexpressing the antibody or protein of interest can be identified.

[0207] kit The present invention also provides kits for use in the instant methods.The kits of the present invention include one or more containers containing the CD123 antibody or CD123 ADC described herein and instructions for use according to any of the methods of the present invention described herein.Generally, these instructions include instructions for administration of the CD123 antibody or CD123 ADC for the above therapeutic treatment.

[0208] The instructions for use of the CD123 antibodies or CD123 ADCs described herein generally include information regarding dosages, administration schedules, and routes of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages), or small unit doses. The instructions provided in the kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0209] The kit of the present invention is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Packaging for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., an atomizer), or an infusion device, such as a minipump, is also contemplated. The kit can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a CD123 antibody. The container can further contain a second pharma- ceutically active agent.

[0210] The kit can optionally provide additional components, such as buffers and instructional information. Typically, the kit comprises a container and a label or package insert(s) on or associated with the container.

[0211] biological deposit Representative material of the invention was deposited with the American Type Culture Collection (ATCC) on June 29, 2017. The vector having ATCC Accession Number PTA-124283 is a polynucleotide encoding a humanized CD123 antibody heavy chain sequence, and the vector having ATCC Accession Number PTA-124284 is a polynucleotide encoding a humanized CD123 antibody light chain sequence. The deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Regulations Thereunder (Budapest Treaty), which ensures maintenance of a viable culture of the deposit for 30 years from the date of deposit. The deposit will be made available by ATCC under the terms of the Budapest Treaty, and is considered to be subject to an agreement between Pfizer Inc. and ATCC, which assures permanent and unlimited availability to the public of the progeny of the cultures of the deposit upon the issuance of the relevant U.S. patent or upon the laying open to the public of any U.S. or foreign patent application, whichever occurs first, and assures availability of the progeny to those persons determined by the Director of the United States Patent and Trademark Office to be entitled thereto in accordance with 35 U.S.C. 122 and the Director's regulations thereunder (including 37 CFR 1.14, and particularly as related to 886 OG 638).

[0212] The assignee of the present application has agreed that if a culture of the deposited material should die or be lost or destroyed when cultured under appropriate conditions, the material will be promptly replaced with another of the same material upon notice. Availability of the deposited material is not to be construed as a license to practice the invention in violation of rights granted under the authority of any government pursuant to its patent laws. EXAMPLES

[0213] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing and fall within the scope of the appended claims.

[0214] Example 1: In vitro cytotoxicity of CD123 antibody drug conjugates This example illustrates the cytotoxicity of various CD123 ADCs. In this study, the cytotoxicity of various CD123 ADCs was tested using a 2D in vitro cytotoxicity assay in the following AML cell lines: MOLM13, MV411, JVM3, Granata519, OCI-AML3, NB4 and HL60. Table 4 shows the expression levels of CD123 for each of the respective cell lines used.

[0215] [Table 5]

[0216] The first group of ADCs tested were CD123 ADCs conjugated to CPIs with AcLysValCitPABC linkers. These ADCs were 18G3-CPI, 16D6-CPI, 3D1-CPI and 20D7-CPI, each of which has a drug:antibody ratio (DAR) of about 2. The preparation of the CD123 ADCs is described in detail in Example 5 below. A control IgG (an IgG that does not bind to CD123) with a CPI drug with a DAR of about 2 ("Neg.8.8") was used as a negative control.

[0217] For 2D in vitro cytotoxicity assay, AML cells were incubated for 96 hours with the following doses of ADC or control: 100ng / ml, 25ng / ml, 6.25ng / ml, 1.56ng / ml, 0.39ng / ml, 0.09ng / ml, 0.024ng / ml, 0.006ng / ml, 0.002ng / ml and 0.0004ng / ml. Cell viability was measured using CelltiterGlo (Promega, Madison, WI) and luminescence was determined using a Victor plate reader (Perkin Elmer, Waltham, MA). Calculations of 50% inhibition values ​​(IC50) were generated using XLfit (IDBS, Boston, MA) 4-parameter curve fitting. Results are summarized in Table 5.

[0218] [Table 6]

[0219] The results show that CD123-CPIs are cytotoxic to cells expressing CD123, and that they are more cytotoxic than non-specific CP1 (Neg8.8-CPI). For example, in MOLM13 cells, the IC50s of CD123 ADCs 18G3-CPI, 16D6-CPI and 3D1-CPI are 0.25±0.04ng / ml, 0.22±0.05ng / ml, 0.33±0.06ng / ml and 0.32±0.14ng / ml, respectively. In contrast, the IC50 of Neg.8.8-CPI was >88.43±18.66 (Table 5, last row). Neg.8.8-CPI, with a DAR of about 2, was substantially less active at the highest dose tested (Table 5, last row). The IC50 values ​​of the CD123 ADCs shown correlate well with the level of CD123 expression on the cells (Tables 4 and 5). For example, the IC50 of 18G3-CPI in MV411 cells, which express high levels of CD123, is 0.41±0.07. In contrast, the IC50 of 18G3-CPI in NB4 cells, which express low levels of CD123, is 63.92±36.08, and the IC50 of 18G3-CPI in HL60 cells, which do not express CD123, is greater than 84.4±33.30.

[0220] When CD123-18G3 ADCs are conjugated to different drugs, both iPr-calicheamicin or CTI (glucuronide), have similar cytotoxic effects. The results of the cytotoxicity assay for 18G3 conjugated to iPr-calicheamicin ("iPr") are summarized in Table 6. The results of the cytotoxicity assay for 18G3 conjugated to CTI (glucuronide) are summarized in Table 7.

[0221] [Table 7]

[0222] [Table 8]

[0223] These data demonstrate that CD123-ADCs with a DAR of approximately 2 are active and induce cell death in cancer cell lines expressing CD123, but are inactive in cells that do not express CD123, demonstrating the potency and specificity of these ADCs.

[0224] Example 2: Interleukin-3 (IL-3) signaling pathway and cytotoxicity of CD123 antibodies and ADCs This example illustrates the ability of CD123 antibodies to block IL-3 signaling and the cytotoxicity of CD123 ADCs.

[0225] This study was performed to determine whether any of the CD123 antibody clones competitively bind to the IL-3 binding site in CD123 / IL-3Rα expressing TF-1 cells. CD123 / IL-3Rα expressing TF-1 cells were co-incubated with 20 ng / ml IL-3 and the following CD123 antibodies: 3D1, 18G3 and 16D6 at doses of 100 ng / ml, 25 ng / ml, 6.25 ng / ml, 1.56 ng / ml, 0.39 ng / ml, 0.09 ng / ml, 0.024 ng / ml, 0.006 ng / ml, 0.002 ng / ml and 0.0004 ng / ml. The CD123 antibody 7G3 was used as a benchmark antibody shown to block the IL-3 signaling pathway, and the Neg 8.8 antibody was used as a negative control. For the CTG assay to measure cell viability, cells were treated for 4 days at 37°C. After the treatment period, cells were harvested and proteins were prepared. STAT5, phosphorylated STAT5 and actin levels were analyzed using Western blot analysis. The results of the Western blot are shown in Figure 1.

[0226] The presence of the downstream signaling protein phosphorylated STAT5 ("phospho-Stat5") indicates activation of the IL-3 signaling pathway. Treatment of TF-1 cells with IL-3 alone or IL-3 plus Neg.8.8 control antibody ("8.8") activated IL-3 signaling, as indicated by the presence of phospho-Stat5 (Figure 1). Treatment with CD123 antibody 7G3 blocks IL-3 signaling. CD123 antibody 3D1 also blocked this pathway. In contrast, CD123 antibodies 18G3 and 16D6 did not block IL-3-mediated STAT5 phosphorylation.

[0227] The cytotoxicity of CD123-18G3-CPI and CD123-16D6-CPI was still maintained in the presence of IL-3 (Table 8).

[0228] [Table 9]

[0229] Example 3: In vivo efficacy of CD123-ADC This example illustrates the in vivo efficacy of CD123 ADCs. The antitumor activity of CD123 ADCs was tested in vivo using acute myeloid leukemia (AML) cell line xenograft models. For each model described below, the first dose was given on day 0. Tumors were measured at least once a week and their volumes were calculated using the formula: tumor volume (mm 3 ) = 0.5 × (tumor width 2 ) (tumor length). Mean tumor volume (± standard error of the mean) for each treatment group was calculated including 10 animals. All animal studies were performed in an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility under the guidelines of the Institutional Animal Care and Use Committee and appropriate animal study approvals. CD123 ADC demonstrated high efficacy in a dose-dependent manner in cell lines with assorted genetic mutations or overexpressed genes and / or proteins.

[0230] AH1 MOLM13 AML xenograft The antitumor activity of CD123 ADCs was examined against in vivo growth of human tumors in NOD / SCID immunodeficient mice. For the subcutaneous (sc) AML model, 5×10 6 MOLM13 cells were implanted subcutaneously into the flanks of female mice. Tumors grew to 200 mm 3 Upon reaching a mean volume of 1000 mg / kg, animals were staged to ensure uniformity of tumor size among various treatment groups. MOLM13 AML sc xenograft models were intravenously administered PBS vehicle, CD123-CPI (18G3, 16D6 or 3D1) at 0.3 mg / kg or 1 mg / kg, or control Neg-8.8-CPI at 0.3 mg / kg or 1 mg / kg, four times every four days (Q4dx4). Data are summarized in Table 9.

[0231] [Table 10-1]

[0232] [Table 10-2]

[0233] CD123-CPI significantly suppressed tumors compared to Neg.8.8-CPI (Table 9). At a dose of 0.3 mg / kg, all three CD123 ADCs: 18G3-CPI, 16D6-CPI, and 3D1-CPI were cytotoxic. By day 17, 10 of 10 animals in each group showed tumor regression. All of these mice remained tumor-free at least until the end of the study, day 60. At the 1 mg / kg dose of CD123 ADC, tumor regression occurred earlier, by day 12.

[0234] These data demonstrate that CD123-CPI suppresses the growth of MOLM13 AML xenograft tumors. To test the efficacy of CD123-ADC at lower doses, an in vivo efficacy study was performed using the same MOLM13 model. Animals were treated as described above. As shown below in Table 10, CD123-CPI at 0.1 mg / kg suppressed tumor growth in all mice very effectively and in a dose-dependent manner (Table 10, center column "18G3-CPI" and "16D6-CPI"). In contrast, tumors in mice treated with Neg.8.8-CPI continued to grow (Table 10, right column "Neg.8.8-CPI").

[0235] [Table 11-1]

[0236] [Table 11-2]

[0237] To test the efficacy of CD123-18G3-CTI, in vivo efficacy test was carried out using the same MOLM13 model.Animals were treated as described above.As shown in Table 11, treatment with CD123-18G3-CTI suppresses tumor growth in mice in a dose-dependent manner.In contrast, tumors in mice treated with Neg.8.8-CTI continue to grow.

[0238] [Table 12-1]

[0239] [Table 12-2]

[0240] BH2 MV4-11 AML xenograft The effect of CD123 ADCs on human tumor growth was evaluated in immunodeficient mice. For a subcutaneous (sc) AML model, 5×10 6 MV4-11 cells were implanted subcutaneously into the flank of female NOD-SCID mice. Tumors were 200 mm 3 Upon reaching a mean tumor volume of 1000 mg / kg, animals were staged to ensure uniformity of tumor size among the various treatment groups. MV4-11 AML sc xenograft animals were administered PBS vehicle, CD123-CPI or 8.8-CPI intravenously every 4 days for 4 doses (Q4dx4) at the following doses: 0.1, 0.3 and 0.6 mg / kg. Data are summarized in Table 12. CD123-18G3-H16-CPI was compared against Neg 8.8-CPI and PBS vehicle.

[0241] The 0.6 mg / kg dose of CD123-CPI was the most potent ADC tested in this study, with 10 of the 10 animals still on study remaining tumor free by day 65. Even at the 0.3 mg / kg dose, 9 of the 10 mice showed tumor regression around day 25 and remained tumor free until the end of the study on day 65. The data demonstrate that CD123-CPI inhibits the growth of MV4-11 xenograft tumors.

[0242] [Table 13]

[0243] To test the efficacy of CD123-CTI, in vivo efficacy test was carried out using the same MV4-11 model.Animals were treated as described above.As shown in Table 13, CD123-CTI also suppresses tumor growth in mice in a dose-dependent manner, while the tumors in mice treated with Neg.8.8-CTI continue to grow.

[0244] [Table 14-1]

[0245] [Table 14-2]

[0246] These results demonstrate that CD123-ADCs are highly effective in treating tumors. Example 4: In vivo efficacy of CD123-ADC This example illustrates the in vivo efficacy of CD123-ADC using AML patient-derived disseminated xenografts (AML PDX).

[0247] The efficacy of CD123-CPI was tested against in vivo growth in immunodeficient mice in a disseminated model established with patient bone marrow cells obtained following appropriate consent procedures. Table 14 provides a summary of the patient samples used in this study.

[0248] [Table 15]

[0249] For that test, 1.0 x 10 6 Bone marrow cells from patients were injected intravenously into the lateral tail vein of irradiated NSG mice. The study was staged and randomized based on engraftment of human CD45+ / CD123+ / CD33+ cells (12-55% in peripheral blood as measured by flow cytometry staining). AML PDX mice were intravenously administered PBS vehicle or 18G3-CPI twice every 7 days (Q7dx2). Peripheral blood and bone marrow were collected from sacrificed mice approximately 3-5 days after the second / last dose. Tumor cells remaining in bone marrow and blood of sacrificed animals were analyzed by flow cytometry. The results are summarized in the graph in Figure 2 and Table 15. In Table 15, the numbers represent the percentage of remaining tumor cells in peripheral blood and bone marrow.

[0250] Each study has approximately 6-10 mice per group. The data demonstrate that CD123 ADCs are effective in reducing tumor cell numbers in both peripheral blood and bone marrow in a dose-dependent manner (Figure 2 and Table 15).

[0251] [Table 16-1]

[0252] [Table 16-2]

[0253] To determine whether CD123-18G3-CPI can increase the overall survival of tumor-bearing mice, a survival study was performed with PDX2407 when peripheral blood showed high engraftment, i.e., about 32% on average. Mice were intravenously administered PBS vehicle or CD123-CPI twice every 7 days (Q7dx2) and monitored daily. If mice showed clinical signs such as lethargy and weight loss, they were euthanized (under the Institutional Animal Care and Use Committee guidelines, in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care).

[0254] The results are summarized in the graph of Figure 3. Treatment with CD123-18G3-CPI extends the overall survival of tumor-bearing animals in a dose-dependent manner. Specifically, treatment with CD123-18G3-CPI at a dose of 0.1 mg / kg extends survival by about 25%, and treatment with CD123-18G3-CPI at a dose of 0.3 mg / kg approximately doubles the length of survival (Figure 3).

[0255] These results demonstrate that treatment with CD123-ADC induces regression of AML, inhibits progression, and prolongs survival. Example 5: Preparation of CD123 antibody drug conjugates (ADCs) This example describes the conjugation and preparation of h18G3-AcLysValCitPABC-DMAE-CO_CPI-000638314, a CD123 ADC (also referred to herein as "18G3-CPI" or "CD123-18G3-CPI").

[0256] 18G3-CPI is an ADC composed of CD123 humanized mAb 18G3 (see "h18G3" in Tables 2.0 and 2.1 above), an AcLysValCitPABC linker, and a cyclopropylpyrroloindoline (CPI) drug. K222R, E295L, Q295L, Y296Q, N297G mutations were introduced in the upper hinge and Fc to allow site-specific conjugation of the CPI drug catalyzed by transglutaminase. The light and heavy chain amino acid sequences of the CD123-18G3-H16-N60G-K222R humanized IgG1 antibody (referred to herein as "h18G3") are as follows: Light chain:

[0257] [ka]

[0258] Heavy Chain:

[0259] [ka]

[0260] The H16 site-specific mutation sequence is in bold italics above, and contains five H16 site-specific transglutaminase mutations K222R (in hinge), E294L, Q295L, Y296Q and N297G. The LLQG sequence is a recognition tag for transglutaminase, and the linker agent is conjugated at Q296. The G in CDR-H2 (underlined) is the N60G mutation.

[0261] The linker agent, AcLysValCitPABC-DMAE-CO_CPI, is site-specifically conjugated at amino acid Q296. The structure of the AcLysValCitPABC-DMAE-CO_CPI-000638314 linker agent is shown below.

[0262] [ka]

[0263] h18G3 was conjugated to AcLysValCitPABC-DMAE-CO_CPI-000638314 linker agent via the use of bacterial transglutaminase (Sigma, 45 U / mg protein). Specifically, the antibody was exchanged into a buffer solution (pH 7) containing 100 mM KPO4, 200 mM NaCl. The linker agent was added at a 10-fold molar excess over the antibody in the presence of 7.5% (v / v) dimethyl sulfoxide (DMSO). The enzymatic reaction was initiated by the addition of 1 U of bacterial transglutaminase per mg of antibody and incubated overnight at 25°C with continuous mixing.

[0264] The reaction mixture was incubated with 15% isopropyl alcohol for 30 minutes at room temperature. It was then diluted into 4 volumes of 1M KPO4 buffer and purified via hydrophobic interaction chromatography (HIC) using a Butyl-Sepharose HP column (GE Lifesciences). The method utilized 1M KPO4, 50 mM Tris, pH 7 for binding, and the ADC was eluted with 50 mM Tris, pH 7 over 10 CV. The HIC purified compound was dialyzed into a final buffer of 20 mM histidine, 85 mg / mL sucrose, pH 5.8. The ADC was further characterized by SEC for purity and reversed-phase chromatography to calculate the drug-antibody ratio. Protein concentration was determined by UV spectrophotometry.

[0265] The lead antibody has very good expression up to 700mg / L when evaluated in CHO pools, with 88% yield recovery and 99% purity after three steps of purification. 18G3 performed very well in conjugation as assessed by achieving a DAR of 1.9-2.0 and 55-60% conjugation yield after purification. The resulting ADC, CD123-18G3-H16-N60G-K222R-hG1-(Q)AcLysValCitPABC-DMAE-CO_CPI, showed good thermal stability and molecular integrity.

[0266] Example 6: Method for conjugating antibodies to AcLysValCitPABC-DMAE-CO_CPI-000638314 using transglutaminase This example describes the conjugation of an antibody to the linker agent AcLysValCitPABC-DMAE-CO_CPI-000638314 (also referred to herein as AcLysPABC-CPI-8314).

[0267] Conjugation of CPI linker-cytotoxic drugs. Conjugation of AcLysPABC-CPI-8314 (structure shown above in Example 5) to the H16 site of the antibody was optimized by varying a variety of different parameters, such as molar and salt composition, pH, enzyme concentration, time and temperature (Table 16). Briefly, the antibody was buffer exchanged into the appropriate buffer and pH in each of the conditions shown in Table 16. A 10-fold molar excess of AcLysPABC-CPI-8314 linker drug was added to the antibody, and 5-10% dimethyl sulfoxide was added to solubilize the linker drug in the reaction mixture. After addition of bacterial transglutaminase (0.5-5 U / mg antibody), the reaction mixture was continuously mixed for the specified time and temperature. Unreacted linker drug and transglutaminase enzyme were removed by either size exclusion chromatography and / or hydrophobic interaction chromatography. Drug-antibody ratios were calculated by LCMS or RP-HPLC.

[0268] Conjugation efficiency as measured by antibody drug ratio (DAR) for various conditions is shown in Table 16. For example, when the antibody was exchanged into a pH 7 buffer containing 30 mM KPO4, 150 mM NaCl, a DAR of 1.6 was achieved.

[0269] Antibodies targeting CD33, CD123, Her2, PRLR, CD22 and other antigens (including antigens) were conjugated to the linker agent AcLysValCitPABC-DMAE-CO_CPI-000638314 using these optimized conditions.

[0270] [Table 17]

[0271] Although the disclosed teachings have been described with reference to various applications, methods, kits and compositions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the invention as claimed below. The foregoing examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings given herein. Although the present teachings have been described with reference to these exemplary embodiments, those skilled in the art will readily appreciate that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the present teachings.

[0272] All references cited herein, including patents, patent applications, articles, textbooks, etc., and references cited therein, are hereby incorporated by reference in their entirety to the extent that they are not already cited. In the event that one or more of the incorporated literature and similar materials differs from or conflicts with this application (including, but not limited to, defined terms, term usage, techniques described, etc.), this application controls.

[0273] The foregoing description and examples detail certain specific embodiments of the invention and describe the best mode contemplated by the inventors. However, no matter how detailed the foregoing may appear in the text, it will be understood that the invention can be practiced in many ways and that the invention should be construed according to the appended claims and any equivalents thereof. Without being limited thereto, the present invention includes the following aspects. [Embodiment 1] An isolated antibody that specifically binds to CD123, the antibody comprising a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs) of VH comprising the amino acid sequence of SEQ ID NO:6, 24, 32, 44, 51 or 64, and a light chain variable region (VL) comprising three CDRs of VL comprising the amino acid sequence of SEQ ID NO:17, 28, 39, 48, 57, or 71. [Aspect 2] The antibody according to aspect 1, wherein the VH comprises: (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 33, 52 or 65; (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 7, 33, 52 or 65; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9, 35, 46 or 67; and / or the VL region comprises (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18, 40, 58 or 72; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19, 42, 60 or 74; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20, 42, 60 or 74. [Aspect 3] An antibody described in aspect 2, wherein the VH comprises (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO:7; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO:25; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO:9; and / or the VL comprises (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO:18; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO:19; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO:20. [Embodiment 4] An antibody according to embodiment 3, wherein the VH comprises the sequence shown in SEQ ID NO:24 or a variant thereof having one or several conservative amino acid substitutions at residues not within the CDRs, and / or the VL comprises the amino acid sequence shown in SEQ ID NO:28 or a variant thereof having one or several amino acid substitutions at amino acids not within the CDRs. [Embodiment 5] An antibody according to embodiment 3, wherein the antibody comprises a light chain comprising the sequence shown in SEQ ID NO:30 and a heavy chain comprising the sequence shown in SEQ ID NO:27. [Embodiment 6] An isolated antibody that specifically binds to CD123, the antibody comprising a heavy chain variable region produced by an expression vector having ATCC accession number PTA-124283 and a light chain variable region produced by an expression vector having ATCC accession number PTA-124284. [Aspect 7] An antibody that specifically binds to CD123 and competes with the antibody of any one of Aspects 1 to 6 for binding to the CDR. [Embodiment 8] The antibody according to any one of embodiments 1 to 7, wherein the antibody comprises an engineered acyl donor glutamine-containing tag at a specific site. [Aspect 9] The antibody according to aspect 8, wherein the acyl donor glutamine-containing tag is selected from the group consisting of Q, LQG, LLQGG (SEQ ID NO: 77), LLQG (SEQ ID NO: 78), LSLSQG (SEQ ID NO: 79), GGGLLQGG (SEQ ID NO: 80), GGGLLQGG (SEQ ID NO: 81), GGGLLQGG (SEQ ID NO: 82), GGGLLQGG (SEQ ID NO: 83), GGGLLQGG (SEQ ID NO: 84), GGGLLQGG (SEQ ID NO: 85), GGGLLQGG (SEQ ID NO: 86), GGGLLQGG (SEQ ID NO: 87), GGGLLQGG (SEQ ID NO: 88), GGGLLQGG (SEQ ID NO: 89), GGGLLQGG (SEQ ID NO: 90), GGGLLQGG (SEQ NO:80), GLLQG(SEQ ID NO:81), LLQ, GSPLAQSHGG(SEQ ID NO:82), GLLQGGG(SEQ ID NO:83), GLLQGG(SEQ ID NO:84), GLLQ(SEQ ID NO:85), LLQLLQGA(SEQ ID NO:86), LLQGA(SEQ ID NO:87), LLQYQGA(SEQ ID NO:88), LLQGSG(SEQ ID NO:89), LLQYQG(SEQ ID NO:90), LLQLLQG(SEQ ID NO:91), SLLQG(SEQ ID NO:92), LLQLQ(SEQ ID NO:93), LLQLLQ(SEQ ID NO:94), LLQGR(SEQ ID NO:95), LLQGPP(SEQ ID NO:96), LLQGPA(SEQ LLQGPGK(SEQ ID NO:100), LLQGPG(SEQ ID NO:101), LLQGP(SEQ ID NO:102), LLQP(SEQ ID NO:103), LLQPGK(SEQ ID NO:104), LLQAPGK(SEQ ID NO:105), LLQGAPG(SEQ ID NO:106), LLQGAP(SEQ ID NO:107) and LLQLQG(SEQ ID NO:108). [Aspect 10] An antibody according to aspect 11, wherein the acyl donor glutamine-containing tag is LLQG (SEQ ID NO:78). [Embodiment 11] An antibody according to embodiment 9 or 10, wherein the antibody further comprises an amino acid modification at position 222, 340, or 370. [Aspect 12] The antibody according to aspect 11, wherein the amino acid modification is a substitution of lysine with arginine. [Aspect 13] The antibody according to aspect 12, wherein the amino acid modification is K222R. [Aspect 14] The antibody according to any one of aspects 1 to 12, wherein the antibody comprises a linker. [Aspect 15] An antibody according to aspect 14, wherein the linker is a cleavable linker. [Aspect 16] An antibody according to aspect 14 or 15, wherein the linker is selected from the group consisting of Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2- An antibody selected from the group consisting of Val-Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine, and Ac-Lys-putrescine. [Aspect 17] An antibody according to aspect 16, wherein the linker is Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl). [Aspect 18] A conjugate of the antibody according to any one of aspects 1 to 17, wherein the antibody is conjugated to a drug. [Embodiment 19] The conjugate according to embodiment 18, wherein the drug is selected from the group consisting of a cytotoxic drug, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide. [Embodiment 20] A conjugate according to embodiment 19, wherein the cytotoxic drug is selected from the group consisting of anthracyclines, auristatins, camptothecins, combretastatins, CBI dimers, cyclopropylpyrroloindoline (CPI) dimers, CTI dimers, dolastatins, duocarmycins, enediynes, geldanamycins, indolinobenzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterins, spliceostatins, pladienolides, and stereoisomers, isosteres, analogs, or derivatives thereof. [Embodiment 21] A conjugate according to embodiment 19 or 20, wherein the cytotoxic drug is a CPI dimer. [Embodiment 22] A conjugate according to embodiment 21, wherein the CPI dimer is C31H31Cl2N4O7P, or a pharma- ceutically acceptable salt or solvate thereof, including C31H31Cl2N4O7P·C2HF3O2. [Aspect 23] A conjugate of the antibody according to any one of Aspects 19 to 21, the conjugate comprising the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic drug). [Aspect 24] A conjugate according to aspect 23, wherein the acyl donor glutamine-containing tag comprises the amino acid sequence LLQG (SEQ ID NO:78) and the linker comprises acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl. [Embodiment 25] A conjugate according to embodiment 23 or 24, wherein the acyl donor glutamine-containing tag is inserted into the antibody at positions E294 to N297. [Embodiment 26] The conjugate according to any one of embodiments 18 to 25, wherein the conjugate comprises an amino acid substitution of lysine to arginine at position 222 of the antibody according to the EU index numbering of Kabat. [Aspect 27] A pharmaceutical composition comprising a therapeutically effective amount of the antibody according to any one of aspects 1 to 17 or the conjugate according to any one of aspects 18 to 26, and a pharma- ceutically acceptable carrier. [Embodiment 28] An isolated polynucleotide comprising a nucleotide sequence encoding the antibody according to any one of embodiments 1 to 17. [Aspect 29] A vector comprising the polynucleotide described in Aspect 28. [Aspect 30] An isolated host cell that recombinantly produces the antibody according to any one of aspects 1 to 17. [Aspect 31] A method for producing an antibody, comprising culturing a host cell according to aspect 30 under conditions which result in the production of the antibody, and isolating the antibody from the host cell or culture. [Aspect 32] A method for treating a condition associated with cells expressing CD123 in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition described in aspect 27. [Aspect 33] The method according to aspect 32, wherein the condition is cancer. [Embodiment 34] The method according to embodiment 33, wherein the cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, and / or other myelomas. macroglobulienemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmactyic lymphoma lymphoma), Waldenström macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma with inflammation, intravascular large cell The method wherein the cancer is selected from the group consisting of B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated monomulticentric Castleman disease, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with features intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell associated lymphomas. [Aspect 35] The method according to aspect 34, wherein the cancer is AML. [Aspect 36] A method for suppressing tumor growth or progression in a subject having malignant cells expressing CD123, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described in aspect 27. [Aspect 37] A method for suppressing metastasis of malignant cells expressing CD123 in a subject, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition described in Aspect 27. [Aspect 38] A method for inducing tumor regression in a subject having malignant cells expressing CD123, comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition described in aspect 27. [Embodiment 39] A method for conjugating an antibody to AcLysValCitPABC-DMAE-CO_CPI-000638314 (AcLysPABC-CPI-8314), comprising the steps of: preparing a composition comprising an antibody and AcLysPABC-CPI-8314 in a buffer comprising 30-100 mM KPO4 and 150-200 mM NaCl; adding bacterial transglutaminase to the composition; and incubating the composition to allow conjugation of the antibody to the AcLysPABC-CPI-8314; The method includes: [Embodiment 40] The method described in embodiment 39, wherein the composition has a pH of 7. [Aspect 41] A method according to aspect 39 or 40, wherein the composition contains 0.5 to 2 units (U) of bacterial transglutaminase per 1 mg of antibody. [Aspect 42] The method according to any one of Aspects 39 to 41, wherein the composition comprises 1 U of bacterial transglutaminase per 1 mg of antibody. [Aspect 43] The method according to any one of Aspects 39 to 42, wherein the AcLysPABC-CPI-8314 is present in a 10-fold molar excess relative to the antibody. [Aspect 44] A method according to any one of aspects 39 to 43, wherein the composition is incubated overnight at 25°C with continuous mixing. [Aspect 45] The method according to any one of aspects 39 to 44, wherein the composition further comprises 7.5% (v / v) dimethyl sulfoxide (DMSO). [Embodiment 46] The method according to any one of embodiments 39 to 45, wherein the buffer solution comprises 30 mM KPO4 and 150 mM NaCl. [Embodiment 47] The method according to any one of embodiments 39 to 45, wherein the buffer solution comprises 100 mM KPO4 and 200 mM NaCl.

Claims

1. An isolated antibody that specifically binds to CD123, the antibody comprising: (i) a complementarity determining region (CDR) 1 (also known as CDR1) of VH comprising the amino acid sequence of SEQ ID NO: 33; (ii) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 34 or 45; and (iii) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 35 or 46 A heavy chain variable region (VH), and (i) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 40; (ii) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 41; and (iii) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 42 A light chain variable region (VL) comprising Including, antibody.

2. The antibody of claim 1, wherein the VH comprises an amino acid sequence of SEQ ID NO:44 or a variant thereof having at least one conservative amino acid substitution at a residue that is not within the CDRs; and / or An antibody wherein the VL comprises the amino acid sequence of SEQ ID NO:48 or a variant thereof having at least one conservative amino acid substitution at a residue that is not within a CDR.

3. The antibody of claim 2, a light chain comprising the amino acid sequence of SEQ ID NO: 49; and An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:

47.

4. The antibody of claim 1, and / or the VH comprises the amino acid sequence of SEQ ID NO: 32 or a variant thereof having at least one conservative amino acid substitution at a residue that is not within a CDR; An antibody wherein the VL comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least one conservative amino acid substitution at a residue that is not within a CDR.

5. The antibody of claim 4, a light chain comprising the amino acid sequence of SEQ ID NO: 43; and An antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:

37.

6. The antibody described in claim 1, wherein the antibody comprises an engineered acyl donor glutamine-containing tag at a specific site.

7. The antibody of claim 6, wherein the acyl donor glutamine-containing tag is selected from the group consisting of Q, LQG, LLQGG (SEQ ID NO: 77), LLQG (SEQ ID NO: 78), LSLSQG (SEQ ID NO: 79), GGGLLQGG (SEQ ID NO: 80), GLLQG (SEQ ID NO: 81), LLQ, GSPLAQSHGG (SEQ ID NO: 82), GLLQGG (SEQ ID NO: 83), GLLQGG (SEQ ID NO: 84), GLLQ (SEQ ID NO: 85), LLQLLQGA (SEQ ID NO: 86), LLQGA (SEQ ID NO: 87), LLQYQGA (SEQ ID NO: 88). NO: 88), LLQGSG (SEQ ID NO: 89), LLQYQG (SEQ ID NO: 90), LLQLLQG (SEQ ID NO: 91), SLLQG (SEQ ID NO: 92), LLQLQ (SEQ ID NO: 93), LLQLLQ (SEQ ID NO: 94), LLQGR (SEQ ID NO: 95), LLQGPP (SEQ ID NO: 96), LLQGPA (SEQ ID NO: 97), GGLLQGPP (SEQ ID NO: 98), GGLLQGA (SEQ ID NO: 99), LLQGPGK (SEQ ID NO: 100), LLQGPG (SEQ ID LLQGP (SEQ ID NO:101), LLQP (SEQ ID NO:102), LLQPGK (SEQ ID NO:104), LLQAPGK (SEQ ID NO:105), LLQGAPG (SEQ ID NO:106), LLQGAP (SEQ ID NO:107), and LLQLQG (SEQ ID NO:108).

8. The antibody of claim 6, wherein the acyl donor glutamine-containing tag is LLQG (SEQ ID NO: 78).

9. The antibody described in claim 1, further comprising an amino acid modification at position 222, 340, or 370 according to the Kabat EU index numbering.

10. The antibody described in claim 9, wherein the amino acid modification is a substitution of lysine with arginine.

11. The antibody described in claim 10, wherein the amino acid modification is K222R.

12. The antibody of claim 1, wherein the antibody comprises a linker.

13. The antibody according to claim 12, wherein the linker is selected from the group consisting of Ac-Lys-Gly (acetyl-lysine-glycine), aminocaproic acid, Ac-Lys-β-Ala (acetyl-lysine-β-alanine), amino-PEG2 (polyethylene glycol)-C2, amino-PEG3-C2, amino-PEG6-C2, Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl), amino-PEG6-C2-Val -Cit-PABC, aminocaproyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propanoyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine and Ac-Lys-putrescine.

14. The antibody of claim 13, wherein the linker is Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl).

15. A conjugate of an antibody according to any one of claims 1 to 14, wherein the antibody is conjugated to a drug.

16. The conjugate of claim 15, wherein the agent is selected from the group consisting of a cytotoxic agent, an immunomodulatory agent, an imaging agent, a therapeutic protein, a biopolymer and an oligonucleotide.

17. The conjugate of claim 15, wherein the drug is a CPI dimer.

18. The conjugate of claim 15, The conjugate comprises the formula: antibody-(acyl donor glutamine-containing tag)-(linker)-(cytotoxic agent), the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 47 and a light chain comprising the amino acid sequence of SEQ ID NO: 49; the acyl donor glutamine-containing tag comprises the amino acid sequence LLQG (SEQ ID NO: 78); the linker comprises acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl, and The conjugate, wherein the cytotoxic agent is a CPI dimer.

19. A pharmaceutical composition comprising a therapeutically effective amount of an antibody described in any one of claims 1 to 14 or a conjugate described in claim 15, and a pharma- ceutically acceptable carrier.

20. An isolated polynucleotide comprising a nucleotide sequence encoding an antibody described in any one of claims 1 to 14.

21. A vector comprising the polynucleotide described in claim 20.

22. An isolated host cell that recombinantly produces an antibody according to any one of claims 1 to 14.

23. A method for producing an antibody, the method comprising culturing a host cell described in claim 22 under conditions that result in the production of the antibody, and isolating the antibody from the host cell or culture.

24. A method for conjugating an antibody according to any one of claims 1 to 14 to AcLysValCitPABC-DMAE-CO_CPI-000638314 (AcLysPABC-CPI-8314), the method comprising the steps of: preparing a composition comprising an antibody and AcLysPABC-CPI-8314 in a buffer comprising 30-100 mM KPO 4 and 150-200 mM NaCl; adding bacterial transglutaminase to the composition; and incubating the composition to allow conjugation of the antibody to the AcLysPABC-CPI-8314; The method includes:

25. Use of an antibody described in any one of claims 1 to 14 or a conjugate described in claim 15, characterized in that the use is for the preparation of a pharmaceutical composition.

26. The pharmaceutical composition for treating a condition associated with cells expressing CD123, To inhibit tumor growth or progression in a subject having malignant cells that express CD123, To inhibit metastasis of malignant cells expressing CD123 in a subject; and / or 26. The use according to claim 25 for inducing tumor regression in a subject having malignant cells expressing CD123.

27. The use of claim 26, wherein the condition is cancer.

28. The cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, and / or other myelomatous myelomatous leukemia. macroglobulienemia), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma lymphoma), Waldenström macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (lower limb type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma with inflammation, intravascular large B-cell lymphoma 28. The use of claim 27, wherein the cancer is selected from the group consisting of diffuse large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated monomulticentric Castleman disease, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, and other B-cell-associated lymphomas.

29. A pharmaceutical composition comprising an antibody described in any one of claims 1 to 14 or a conjugate described in claim 15.

30. A method for treating a condition associated with cells expressing CD123, comprising administering to said patient a To inhibit tumor growth or progression in a subject having malignant cells that express CD123, To inhibit metastasis of malignant cells expressing CD123 in a subject; and / or 30. The pharmaceutical composition of claim 29 for inducing tumor regression in a subject having malignant cells that express CD123.

31. The pharmaceutical composition of claim 30, wherein the condition is cancer.

32. The cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), hairy cell leukemia, B-cell non-Hodgkin's lymphoma (NHL), multiple myeloma, malignant plasma cell neoplasm, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), follicular lymphoma, Burkitt's lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's macroglobulinemia, and / or other myelomatous myelomatous leukemia. macroglobulienemia), diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma lymphoma), Waldenström macroglobulinemia, nodal marginal zone B-cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (lower limb type), EBV-positive diffuse large B-cell lymphoma of the elderly, diffuse large B-cell lymphoma with inflammation, intravascular large B-cell lymphoma 32. The pharmaceutical composition of claim 31, wherein the cancer is selected from the group consisting of ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated monomulticentric Castleman disease, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-associated lymphomas.

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