Antibodies and antibody-drug conjugates specific for CD123 and uses thereof

Abstract

The present invention provides antibodies that specifically bind to CD123. The invention further relates to immunoconjugates (e.g., antibody-drug conjugates or ADCs) comprising these antibodies, nucleic acids encoding the antibodies and methods of obtaining these antibodies. The invention further relates to therapeutic methods of using these antibodies and ADCs to treat disorders associated with cells expressing CD123, such as cancer or autoimmune diseases.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880069901.2, entitled “Antibodies and Antibody-Drug Conjugates Specific to CD123 and Uses Thereof”, filed on October 16, 2018.

[0002] References to sequence listings

[0003] This application is submitted electronically via EFS-Web, including an electronically submitted sequence listing in .txt format. The .txt file contains a sequence listing titled "PC72333_SEQ_LIST_ST25.txt", created on October 27, 2017, and is 78KB in size. The sequence listing contained in this .txt file is part of this specification and is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention relates to antibodies, such as full-length antibodies specifically binding to CD123. The present invention further relates to a conjugate comprising the CD123 antibody (e.g., antibody-drug conjugate or "ADC"), a composition comprising the CD123 antibody or its conjugate, and a method for treating a disorder (e.g., cancer) associated with cells expressing CD123 using the CD123 antibody or its conjugate. Background Art

[0005] 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 IL-3 signals. The biological effect of IL-3 is to stimulate the survival and proliferation of pluripotent cells. It is believed that CD123 is involved in stimulating the proliferation of acute myeloid leukemia (AML) cells and has a direct function in tumor biology. CD123 is often expressed on leukemia stem cells (LSC), a cell population associated with patient relapse. In normal human tissues, CD123 expression is mostly limited to hematopoietic cells, especially plasmacytoid dendritic cells (pDC), which constitute <0.4% of human peripheral blood mononuclear cells. As a component of innate immunity, pDC produces large amounts of type 1 interferon (IFN-α / β) in response to viral and bacterial stimulation. Importantly, CD123 is not expressed on hematopoietic stem cells.

[0006] New cases of leukemia, lymphoma, and myeloma are expected to account for 10.2% of the 1,688,780 estimated new cancer cases diagnosed in the United States in 2017. CD123 is expressed on cancer cells of a variety of hematological malignancies, including acute myeloid leukemia (AML), where its expression is >80%. Examples of hematological cancer cells expressing CD123 include blasts and leukemic stem cells. Diseases associated with CD123 expression include AML, myelodysplastic syndrome (MDS; low and high risk), acute lymphocytic leukemia (ALL, all subtypes), diffuse large B-cell lymphoma (DLBCL), chronic myeloid leukemia (CML), and blastic plasmacytoid dendritic cell neoplasm (BPDCN).

[0007] Currently, treatments for these diseases include more than 50 individual drugs, with others being studied and clinically tested. Radiation therapy is also often used to treat blood cancers, sometimes administered along with drug therapy. Immunotherapy, gene therapy, and personalized medicine are also used. However, these therapies can have significant side effects and adverse reactions. Therefore, there is a need to provide new and improved treatments for blood cancers that express CD123 (IL-3Rα). Summary of the invention

[0008] The invention disclosed herein relates to antibodies that bind to CD123 and conjugates comprising antibodies that bind to CD123, such as antibody-drug conjugates (ADCs), and methods of making and using these antibodies and ADCs to treat disorders.

[0009] Provided herein are antibodies that specifically bind to CD123. In some embodiments, the present invention provides an isolated antibody that specifically binds to CD123, wherein the antibody comprises: a heavy chain variable region (VH) and a light chain variable region (VL), the heavy chain variable region comprising three complementary determining regions (CDRs) of the following VH, the VH comprising SEQ ID NO:6, 24, 32, 44, 51 or 64 amino acid sequences, the light chain variable region comprising three CDRs of the following VL, the VL comprising SEQ ID NO:17, 28, 39, 48, 57 or 71 amino acid sequences. In some embodiments, the VH may comprise: (i) VH CDR1 comprising SEQ ID NO:7, 33, 52 or 65 amino acid sequences; (ii) VH CDR2 comprising SEQ ID NO:8, 25, 34, 45, 53 or 66 amino acid sequences; and (iii) VH CDR3 comprising SEQ ID NO:9, 35, 46 or 67 amino acid sequences. In some embodiments, the VL region may comprise: (i) a VL CDR1 comprising an amino acid sequence of SEQ ID NO: 18, 40, 58 or 72; (ii) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 19, 42, 60 or 74; and (iii) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 20, 42, 60 or 74. In some embodiments, the VH may comprise: (i) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 7; (ii) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 25; and (iii) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 9. In some embodiments, the VL may comprise: (i) VL CDR1 comprising the amino acid sequence of SEQ ID NO: 18; (ii) VL CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and (iii) VLCDR3 comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH may comprise the sequence shown in SEQ ID NO: 24 or a variant thereof having one or more conservative amino acid substitutions in residues not within the CDRs, and / or the VL may comprise the amino acid sequence shown in SEQ ID NO: 28 or a variant thereof having one or more amino acid substitutions in amino acids not within the CDRs. In some embodiments, the antibody may comprise a light chain and a heavy chain, the light chain comprising the sequence shown in SEQ ID NO: 30, and the heavy chain comprising the sequence shown in SEQ ID NO: 27.

[0010] Also provided is an isolated antibody that specifically binds to 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.

[0011] Also provided is an isolated antibody that specifically binds to CD123 and competes for binding to CD123 with an antibody comprising the following heavy chain variable region (VH) and VL, wherein the heavy chain variable region comprises the three CDRs of the following VH, wherein the VH comprises the amino acid sequence of SEQ ID NO: 6, 24, 32, 44, 51 or 64; and the VL comprises the three CDRs of the following VL, wherein the VL comprises the amino acid sequence of SEQ ID NO: 17, 28, 39, 48, 57 or 71.

[0012] Also provided is an isolated CD123 antibody comprising: a VH comprising the amino acid sequence of SEQ ID NOs: 7, 8 and 9 and a VL comprising the amino acid sequence of SEQ ID NOs: 18, 19 and 20.

[0013] 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.

[0014] Also provided is an isolated CD123 antibody comprising: 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.

[0015] Also provided is an isolated CD123 antibody comprising: 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.

[0016] Also provided is an isolated CD123 antibody comprising: a VH comprising the amino acid sequence of SEQ ID NOs: 52, 53 and 54 and a VL comprising the amino acid sequence of SEQ ID NOs: 58, 59 and 60.

[0017] Also provided is an isolated CD123 antibody comprising: a VH comprising the amino acid sequence of SEQ ID NOs: 65, 66 and 67 and a VL comprising the amino acid sequence of SEQ ID NOs: 72, 73 and 74.

[0018] In some embodiments, a CD123 antibody as described herein may comprise a tag engineered to contain an acyl donor glutamine at a specific site. In some embodiments, the tag can comprise an amino acid sequence 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), 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), In some embodiments, the glutamine-containing tag is LLQG (SEQ ID NO: 78).

[0019] In some embodiments, the CD123 antibodies described herein may include amino acid modifications at positions 222, 340 or 370. In some embodiments, the amino acid modifications may be substitutions of lysine to arginine. In some embodiments, the amino acid modifications may be K222R.

[0020] In some embodiments, the CD123 antibodies described herein may include a joint. In some embodiments, the joint may be cleavable. In some embodiments, the joint may be selected from: 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, aminohexanoyl-Val-Cit-PABC, [(3R,5R)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine and Ac-Lys-putrescine. In some embodiments, the linker may be Ac-Lys-Val-Cit-PABC (acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl).

[0021] In some embodiments, the antibodies described herein comprise a constant region. In some embodiments, the antibodies are humanized antibodies. In some embodiments, the antibodies are of human IgG1, IgG2 or IgG2Δa, IgG3 or IgG4 subtype. In some embodiments, the antibodies are IgG1 antibodies. In some embodiments, the antibodies comprise an N60G mutation.

[0022] A conjugate is also provided, comprising a CD123 antibody as described herein conjugated to an agent. In some embodiments, the agent may be selected from: a cytotoxic agent, an immunomodulator, an imaging agent, a therapeutic protein, a biopolymer, and an oligonucleotide. In some embodiments, the agent may be a cytotoxic agent. In some embodiments, the cytotoxic agent may be selected from anthracyclines, auristatins, camptothecins, combretastatins, CBI dimers, cyclopropylpyrroloindoline (CPI) dimers, CTI dimers, dolastatins, duocarmycins, enediynes, geldanamycins, indolin-benzodiazepine dimers, maytansines, puromycins, pyrrolobenzodiazepine dimers, taxanes, vinca alkaloids, tubulysins, hemiasterlins, spliceostatins, pladienolides, and stereoisomers, isosteres, analogs, or derivatives thereof. In some embodiments, the cytotoxic agent may be a CPI dimer. In some embodiments, the CPI dimer is CPI-8314. In some embodiments, the CPI dimer may have the following structure:

[0023]

[0024] In some embodiments, the cytotoxic agent may have the 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]pentan-1-yl)carbonyl]-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-yl dihydrogen phosphate. In some embodiments, the cytotoxic agent may be C31H31Cl2N4O7P or a pharmaceutically acceptable salt or solvate. In some embodiments, the cytotoxic agent may be in the form of a trifluoroacetic acid (TFA) salt: C31H31Cl2N4O7P.C2HF3O2.

[0025] In other embodiments, the cytotoxic agent can be 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 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(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-oxopropyl)- butyl}-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}-3-methoxy-5-methyl-1-oxohept-4-yl]-N-methyl-L-valinamide amide) 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-oxohept-4-yl]-N-methyl-L-valinamide).

[0026] In some embodiments, the conjugate may comprise the formula: antibody-(tag containing acyl donor glutamine)-(linker)-(cytotoxic agent). In some embodiments, the tag containing acyl donor glutamine may comprise the amino acid sequence LLQG (SEQ ID NO: 78) and the linker may comprise acetyl-lysine-valine-citrulline-p-aminobenzyloxycarbonyl. In some embodiments, the tag containing acyl donor glutamine may be inserted at antibody position E294-N297. In some embodiments, the conjugate may further comprise an amino acid substitution of lysine to arginine (K222R) at antibody position 222 numbered according to the EU index of Kabat.

[0027] 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 pharmaceutically acceptable carrier.

[0028] Also provide isolated polynucleotides, it comprises the nucleotide sequence of coding CD123 antibody described herein.Also provide carrier, it comprises this polynucleotide.

[0029] Also provide isolated host cells, it recombinantly produces any one of the CD123 antibodies described herein.Also provide a method for producing an antibody, including culturing such host cells under conditions leading to the production of the antibody and separating the antibody from the host cell or culture.

[0030] Also provided is a method for treating a disorder associated with a cell expressing CD123 in a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition to a subject in need thereof, the pharmaceutical composition comprising any one of the CD123 antibodies described herein or a conjugate of any of the antibodies and a pharmaceutically acceptable carrier. In some embodiments, the disorder is cancer. In some embodiments, the cancer may be a cancer 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 myeloid leukemia (CML), follicular lymphoma, Burkitt's lymphoma (Burkitt's lymphoma), marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphoblastic lymphoma, myeloid leukemia, Waldenstrom's 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, Waldenstrom's macroglobulinemia 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, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric 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 classic Hodgkin lymphoma, and other B-cell-related lymphomas. In some embodiments, the cancer is AML.

[0031] Also provided is a method of inhibiting tumor growth or progression in a subject having malignant cells expressing CD123, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any one of the CD123 antibodies provided herein or a conjugate of any one of the antibodies and a pharmaceutically acceptable carrier.

[0032] Also provided is a method for 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 one of the CD123 antibodies provided herein or a conjugate of any one of the antibodies and a pharmaceutically acceptable carrier.

[0033] Also provided is a method of inducing tumor regression in a subject having malignant cells expressing CD123, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising any one of the CD123 antibodies provided herein or a conjugate of any one of the antibodies and a pharmaceutically acceptable carrier.

[0034] On the other hand, 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 (e.g., cancer or autoimmune disease) associated with CD123 expression in a subject in need. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) comprises a CD123 antibody or CD123 ADC described herein for suppressing tumor growth or progression in a subject with malignant cells expressing CD123. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) is provided, comprising a CD123 antibody or CD123 ADC described herein for suppressing malignant cell metastasis expressing CD123 in a subject in need. In some embodiments, 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 with malignant cells expressing CD123.

[0035] In another aspect, the present invention provides CD123 antibodies or CD123 ADC as described herein for treating a disorder (e.g., cancer or autoimmune disease) associated with CD123 expression in a subject in need thereof. In some embodiments, CD123 antibodies or CD123 ADC as described herein are provided for suppressing tumor growth or progression in a subject with malignant cells expressing CD123. In some embodiments, CD123 antibodies or CD123 ADC as described herein are provided for suppressing the transfer of malignant cells expressing CD123 in a subject in need thereof. In some embodiments, CD123 antibodies or CD123 ADC as described herein are provided for inducing tumor regression in a subject with malignant cells expressing CD123.

[0036] On the other hand, the present invention provides the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for treating disorders (such as cancer or autoimmune diseases) associated with CD123 expression. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inhibiting tumor growth or progression. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inhibiting the metastasis of malignant cells expressing CD123. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inducing tumor regression.

[0037] Also provided herein is a method of 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 to 100 mM KPO4 and 150 to 200 mM NaCl; adding bacterial transglutaminase to the composition; and incubating the composition to allow the antibody to be conjugated to AcLysPABC-CPI-8314. In some embodiments, the pH of the composition is 7. In some embodiments, the composition comprises 0.5 to 2 units (U) of bacterial transglutaminase per mg of antibody. In some embodiments, the composition comprises 1U of bacterial transglutaminase per mg of antibody. In some embodiments, AcLysPABC-CPI-8314 is present in a 10-fold molar excess to 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) dimethyl sulfoxide (DMSO). In some embodiments, the buffer comprises 30mM KPO4 and 150mM NaCl. In some embodiments, the buffer comprises 100mM KPO4 and 200mM 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

[0039] Figure 1 Depicted are the results of Western blot analysis of cells treated with IL-3 plus antibodies ("Ab"). 7G3 is a benchmark antibody and 3D1, 18G3 and 16D6 are CD123 antibodies; 8.8 is a negative control antibody that does not bind CD123. STAT5, phosphorylated STAT5 and actin levels were analyzed.

[0040] Figure 2 Depicted are representative flow cytometry analyses showing the percentage of tumor cells remaining in the peripheral blood (left panel) and bone marrow (right panel) in animal models following treatment with vehicle or CD123-ADC (CD123-18G3-CPI).

[0041] Figure 3 Depicted graphs summarize the length of survival (in days) of animals treated with vehicle or the indicated doses of CD123 ADC. DETAILED DESCRIPTION OF THE INVENTION

[0043] The invention disclosed herein provides antibodies and antibody-drug conjugates (ADC) that specifically bind CD123 (e.g., human CD123). The present invention also provides polynucleotides encoding these antibodies, compositions comprising these antibodies, and methods for preparing and using these antibodies. The present invention also provides methods for treating conditions such as cancer or autoimmune diseases associated with CD123 expression in a subject.

[0044] General techniques

[0045] 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 the art. These techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JECellis, ed., 1998) Academic Press; Animal Cell Culture(RIFreshney,ed.,1987);Introduction to Cell and Tissue Culture(JPMather and PERoberts,1998)Plenum Press;Cell and Tissue Culture:Laboratory Procedures(A.Doyle,JBGriffiths,and DGNewell,eds.,1993-1998)J.Wiley and Sons;Methods inEnzymology(Academic) Press, Inc.); Handbook of Experimental Immunology (DMWeirand CCBlackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JMMiller and MPCalos, eds., 1987); Current Protocols in Molecular Biology (FMAusubelet al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1987);, 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach approach (P.Shepherd and C.Dean, eds., Oxford University Press, 2000); Using antibodies: alaboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M.Zanetti and JDCapra, eds., Harwood Academic Publishers, 1995).

[0046] definition

[0047] "Antibodies" are immunoglobulin molecules that can specifically bind to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term covers not only complete polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv), single-chain antibodies (ScFv) and domain antibodies (including, for example, shark and camel antibodies), and fusion proteins comprising antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites. Antibodies include antibodies of any type, such as IgG, IgA or IgM (or their subtypes), and the antibodies do not need to be of any particular type. Immunoglobulins can be divided into different types based on the antibody amino acid sequence of the constant region of their heavy chains. There are five major immunoglobulin types: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subtypes (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0048] As used herein, the antibody term "antigen binding fragment" or "antigen binding portion" refers to one or more fragments of a complete antibody that retain the ability to specifically bind to a given antigen (e.g., CD123). The antigen binding function of an antibody can be achieved by a fragment of a complete antibody. Examples of binding fragments encompassed by the antibody term "antigen binding fragment" include Fab, Fab', F(ab')2, Fd fragments consisting of a heavy chain variable region (VH) and a CH1 domain, Fv fragments consisting of the VL and VH domains of a single antibody arm, single domain antibody (dAb) fragments (Ward et al., Nature 341: 544-546, 1989) and isolated complementary determining regions (CDRs).

[0049] The antibody, ADC or polypeptide that "preferentially binds" or "specifically binds" (used interchangeably herein) to a target (e.g., CD123 protein) is a term well known in the art, and the method for determining such specificity or preferential binding is also well known in the art. If the reaction or association of a molecule with a specific cell or substance is more frequent, faster, longer lasting, and / or with higher affinity than other cells or substances, the molecule is referred to as exhibiting "specific binding" or "preferential binding". If an antibody is combined with a target with higher affinity, avidity, faster and / or longer duration than with other substances, the antibody "specifically binds" or "preferentially binds" to the target. For example, an antibody that specifically or preferentially binds to a CD123 epitope refers to an antibody that binds to this epitope with higher affinity, avidity, faster and / or longer duration than with other CD123 epitopes or non-CD123 epitopes. It is also understood by reading this definition that, for example, an antibody (or portion 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 (although it may include) exclusive binding. In general (but not necessarily), reference to binding refers to preferential binding.

[0050] The "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain (whether referred to alone or in combination). As is known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) and three complementarity determining regions (CDRs) connecting the four framework regions. The CDRs in each chain are brought together by the FRs and form the antigen binding site of the antibody with the CDRs of the other chain. There are at least two techniques for determining CDRs: (1) methods based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al., 1997, J. Molec. Biol. 273: 927-948). As used herein, CDRs may refer to CDRs defined by either method or a combination of both methods.

[0051] The "CDRs" of a variable domain are amino acid residues located within the variable region that are identified according to the Kabat definition, the Chothia definition, the cumulative definition of both Kabat and Chothia, the AbM definition, the contact definition and / or the conformational definition, or any CDR determination method known in the art. Antibody CDRs may 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., Public Health Service, NIH, Washington DC. The locations of CDRs may also be identified as structural loop structures first described by Clothia et al. See, e.g., Chothia et al., Nature 342:877-883, 1989. Other methods of identifying CDRs include the "AbM definition," which is a compromise of Kabat and Chothia derived from the use of Oxford Molecular's AbM antibody modeling software (now ), or the "contact definition" of CDRs based on antigen contact observation as described in MacCallum et al., J. Mol. Biol. 262: 732-745, 1996. In another method referred to herein as the "conformational definition" of CDRs, the positions of CDRs may be identified as residues that contribute enthalpy to antigen binding. See, for example, Makabe et al., Journal of Biological Chemistry, 283: 1156-1166, 2008. Other CDR boundary definitions may still not strictly adhere to one of the above methods, but will overlap with at least part of the Kabat CDRs, although they may be shortened or extended based on predictions or experimental results that specific residues or residue groups or even entire CDRs do not significantly affect antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art (including a combination of methods). The methods used herein can utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, contact and / or conformational definitions.

[0052] The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to amino acid chains of any length, preferably relatively short (e.g., 10 to 100 amino acids). The chain may be linear or branched, it may contain modified amino acids and / or may be interrupted by non-amino acids. The term also encompasses amino acid chains modified by natural or human intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as polypeptides containing other modifications known in the art. It should be understood that the polypeptide can exist as a single chain or an associated chain.

[0053] The antibodies of the present invention can be produced using techniques well known in the art, such as recombinant technology, phage display technology, synthetic technology, or a combination of these technologies, or other technologies readily known in the art (see, for example, Jayasena, SD, Clin. Chem., 45: 1628-50, 1999 and Fellouse, FA, et al, J. MoI. Biol., 373 (4): 924-40, 2007).

[0054] As is known in the art, "polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a chain of nucleotides of any length, including DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. A polynucleotide may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be made before or after assembly of the chain. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as, for example, with uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing side groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified bonds (e.g., alpha anomeric nucleic acids, etc.), and unmodified polynucleotide forms. In addition, any hydroxyl group normally present in carbohydrates may be substituted, for example, with phosphonic acid groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional bonds with other nucleotides, or may be conjugated to a solid support. The 5' and 3' end OH can be phosphorylated or replaced by an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized as standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose sugars commonly known in the art, including, for example, 2'-O-methyl-ribose, 2'-O-allyl ribose, 2'-fluoro-ribose or 2'-azido-ribose, carbocyclic sugar analogs, α or β anomeric sugars, diastereomer sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleosides. One or more phosphodiester bonds can be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2 ("methylal"), wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1 to 20 carbons), optionally containing an ether (-O-) linkage, an aryl, an alkenyl, a cycloalkyl, a cycloalkenyl or an araldyl. All bonds in a polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0055] 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, alone or in combination.

[0056] As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free of 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.

[0057] "Host cell" includes an individual cell or cell culture that can or has received a vector for the introduction of a polynucleotide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutation. Host cells include cells transfected in vivo with a polynucleotide of the invention.

[0058] As is known in the art, the term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the immunoglobulin heavy chain Fc region may vary, the human IgG heavy chain Fc region is generally defined as a fragment from the amino acid residue at the Cys226 or Pro230 position to its carboxyl terminus. The numbering of the residues in the Fc region is the EU index numbering as described by Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally includes two constant regions, CH2 and CH3.

[0059] As used herein, the term "competition" with respect to antibodies refers to the binding of a first antibody or its antigen-binding fragment (or portion) to an epitope in a manner sufficiently similar to the binding of a second antibody or its antigen-binding portion, such that the binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody to its cognate epitope in the absence of the second antibody. Alternatively, it may be another case, but not necessarily, that the binding of the second antibody to its epitope is also detectably reduced due to the presence of the first antibody. In other words, the first antibody can inhibit the binding of the second antibody to its epitope, but the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, when various antibodies detectably inhibit the binding of other antibodies to their cognate epitopes or ligands to the same, higher or lower extent, the antibodies are said to "cross-compete" with each other for binding to their respective epitopes. The present invention encompasses both competing antibodies and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), the skilled artisan will appreciate that based on the teachings provided herein, competing and / or cross-competing antibodies are encompassed by the invention and can be used in the methods disclosed herein.

[0060] A "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but still retains at least one effector function of the native sequence Fc region. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of a parent polypeptide, for example, about 1 to about 10 amino acid substitutions in the native sequence Fc region or the Fc region of a parent polypeptide, and preferably about 1 to about 5 amino acid substitutions. The variant Fc region herein will preferably have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region of a parent polypeptide, most preferably at least about 90% sequence identity, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity.

[0061] As used herein, the term "CD123" refers to any form of CD123 and variants thereof that retain at least part of the CD123 activity. Unless otherwise specified, such as specifically referring to human CD123, CD123 includes the natural sequence CD123 of all mammalian species, such as humans, dogs, cats, horses and cattle. Exemplary CD123 sequences are shown in Table 1.

[0062] Table 1

[0063]

[0064]

[0065] As used herein, "CD123 antibody" refers to an antibody that specifically binds to CD123 and regulates biological activity and / or downstream events mediated by CD123. In some embodiments, the CD123 antibody is an antagonist antibody. CD123 antagonists include (including significantly to any extent) blocking, antagonizing, inhibiting or reducing antibodies for CD123 biological activity, and the biological activity includes downstream events mediated by CD123, such as IL-3 binding and / or downstream signaling, STAT5 phosphorylation and pluripotent cell survival. Examples of CD123 antibodies and CD123 antibody-drug conjugates ("CD123 ADC") are provided herein.

[0066] As used herein, "treatment" is a method for obtaining beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical results include but are not limited to the following one or more: reduce (or destroy) the proliferation of tumors or cancer cells, inhibit the metastasis of neoplastic cells, alleviate CD123 related diseases (such as cancer or autoimmune diseases), reduce the symptoms caused by CD123 related diseases (such as cancer or autoimmune diseases), increase the quality of life of people suffering from CD123 related diseases (such as cancer or autoimmune diseases), reduce the dosage of other drugs required for the treatment of CD123 related diseases (such as cancer or autoimmune diseases), delay the progress of CD123 related diseases (such as cancer or autoimmune diseases), cure CD123 related diseases (such as cancer or autoimmune diseases) and / or extend the survival of objects with CD123 related diseases (such as cancer or autoimmune diseases).

[0067] "Improvement" refers to the alleviation or improvement of one or more symptoms compared to not administering CD123 antibody or CD123 ADC. "Improvement" also includes shortening or reducing the duration of symptoms.

[0068] As used herein, the "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition is an amount sufficient to affect any one or more beneficial or desired results. For preventive use, beneficial or desired results include eliminating or reducing risk, reducing severity or delaying the onset of disease, including biochemical, histological and / or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes manifested during disease development. For therapeutic use, beneficial or desired results include clinical results such as the following: reducing the occurrence or improving one or more symptoms of a variety of CD123-related diseases or conditions (such as, for example, but not limited to cancer), reducing the dose of other drugs required for the treatment of the disease, improving the effect of other drugs and / or delaying the progression of CD123-related diseases of the subject. The effective dose can be administered in one or more administrations. For the purposes of the present invention, the effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly complete preventive or therapeutic treatment. As clinically understood, the effective dose of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or accomplished when combined with one or more other agents.

[0069] An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, farm animals, competition animals, pets, primates, horses, dogs, cats, mice, and rats.

[0070] As used herein, "vector" refers to a construct that can deliver and preferably express one or more genes or sequences 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.

[0071] As used herein, an "expression control sequence" is a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter such as a constitutive or inducible promoter or an enhancer. An expression control sequence is operably linked to a nucleic acid sequence to be transcribed.

[0072] As used herein, "pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, enables the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005).

[0073] As used herein, the term "a tag containing the acyl donor glutamine" or "glutamine tag" refers to a polypeptide or protein containing one or more Gln residues that act as an amine acceptor for transglutaminase. See, e.g., PCT Publication Nos. WO2012059882 and WO2015015448, which are incorporated herein by reference in their entirety.

[0074] As used herein, the term “k on " refers to the rate constant for the association of an antibody with an antigen. In particular, the rate constant (k on and k off ) and equilibrium dissociation constants are measured using IgG and CD123 protein (e.g., CD123-Fc fusion protein).

[0075] As used herein, the term “k off ” refers to the rate constant for the dissociation of an antibody from the antibody / antigen complex.

[0076] As used herein, the term "K D ” refers to the equilibrium dissociation constant of the antibody-antigen interaction.

[0077] When "about" a value or parameter is mentioned herein, it includes (and describes) embodiments involving the value or parameter itself. For example, description referring to "about X" includes description of "X". Numerical ranges include the numbers defining the range.

[0078] As used herein, CPI refers to 1,2,8,8a-tetrahydrocyclopropyl[c]pyrrolo[3,2-e]indol-4(5H)-one or a substituted or derivatized form thereof. CPI may also refer to the seco form of CPI or seco-CPI, which is also known as 8-(chloromethyl)-1-methyl-3,6,7,8-tetrahydropyrrolo[3,2-e]indol-4-ol or a substituted or derivatized form (or forms).

[0079] It should be understood that whenever an embodiment herein is described with the language "comprising," it also provides other similar embodiments described with the language "consisting of" and / or "consisting essentially of."

[0080] Where aspects or embodiments of the invention are described in terms of Markush groups or other selective groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the group and all possible subgroups within the main group, and also includes the main group without one or more of the group members. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

[0081] 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 the invention belongs. In the event of a conflict, the present specification (including definitions) shall control. In this specification and claims, the term "comprising" or variations such as "including" or "comprising" will be understood to mean including the integer or group of integers being recited, but not excluding any other integer or group of integers. Unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0082] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0083] CD123 antibodies and antibody-drug conjugates

[0084] 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, characterized by any one or more of the following features: (a) treating, preventing, ameliorating one or more symptoms of a disorder associated with malignant cells expressing CD123 in a subject (e.g., cancers such as but not limited to AML, B-ALL, HCL, etc.); (b) inhibiting tumor growth or progression in a subject (having a malignant tumor expressing CD123); (c) inhibiting metastasis of cancer (malignant) cells expressing CD123 in a subject (having one or more malignant cells expressing CD123); (d) inducing regression (e.g., long-term regression) of a tumor expressing CD123; and (e) exerting cytotoxic activity in malignant cells expressing CD123.

[0085] The antibody used in the present invention can cover monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugated antibodies, single-chain antibodies (ScFv), mutants thereof, fusion proteins (e.g., domain antibodies) comprising antibody portions, humanized antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites with desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody can be mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, CD123 antibodies described herein are monoclonal antibodies. For example, CD123 antibodies can be humanized monoclonal antibodies, human antibodies, or chimeric monoclonal antibodies.

[0086] In some embodiments, the antibody may include a modified constant region, such as, for example, but not limited to, a constant region with an increased potential for inducing an immune response. For example, the constant region may be modified to have an increased affinity for Fcγ receptors (e.g., FcγRI, FcγRIIA, or FcγIII). In some embodiments, the antibody may include an immunologically inert constant region, i.e., a constant region with a reduced potential for inducing an immune response. In some embodiments, the constant region is modified as described in Eur. J. Immunol., 29: 2613-2624, 1999; PCT Application No. PCT / GB99 / 01441 and / or UK Patent Application No. 98099518. The Fc may be human IgG1, human IgG2, human IgG3, or human IgG4. The Fc may be human IgG2 containing mutations A330P331 to S330S331 (IgG2Δa), wherein the amino acid residues are numbered with reference to the wild-type IgG2 sequence. Eur. J. Immunol., 29: 2613-2624, 1999. In some embodiments, the antibody comprises a constant region of IgG4 comprising the following mutations (Armour et al., Molecular Immunology 40 585-593, 2003): E233F234L235 to P233V234A235 (IgG4Δc), wherein the numbering is with reference to wild-type IgG4. In another embodiment, the Fc is human IgG4 E233F234L235 to P233V234A235 and has a deletion G236 (IgG4Δb). In another embodiment, the Fc is any human IgG4 Fc (IgG4, IgG4Δb or IgG4Δc) containing the hinge stabilizing mutation S228 to P228 (Aalberse et al., Immunology 105, 9-19, 2002). In another embodiment, the Fc may be aglycosylated Fc.

[0087] In some embodiments, the constant region can be deglycosylated by mutating the oligosaccharide attachment residue (e.g., Asn297) and / or flanking residues in the constant region that are part of a glycosylation recognition sequence. In some embodiments, the N-linked glycosylation of the constant region is enzymatically deglycosylated. The N-linked glycosylation of the constant region can be deglycosylated enzymatically or by expression in a glycosylation-deficient host cell.

[0088] One method of determining the binding affinity of an antibody to CD123 is to measure the binding affinity of a monofunctional Fab fragment of the antibody. To obtain a monofunctional Fab fragment, the antibody (e.g., IgG) can be cleaved by papain or recombinantly expressed. The affinity of the CD123 Fab fragment of the antibody can be measured by surface plasmon resonance (Biacore) equipped with a pre-immobilized streptavidin sensor chip (SA) or anti-mouse Fc or anti-human Fc. TM 3000 TM Surface plasmon resonance (SPR) system, Biacore TM , INC, Piscataway NJ) using HBS-EP running buffer (0.01M HEPES, pH7.4, 0.15NaCl, 3mM EDTA, 0.005% v / v surfactant P20). Biotinylated or Fc-fused human CD123 can be diluted to a concentration of less than 0.5μg / mL with HBS-EP buffer and injected through each chip channel using different contact times to achieve two antigen density ranges, 50 to 200 response units (RU) for detailed kinetic studies or 800 to 1,000RU for screening assays. Regeneration studies showed that 25mM NaOH in 25% v / v ethanol effectively removed bound Fab while maintaining the activity of CD123 on the chip for more than 200 injections. Typically, serial dilutions of purified Fab samples (concentrations from 0.1 to 10 times the estimated K D The concentration of the Fab protein was determined by ELISA and / or SDS-PAGE electrophoresis using Fab of known concentration (as determined by amino acid analysis) as a standard. The data were globally fitted to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6.99-110) using the BIAevaluation program to simultaneously obtain the kinetic association rate (k on ) and dissociation rate (k off ). Equilibrium dissociation constant (K D ) value in k off / k onCalculation. This method is suitable for determining the binding affinity of an antibody to any CD123, including human CD123, CD123 of another mammal (such as mouse CD123, rat CD123 or primate CD123), and different forms of CD123 (such as glycosylated CD123). The binding affinity of an antibody 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 (such as human CD123 (e.g., SEQ ID NO: 1) D ) can be from about 0.002 nM to about 6500 nM. In 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 any of: 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 some 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.

[0089] The CD123 antibody described herein can be prepared by any method known in the art. For the production of hybridoma cell lines, the approach and scheme of the immunization host animal are generally consistent with established and conventional antibody stimulation and production techniques, as further described herein. The general techniques for producing human and mouse antibodies are known in the art and / or described herein.

[0090] In some embodiments, the CD123 antibody may comprise a heavy chain variable region (VH) comprising three CDRs of the following VH, the VH comprising the amino acid sequence of SEQ ID NO: 6, 24, 32, 44, 51 or 64. In some aspects of the invention, the antibody comprises a light chain variable region (VL) comprising three CDRs of the following VL, the VL comprising the amino acid sequence of SEQ ID NO: 17, 28, 39, 48, 57 or 71. In some aspects of the invention, the antibody comprises a VH and a VL, the VH comprising three CDRs of the following VH, the VH comprising the amino acid sequence of SEQ ID NO: 6, 24, 32, 44, 51 or 64, the VL comprising three CDRs of the following VL, the VL comprising the amino acid sequence of SEQ ID NO: 17, 28, 39, 48, 57 or 71. In other embodiments, the CD123 antibody may comprise: a VH comprising an amino acid sequence of SEQ ID NOs: 7, 8 and 9 and a VL comprising an amino acid sequence of SEQ ID NOs: 18, 19 and 20. In other embodiments, the CD123 antibody may comprise: a VH comprising an amino acid sequence of SEQ ID NOs: 7, 25 and 9 and a VL comprising an amino acid sequence of SEQ ID NOs: 18, 19 and 20. In other embodiments, the CD123 antibody may comprise: a VH comprising an amino acid sequence of SEQ ID NOs: 33, 34 and 35 and a VL comprising an amino acid sequence of SEQ ID NOs: 40, 41 and 42. In other embodiments, the CD123 antibody may comprise: a VH comprising an amino acid sequence of SEQ ID NOs: 33, 45 and 46 and a VL comprising an amino acid sequence of SEQ ID NOs: 40, 41 and 42. In other embodiments, the CD123 antibody may comprise: a VH comprising the amino acid sequence of SEQ ID NOs: 52, 53 and 54 and a VL comprising the amino acid sequence of SEQ ID NOs: 58, 59 and 60. In other embodiments, the CD123 antibody may comprise: a VH comprising the amino acid sequence of SEQ ID NOs: 65, 66 and 67 and a VL comprising the amino acid sequence of SEQ ID NOs: 72, 73 and 74.

[0091] Representative CD123 antibody heavy chain variable region and light chain variable region can respectively comprise SEQ ID NO:6,24,32,44,51 and 64 and SEQ ID NO:17,28,39,48,57 and 71 amino acid sequences. Representative CD123 antibody heavy chain and light amino chain can respectively comprise SEQ ID NO:15,27,37,47,55 and 69 and SEQ ID NO:23,30,43,49,62 and 76 amino acid sequences. Representative CD123 antibody sequences are shown in Table 2.0.

[0092] Table 2.0 Representative CD123 antibody sequences

[0093]

[0094] 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).

[0095] Table 2.1

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Substitution variants have at least one amino acid residue removed and a different residue inserted in its place in the antibody molecule. The sites of substitution mutagenesis that are of greatest concern include hypervariable regions, but framework changes may also be considered. Conservative substitutions are shown in Table 3 under the heading "conservative substitutions". If this substitution causes a change in biological activity, then Table 3 is referred to as "exemplary substitutions" or the more substantial changes further described below with reference to the amino acid classification may be introduced, and the product is then screened.

[0103] Table 3: Amino Acid Substitutions

[0104]

[0105] Substantial modification of the biological properties of the antibody is accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​substitution, such as β-sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties:

[0106] (1) Non-polar: norleucine, Met, Ala, Val, Leu, Ile;

[0107] (2) Non-electric polarity: Cys, Ser, Thr, Asn, Gln;

[0108] (3) Acidic (negatively charged): Asp, Glu;

[0109] (4) Basic (positively charged): Lys, Arg;

[0110] (5) Residues that affect chain direction: Gly, Pro; and

[0111] (6) Aromaticity: Trp, Tyr, Phe, His.

[0112] Non-conservative substitutions are made by exchanging a member of one of these classes for another class.

[0113] For example, one type of substitution that may occur is to change one or more cysteines that may be chemically reactive in the antibody to another residue such as, but not limited to, alanine or serine. For example, it may be a substitution of a non-canonical cysteine. The substitution may be carried out in the CDR or framework region or constant region of the variable domain of the antibody. In some embodiments, the cysteine ​​is canonical. Any cysteine ​​residue that does not participate in maintaining the proper conformation of the antibody may also be substituted (usually with serine) to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to antibodies to improve their stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

[0114] The present invention also provides a conjugate of a CD123 antibody or its antigen binding fragment as described herein, wherein the antibody is directly or indirectly conjugated to an agent (e.g., a cytotoxic agent) via a joint for targeted immunotherapy (e.g., antibody-drug conjugate or ADC). For example, a cytotoxic agent may be connected or conjugated to the CD123 antibody as described herein for targeted local delivery of the cytotoxic agent portion to a tumor (e.g., a tumor expressing CD123).

[0115] Methods for conjugating cytotoxic or other therapeutic agents to antibodies have been described in many literatures. For example, chemical modification of antibodies can be performed via lysine side chain amines or cysteine ​​sulfhydryls activated by reducing interchain disulfide bonds to allow conjugation reactions to occur. See, for example, Tanaka et al., FEBS Letters 579:2092-2096, 2005 and Gentle et al., Bioconjugate Chem. 15:658-663, 2004. Reactive cysteine ​​residues engineered at specific sites of antibodies have also been described for conjugation of specific drugs with defined stoichiometric amounts. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. The use of a tag containing an acyl donor glutamine or conjugation of endogenous glutamine made reactive (i.e., the ability to form a covalent bond to serve as an acyl donor) by polypeptide engineering in the presence of a transglutaminase and an amine (e.g., a cytotoxic agent containing or linked to a reactive amine) is also described in international patent applications WO2012 / 059882 and WO2015015448, which are incorporated herein by reference in their entirety. Transglutaminases are protein-glutamine gamma-glutamyltransferases (EC 2.3.2.13) that generally catalyze the pH-dependent transamidation of glutamine residues with lysine residues. The transglutaminases used in the invention described herein can be obtained or manufactured from a variety of sources or 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 using transglutaminase to prepare ADCs are described, for example, in U.S. Patent Application Publication No. 20170043033, which is incorporated herein by reference in its entirety.

[0116] ADCs include antibody components that are typically conjugated to a pharmaceutical agent through the use of a linker. In some embodiments, the ADCs disclosed herein include antibodies that are site-specifically conjugated to an amine donor substance (e.g., a small molecule coupled to a linker with an amine donor unit) through an engineered glutamine-containing tag, endogenous glutamine (i.e., non-engineered natural glutamine, such as glutamine in a variable domain, CDR, etc.), and / or reactive endogenous glutamine.

[0117] 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 one or more amino acids in the antibody (e.g., amino acid deletion, insertion, substitution or mutation), by enzymatic deglycosylation, or by reaction with an engineered transglutaminase. Thus, in one aspect, an antibody-drug conjugate (ADC) comprising the formula: Antibody-(T-(XYZ a ) b )c , wherein: T is 1) a glutamine-containing tag engineered at a specific site, 2) endogenous glutamine and / or 3) endogenous glutamine made reactive by antibody engineering or an engineered transglutaminase; X is an amine donor unit; Y is a linker; and Z is an agent moiety; XYZ is an amine donor agent site-specifically conjugated to a glutamine-containing tag, endogenous glutamine and / or 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. The glutamine-containing tag on the antibody described herein, endogenous glutamine and / or reactive glutamine, and the amine donor agent (XYZ) are all substrates for transglutaminase, and the bond between the glutamine-containing tag and / or endogenous / reactive glutamine and the amine donor agent has the formula CH2-CH2-CO-NH-, wherein NH- is connected to the linker and the agent portion.

[0118] In some embodiments, the CD123 antibody or conjugate described herein comprises a tag containing an acyl donor glutamine engineered at a specific site of the antibody (eg, the carboxyl terminus, the amino terminus, or another site of the CD123 antibody). In some embodiments, the tag comprises the amino acid glutamine (Q) or the amino acid sequence 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), LLQLQ (SEQ ID NO:93), LLQLLQ (SEQ ID NO:94), 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 (SEQID 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).

[0119] In some embodiments, a tag containing the acyl donor glutamine comprises, for example, LLQG (SEQ ID NO: 78), which replaces amino acid residues E294-N297 in the antibody heavy chain.

[0120] The present invention also provides an isolated antibody comprising a tag containing an acyl donor glutamine and an amino acid modification at position 222, 340 or 370 (EU numbering system) 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 substitution of lysine to arginine (e.g., K222R, K340R or K370R).

[0121] Agents that can be conjugated to the CD123 antibodies or antigen-binding fragments of the present invention include, but are not limited to, cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers or oligonucleotides.

[0122] Examples of cytotoxic agents include, but are not limited to, anthracyclines, auristatin, dolastatin, combretastatin, duocarmycin, pyrrolobenzodiazepine dimer, indolin-benzodiazepine dimer, enediyne, geldanamycin, maytansine, puromycin, taxanes, vinca alkaloids, camptothecin, tubulysin, hemiaster, spriatostatin, pradienolide, and stereoisomers, isosteres, analogs or derivatives thereof.

[0123] In some aspects, the drug / agent is a cyclopropylpyrroloindoline (CPI) dimer, a CTI dimer or a CBI dimer. CPI dimers induce interchain DNA crosslinking and potent cytotoxicity. PCT International Publication No. WO2015 / 110935 (which is incorporated herein by reference in its entirety) discloses CPI and CBI dimers that can be used for CD123 ADCs of the present invention and provides methods for producing CPI and CBI dimers. For example, the agent (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]pentan-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 following structure:

[0124]

[0125] It has the formula C31H31Cl2N4O7P.

[0126] Anthracyclines are derived from Streptomyces bacteria and have been used to treat a variety of cancers, such as leukemia, lymphoma, breast cancer, uterine cancer, ovarian cancer, and lung cancer. Exemplary anthracyclines include, but are not limited to, daunomycin, doxorubicin (i.e., adriamycin), epirubicin, idarubicin, valrubicin, and mitoxantrone.

[0127] Dolastatin and its peptide analogs and derivatives, auristatin, are highly potent antimitotic agents that have been shown to have anticancer and antifungal activity. See, for example, U.S. Pat. No. 5,663,149 and Pettit et al., Antimicrob. Agents Chemother. 42:2961-2965, 1998. Exemplary dolastatins and auristatins include, but are not limited to, dolastatin 10, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP), MMAD (monomethyl auristatin D or monomethyl dolastatin 10), MMAF (monomethyl auristatin F or N-methyl valine-valine-dolaisoleuine-dolaproine-phenylalanine), MMAE (monomethyl auristatin E or N-methyl valine-valine-dolaisoleuine-dolaproine-norephedrine), 5-benzoylvaleric acid-AE ester (AEVB), and other novel auristatins (as described in U.S. Patent Publication No. 2013 / 0129753). In some embodiments, 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-oxohept-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0128]

[0129] In some embodiments, the auristatin is 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(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), which has the following structure:

[0130]

[0131] In some embodiments, auristatin is 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-oxohept-4-yl]-N-methyl L-valinamide), which has the following structure:

[0132]

[0133] 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-oxohept-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0134]

[0135] 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-oxohept-4-yl]-N-methyl-L-valinamide), which has the following structure:

[0136]

[0137] Camptothecins are cytotoxic quinoline alkaloids that inhibit topoisomerase I. Examples of camptothecins and their derivatives include, but are not limited to, topotecan and irinotecan and their metabolites such as SN-38.

[0138] Combretastatins are natural phenols with tumor angiogenic properties. Exemplary combretastatins and their derivatives include, but are not limited to, combretastatin A-4 (CA-4) and ombrabulin.

[0139] Duocarmycin and CC-1065 are DNA alkylating agents with cytotoxic effects. See Boger and Johnson, PNAS 92: 3642-3649 (1995). Exemplary Duocarmycin and CC-1065 include, but are not limited to, (+)-duocarmycin A and (+)-duocarmycin SA, (+)-CC-1065, and compounds disclosed in international application PCT / IB2015 / 050280, including but not limited to 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}thien-2-yl)carbonyl]-2,3-dihydro-1H-benzo[e]indol-5-yl}oxy)carbonyl](methyl)amino}ethyl)(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide, which has the following structure:

[0140]

[0141] 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]pentan-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-ornithinamide, which has the following structure:

[0142]

[0143] 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-ornithinamide, which has the following structure:

[0144]

[0145] Enediynes are a class of antitumor bacterial products characterized by having nine-membered and ten-membered rings or the presence of conjugated triple-double-triple bond ring systems. Exemplary enediynes include, but are not limited to, calicheamicin, esperamicin, uncialamicin, dynemicin, and their derivatives.

[0146] Geldanamycin is a benzoquinone ansamycin antibiotic that binds to Hsp90 (heat shock protein 90) and has been used as an anti-tumor drug. Exemplary geldanamycins include, but are not limited to, 17-AAG (17-N-allylamino-17-demethoxygeldanamycin) and 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin).

[0147] Hemiaster and its analogs (eg, HTI-286) bind tubulin, disrupt normal microtubule dynamics, and stoichiometrically depolymerize microtubules.

[0148] Maytansine or its derivatives, maytansinoids, inhibit cell proliferation by inhibiting microtubule formation during mitosis by inhibiting tubulin polymerization. See Remillard et al., Science 189: 1002-1005, 1975. Exemplary maytansine and maytansinoids include, but are not limited to, mertansine (DM1) and its derivatives and ansamitocin.

[0149] Pyrrolobenzodiazepine dimers (PBD) and indolin-benzodiazepine dimers (IGN) are antitumor agents containing one or more imine functional groups or their equivalents 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, preferably a purine-guanine-purine sequence. Exemplary PBD and its analogs include, but are not limited to, SJG-136.

[0150] Sepriciostatin and pradienolide are antitumor compounds that inhibit splicing and interact with the spliceosomal SF3b. ​​Examples of sepriciostatins include, but are not limited to, sepriciostatin A, FR901464, and (2S,3Z)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazino-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, which has the following structure:

[0151]

[0152] Examples of pradienolide include, but are not limited to, pradienolide B, pradienolide D, or E7107.

[0153] Taxanes are diterpenes that act as anti-tubulin agents or mitotic inhibitors. Exemplary taxanes include, but are not limited to, paclitaxel (e.g. ) and docetaxel

[0154] Tubulysin is a natural product isolated from a strain of Myxococcus that has been shown to depolymerize microtubules and induce mitotic arrest. Exemplary tubulysins include, but are not limited to, tubulysin A, tubulysin B, and tubulysin D.

[0155] Vinca alkaloids are also anti-tubulin agents. Exemplary vinca alkaloids include, but are not limited to, vincristine, vinblastine, vindesine, and vinorelbine.

[0156] In some embodiments, the cytotoxic agent is selected from: 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-oxohept-4-yl]-N-methyl-L-valinamide), 3377 (N,2-dimethylalanyl-N-{(1S,2R)-4-{(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), 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-oxohept-4-yl]-N-methyl-L-valinamide amide), 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-oxohept-4-yl]-N-methyl L-valinamide), 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}-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-5-methyl-1-oxohept-4-yl]-N-methyl L-valinamide

[00136] The invention relates to (1-{[(2R,3R,5S,6S)-5-{[(2R,3R,5S,6S)-6-{(2E,4E)-5-[(3R,4R,5R,7S)-7-(2-hydrazino-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.

[0157] In some embodiments, the agent is an immunomodulatory agent. Examples of immunomodulators include, but are not limited to, gancyclovier, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrexate, glucocorticoids and their 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, interferon-β, and interferon-γ), a stem cell growth factor designated "S1 factor", erythropoietin, and thrombopoietin, or a combination thereof.

[0158] In some embodiments, the agent moiety is an imaging agent (e.g., a fluorophore or a chelator), such as fluorescein, rose bengal, lanthanide phosphors and their derivatives, or a radioactive isotope bonded to a chelator. Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein phosphoamidate (FAM) (e.g., 5-FAM), eosin, carboxyfluorescein, erythrosine, Alexa Fluor, fluorescein ... (e.g., Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700, or 750), carboxytetramethyl rose bengal (TAMRA) (e.g., 5,-TAMRA), tetramethyl rose bengal (TMR), and sulfonyl rose bengal (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-pentanedioic acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA), and 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) (BAPTA).

[0159] Examples of fluorophores include, but are not limited to, fluorescein isothiocyanate (FITC) (e.g., 5-FITC), fluorescein phosphoramidate (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), carboxytetramethyl rose bengal (TAMRA) (e.g., 5,-TAMRA), tetramethyl rose bengal (TMR), and sulfonyl rose bengal (SR) (e.g., SR101).

[0160] In some embodiments, therapeutic or diagnostic radioisotopes or other markers (e.g., PET or SPECT markers) may be incorporated into a pharmaceutical agent conjugated to a CD123 antibody or antigen-binding fragment as described herein. Examples of radioisotopes or other markers include, but are not limited to 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 Such as 97 Such as 99 Tc, 103 Such as 105 Rh, 105 Such as 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. 131In, 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 or 225 Ac.

[0161] In some embodiments, the agent is a therapeutic protein, including but not limited to toxins, hormones, enzymes, and growth factors.

[0162] Examples of toxin proteins (or polypeptides) include, but are not limited to, diphtheria toxin (e.g., diphtheria toxin A chain), Pseudomonas exotoxin and endotoxin, ricin (e.g., ricin A chain), abrin (e.g., abrin A chain), modeccin (e.g., modeccin A chain), alpha-sarcin, Aleurites fordii protein, dianthin protein, ribonuclease (RNase), DNase I, Staphylococcus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curculin, crotin, Sapaonaria officinalis protein, dapoxetine ... officinalis inhibitors, mitogellin, restrictocin, phenomycin, enomycin, tricothecene, inhibitor cystine knot (ICK) peptides (e.g., ceratotoxin), and conotoxins (e.g., KIIIA or SmIIIa).

[0163] In some embodiments, the medicament is a biocompatible polymer. CD123 antibody or antigen binding fragment described herein can be conjugated with a biocompatible polymer to increase serum half-life and biological activity and / or extend in vivo half-life. Examples of biocompatible polymers include water-soluble polymers, such as polyethylene glycol (PEG) or its derivatives and biocompatible polymers containing zwitterions (e.g., polymers containing phosphorylcholine).

[0164] In some embodiments, the agent is an oligonucleotide, such as an antisense oligonucleotide.

[0165] In another aspect, the invention provides a conjugate of the antibody described herein, wherein the conjugate comprises the following formula: antibody-(tag containing acyl donor glutamine)-(linker)-(cytotoxic agent), wherein the tag containing acyl donor glutamine is engineered at a specific site of the antibody (e.g., the carboxyl terminus of the heavy chain or light chain, after residue T135 of the antibody heavy chain, or 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 such as 0101, 0131 or 3377).

[0166] Examples of linkers containing one or more reactive amines include, but are not limited to, 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 aminoPEG6-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]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, [(3S,5S)-1-{3-[2-(2-aminoethoxy)ethoxy]propionyl}piperidine-3,5-diyl]bis-Val-Cit-PABC, putrescine or Ac-Lys-putrescine.

[0167] In some embodiments, (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]pentan-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-ornithinamide, molecular formula C63H82Cl2N13O15P, having the following structure:

[0168]

[0169] In some embodiments, (linker)-(cytotoxic agent) is in the trifluoroacetate form of C63H82Cl2N13O15P, which has the following structure:

[0170]

[0171] The formula of the trifluoroacetate salt form is C63H82Cl2N13O15P·C2HF3O2.

[0172] In some embodiments, the ADC is 1) antibody-LLQG (SEQ ID NO: 78)-Ac-Lys-Val-Cit-PABC-CPI-8314. In some embodiments, the tag containing the acyl donor glutamine comprises, for example, LLQG (SEQ ID NO: 78), which replaces 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.

[0173] In some embodiments, the present invention encompasses compositions (including pharmaceutical compositions) comprising antibodies or ADCs described herein or antibodies or ADCs prepared by the methods described herein and having the characteristics. As used herein, compositions comprise one or more antibodies or ADCs that bind CD123. These compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients well known in the art, including buffers.

[0174] Methods of using CD123 antibodies and ADCs thereof

[0175] The antibodies and ADCs of the invention can be used in many applications, including but not limited to therapeutic treatments and diagnostic treatments.

[0176] On the one hand, the present invention provides a method for treating a disease associated with CD123 expression in a subject. In some embodiments, the method for treating a disease associated with CD123 expression in a subject comprises administering an effective amount of a composition (e.g., a pharmaceutical composition) to a subject in need of treatment, the composition comprising CD123 antibodies or CD123 ADCs described herein. Diseases associated with CD123 expression include, but are not limited to, abnormal CD123 expression, altered or abnormal CD123 expression, malignant cells expressing CD123, and proliferative diseases (e.g., cancer) or autoimmune diseases.

[0177] Thus, in some embodiments, a method of treating cancer in a subject is provided, the method comprising administering to a subject in need of treatment an effective amount of a composition comprising a CD123 antibody or CD123 ADC described herein. As used herein, cancer can be, for example, but not limited to: 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, 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 myelogenous 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's 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, Waldenstrom's 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, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, HHV8-associated multicentric Kochman disease (HHV8-associated In some embodiments, the cancer is AML, B-ALL, BPDCN, NHL or HCL.

[0178] In some embodiments, a method for inhibiting tumor growth or progression in a subject with malignant cells expressing CD123 is provided, the method comprising administering an effective amount of a composition to a subject in need, the composition comprising a CD123 antibody or CD123 ADC as described herein. In other embodiments, the present invention provides a method for inhibiting cell metastasis expressing CD123 in a subject, the method comprising administering an effective amount of a composition to a subject in need, the composition comprising a CD123 antibody or CD123 ADC as described herein. In other embodiments, the present invention provides a method for inducing regression of malignant cell tumors in a subject, the method comprising administering an effective amount of a composition to a subject in need, the composition comprising a CD123 antibody or CD123 ADC as described herein.

[0179] In some embodiments, a method of treating an autoimmune disease in a subject is provided, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a CD123 antibody or CD123 ADC described herein.

[0180] As used herein, autoimmune diseases include, but are not limited to, 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, gamma globulinemia, 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, autoimmune progesterone dermatitis, autoimmune thrombocytopenic purpura, autoimmune urticaria, autoimmune uveitis, Bechet's disease, Castleman's disease, cold agglutinin disease, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, narcolepsy, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, relapsing polychondritis, rheumatic fever, temporal arteritis, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis and Wegener's granulomatosis.

[0181] On the other hand, the present invention provides an effective amount of a composition (e.g., a pharmaceutical composition) for treating a condition (e.g., cancer or autoimmune disease) associated with CD123 expression in a subject in need, comprising a CD123 antibody or CD123 ADC as described herein. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) for suppressing tumor growth or progression in a subject with malignant cells expressing CD123 is provided, comprising a CD123 antibody or CD123 ADC as described herein. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) for suppressing the transfer of malignant cells expressing CD123 in a subject in need is provided, comprising a CD123 antibody or CD123 ADC as described herein. In some embodiments, an effective amount of a composition (e.g., a pharmaceutical composition) for inducing tumor regression in a subject with malignant cells expressing CD123 is provided, comprising a CD123 antibody or CD123 ADC as described herein.

[0182] On the other hand, the present invention provides CD123 antibodies or CD123 ADCs described herein for treating disorders (e.g., cancer or autoimmune diseases) associated with CD123 expression in subjects in need. In some embodiments, CD123 antibodies or CD123 ADCs described herein are provided for suppressing tumor growth or progression in subjects with malignant cells expressing CD123. In some embodiments, CD123 antibodies or CD123 ADCs described herein are provided for suppressing the transfer of malignant cells expressing CD123 in subjects in need. In some embodiments, CD123 antibodies or CD123 ADCs described herein are provided for inducing tumor regression in subjects with malignant cells expressing CD123.

[0183] On the other hand, the present invention provides the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for treating disorders (e.g., cancer or autoimmune diseases) associated with CD123 expression. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inhibiting tumor growth or progression. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inhibiting the metastasis of malignant cells expressing CD123. In some embodiments, there is provided the use of CD123 antibodies or CD123 ADCs described herein in the preparation of drugs for inducing tumor regression.

[0184] On the other hand, the present invention provides a method for detecting, diagnosing and / or monitoring a disease related to CD123 expression. For example, CD123 antibodies described herein can be labeled with detectable moieties, such as developer and enzyme-substrate markers. Antibodies described herein can also be used for in vivo diagnostic assays such as in vivo development (e.g., PET or SPECT) or staining agents.

[0185] In some embodiments, the methods described herein further comprise the step of treating the subject with other forms of therapy. In some embodiments, the other forms of therapy are other anti-cancer therapies, including but not limited to chemotherapy, radiation therapy, surgery, hormone therapy, and / or other immunotherapies.

[0186] In some embodiments, the other forms of therapy include administering one or more therapeutic agents other than the CD123 antibodies or CD123 ADCs described herein. The one or more therapeutic agents may be, for example, but not limited to: a second antibody (e.g., an anti-VEGF (vascular endothelial growth factor) antibody (e.g., ), anti-HER2 antibodies (e.g. ), anti-CD25 antibody, anti-CD33 antibody, anti-CD20 antibody (e.g. ), anti-mucin-like glycoprotein antibodies, anti-TNF antibodies, anti-PD-1 or PD-L1 antibodies, and / or epidermal growth factor receptor (EGFR) antibodies (e.g. )), angiogenesis inhibitors, cytotoxic agents (e.g., anthracyclines (e.g., daunomycin, doxorubicin, epirubicin, idarubicin, valrubicin, and mitoxantrone), taxanes (e.g., paclitaxel and docetaxel), dolastatin, duocarmycin, enediynes, geldanamycin, maytansine, puromycin, vinca alkaloids (e.g., vincristine), topoisomerase inhibitors (e.g., etoposide), tubulysins, pyrimidine analogs (e.g., fluorouracil), platinum-containing agents (e.g., cisplatin, carboplatin, and oxaliplatin), alkylating agents (e.g., melphalan, cyclophosphamide, or carmustine), and hemiaster), 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 such as ([(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]boronic acid) and other agents such as tamoxifen.

[0187] For example, in some embodiments, a method for treating AML is provided, the method comprising administering to a subject in need of treatment an effective amount of a composition comprising a CD123 antibody or CD123 ADC as described herein and one other therapeutic agent such as a chemotherapeutic agent or thalidomide or a derivative thereof (e.g., lenalidomide). In some embodiments, the one other therapeutic agent is selected from: bortezomib (e.g. ), melphalan, prednisone, doxorubicin, lenalidomide, thalidomide, prednisone, carmustine, etoposide, cisplatin, cyclophosphamide, and vincristine. In some embodiments, the other therapeutic agent is bortezomib (e.g. ), melphalan, or prednisone. In some embodiments, the subject has relapsed or is refractory to prior AML therapy.

[0188] CD123 antibody or CD123 ADC can be administered to an individual via any appropriate route. It will be appreciated by those skilled in the art that the examples described herein are not intended to be limiting but rather an illustration of available technology. Therefore, in some embodiments, CD123 antibody or CD123 ADC is administered to an individual according to known methods, such as intravenous administration, such as push injection or continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, intracranial, percutaneous, subcutaneous, intraarticular, sublingual, intrasynovial, sprayed into, intrathecal, oral, inhaled or topical routes. Administration may be systemic, such as intravenous administration or topical administration. Liquid formulation sprayers (including jet sprayers and ultrasonic sprayers) obtained from commercial routes can be used for administration. Liquid formulations can be directly sprayed, and freeze-dried powders can be administered by spraying after reconstruction. Alternatively, CD123 antibody or CD123 ADC can be atomized or inhaled in the form of freeze-dried and ground powder using fluorocarbon formulations and metered dose inhalers.

[0189] In one embodiment, CD123 antibody or CD123 ADC is administered via a fixed-point or targeted local delivery technique. Examples of fixed-point or targeted local delivery techniques include implantable reservoir sources or local delivery catheters of various Trop antibodies or CD123 ADCs, such as infusion catheters, indwelling catheters, or needle catheters, synthetic grafts, adventitial coatings, shunts and stents or other implantable devices, fixed-point carriers, direct injections or direct applications. See, for example, PCT Publication No. WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0190] A variety of formulations of CD123 antibodies or CD123 ADCs can be used for administration. In some embodiments, CD123 antibodies or CD123 ADCs can be used for separate administration. In some embodiments, CD123 antibodies (or CD123 ADCs) and pharmaceutically acceptable excipients can be a variety of formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that help to administer pharmacologically effective substances. For example, excipients can provide shape or consistency, or as diluents. Suitable excipients include, but are not limited to, stabilizers, wetting agents, emulsifiers, salts for changing permeability, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and non-parenteral drug delivery are described in Remington, The Science and Practice of Pharmacy, 21st Ed., Mack Publishing, 2005.

[0191] In some embodiments, these agents are formulated for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). Thus, these agents may be combined with a pharmaceutically acceptable vehicle such as saline, Ringer's solution, dextrose solution, etc. The specific dosing regimen (i.e., dosage, timing, and repetition) will depend on the specific individual and the individual's medical history.

[0192] CD123 antibody or CD123 ADC described herein can be administered using any appropriate method, including injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). CD123 antibody or CD123 ADC can also be administered by inhalation as described herein. Typically, when CD123 antibody and CD123 ADC are administered, the initial candidate dose may be about 2 mg / kg. For purposes of the present invention, according to the above factors, a typical daily dose may be about 3 μg / kg to 30 μg / kg, to 300 μg / kg, to 3 mg / kg, to 30 mg / kg, to 100 mg / kg or any range above. For example, a dosage 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. When repeatedly administered for several days or longer periods, according to the disease, treatment continues until desired symptom suppression occurs or sufficient treatment levels are reached, such as suppressing or delaying the progression or metastasis of tumor growth / cancer cells. An exemplary dosing regimen includes administering an initial dose of about 2 mg / kg of CD123 antibody or CD123 ADC, followed by a maintenance dose of about 1 mg / kg per week, or a maintenance dose of about 1 mg / kg per week. Another exemplary dosing regimen includes administering an initial dose of about 0.21, about 0.5, or about 0.8 mg / kg per week or every three weeks. Other exemplary dosing regimens include administering a gradual increase in dose (e.g., an initial dose of 1 mg / kg, gradually increasing to administer one or more higher doses per week or longer). Other dosing regimens can also be used according to the pharmacokinetic attenuation pattern that the physician wishes to achieve. For example, in some embodiments, administration is considered to be one to four times per week. In other embodiments, administration is considered to be administered once a month or every other month or every three months. The progress of this treatment can be easily monitored by conventional techniques and determinations. Dosing regimens (including the CD123 antibodies or CD123 ADCs used) can change over time.

[0193] For the purposes of the present invention, the appropriate dose of CD123 antibody or CD123 ADC will depend on the CD123 antibody or CD123 ADC used (or a composition thereof), the type and severity of the symptoms to be treated, whether the agent is administered for therapeutic purposes, previous treatment, the subject's clinical history and response to the agent, the subject's clearance rate of the administered agent, and the considerations of the attending physician. Usually the physician will administer the CD123 antibody or CD123 ADC until a dose that can achieve the desired result is reached. The dose and / or frequency may vary depending on the course of treatment. Empirical considerations such as half-life will generally affect the determination of the dose. For example, antibodies compatible with the human immune system, such as humanized antibodies 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 immune system. The frequency of administration can be determined and adjusted during the course of treatment, usually but not necessarily according to the treatment and / or inhibition and / or improvement and / or delay of symptoms, such as tumor growth inhibition or delay, etc. Alternatively, a sustained continuous release formulation of a CD123 antibody or CD123 ADC may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[0194] In one embodiment, in individuals who have received one or more administrations of CD123 antibodies or CD123 ADCs thereof, the dosage of CD123 antibodies or CD123 ADCs can be determined empirically. Increasing doses of CD123 antibodies or CD123 antagonists are given to individuals. In order to evaluate the efficacy, indicators of the disease can be tracked.

[0195] According to the method of the present invention, the administration of CD123 antibodies or CD123 ADCs can be continuous or intermittent, depending on, for example, the physiological condition of the recipient, whether the purpose of administration is therapeutic or preventive, and other factors known to those skilled in the art. The administration of CD123 antibodies or CD123 ADCs can be substantially continuous for a predetermined period of time or can be a series of spaced doses.

[0196] In some embodiments, there may be more than one CD123 antibody or CD123 ADC. There may be at least one, at least two, at least three, at least four, at least five different or more CD123 antibodies or CD123 ADC. Typically, these CD123 antibodies or CD123 ADC may 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 for an epitope on CD123 and a second CD123 antibody for a different epitope on CD123.

[0197] The therapeutic formulation of the CD123 antibody or CD123 ADC used in the present invention is prepared by mixing an antibody having the desired purity with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005) in the form of a freeze-dried formulation or an aqueous solution for storage. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dose and concentration used, and may include buffers such as phosphates, citrates and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-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 nonionic surfactants such as TWEEN TM 、PLURONICS TM or polyethylene glycol (PEG).

[0198] Liposomes containing CD123 antibodies or CD123 ADCs are prepared by methods known in the art, such as Epstein, et al., Proc. Natl. Acad. Sci. USA 82: 3688, 1985, Hwang, et al., Proc. Natl Acad. Sci. USA 77: 4030, 1980 and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced using a reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of limited pore size to produce liposomes with a desired diameter.

[0199] The active ingredient can also be encapsulated in microcapsules prepared by, for example, coacervation techniques or by interfacial polymerization, such as in hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Remington, The Science and Practice of Pharmacy, 21st Ed., Mack Publishing, 2005.

[0200] Sustained release formulations can be prepared. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which 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 polyvinyl 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 TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[0201] The formulation to be used for in vivo administration must be sterile. This can be easily achieved by, for example, filtration through a sterile filtration membrane. Therapeutic CD123 antibodies or CD123 ADC compositions are typically placed in a container with a sterile interface, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0202] The composition of the present invention may be in unit dosage form, such as tablets, pills, capsules, powders, granules, solutions, suspensions or suppositories, for oral, parenteral or rectal administration, or by inhalation or spraying.

[0203] To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gelatin) and other pharmaceutical diluents (e.g., water) to form a solid preformulated composition containing a homogeneous mixture of the compound of the present invention or its non-toxic pharmaceutically acceptable salt. When referring to these homogeneous preformulated compositions, it is meant that the active ingredient is evenly dispersed in the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulated composition is then subdivided into unit dosage forms of the above-mentioned type, 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 with a long-acting advantage. For example, the tablet or pill may include an inner dosage and an outer dosage component, the latter being in the form of encapsulating the former. The two components can be separated by an enteric layer, which is used to resist disintegration in the stomach so that the inner component can pass intact to the duodenum or be delayed in release. Various materials can be used for the enteric layer or coating layer, including mixtures of various polymeric acids and polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

[0204] Suitable surfactants include, in particular, nonionic agents such as polyoxyethylene sorbitan (e.g. Tween TM 20, 40, 60, 80 or 85) and other sorbitans (such as Span TM 20, 40, 60, 80 or 85). Compositions containing surfactants will conveniently contain 0.05-5% surfactant, and may be 0.1-2.5%. It will be appreciated that other ingredients may be added if desired, such as mannitol or other pharmaceutically acceptable vehicles.

[0205] Suitable emulsions can be prepared using commercially available fat emulsions, such as Intralipid TM , Liposyn TM Infonutrol TM 、Lipofundin TM and Lipiphysan TM The active ingredient may be dissolved in a premixed emulsion composition or may be dissolved in an oil (e.g. soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and mixed with a phospholipid (e.g. lecithin, soybean lecithin or soybean lecithin) and water to form an emulsion. It will be appreciated that other ingredients such as glycerol or glucose may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, e.g. 5-20%. The fat emulsion may comprise fat droplets of 0.1-1.0 μm, particularly 0.1-0.5 μm, and have a pH in the range of 5.5-8.0.

[0206] The emulsion composition can be prepared by mixing CD123 antibody or CD123 ADC with Intralipid TM or those prepared from its ingredients (soybean oil, lecithin, glycerol and water).

[0207] Compositions for inhalation or spraying include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid composition may contain a suitable pharmaceutically acceptable excipient as described above. In some embodiments, the composition is administered by oral or nasal respiratory route to provide a local or systemic effect. Compositions in preferably sterile pharmaceutically acceptable solvents can be atomized by using a gas. Atomized solutions can be inhaled directly from a spray device, or the atomizing device can be connected to a mask, curtain, or intermittent positive pressure breathing machine. Solutions, suspensions, or powder compositions can be administered from a device, preferably orally or nasally, which delivers the formulation in an appropriate manner.

[0208] Composition

[0209] The composition used in the method of the present invention comprises an effective amount of CD123 antibodies or CD123 ADCs described herein. Examples of such compositions and how to prepare are also described in earlier paragraphs and below. In some embodiments, the composition comprises one or more CD123 antibodies or CD123 ADCs. For example, CD123 antibodies recognize human CD123. In some embodiments, CD123 antibodies are human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, CD123 antibodies include constant regions that can induce a desired immune response (such as antibody-mediated lysis or ADCC). In other embodiments, CD123 antibodies include constant regions that do not induce an undesirable or undesirable immune response (such as antibody-mediated lysis or ADCC). In other embodiments, CD123 antibodies include one or more CDRs (such as one, two, three, four, five, or all six CDRs in some embodiments) of an antibody.

[0210] It should be understood that the composition may include more than one CD123 antibody or CD123 ADC (e.g., a mixture of CD123 antibodies recognizing different epitopes of CD123). Other exemplary compositions include more than one CD123 antibody or CD123 ADC recognizing the same epitope, or different species CD123 antibodies or CD123 ADCs binding different epitopes of CD123 (e.g., human CD123).

[0211] In some embodiments, CD123 antibodies can be administered in combination with one or more additional therapeutic agents. These include, but are not limited to, the following: chemotherapeutic agents, vaccines, CAR-T cell-based therapies, radiotherapy, cytokine therapy, vaccines, CD123 bispecific antibodies, other immunosuppressive pathway inhibitors, angiogenesis inhibitors, T cell activators, metabolic pathway inhibitors, mTOR inhibitors, adenosine pathway inhibitors, tyrosine kinase inhibitors, including but not limited to inlyta, ALK inhibitors and sunitinib, BRAF inhibitors, epigenetic modifiers, IDO1 inhibitors, inhibitors or depletion agents of Treg cells and / or myeloid-derived suppressor cells, JAK inhibitors, STAT inhibitors, cell cycle protein dependence Kinase inhibitors, biotherapeutics (including but not limited to antibodies against VEGF, 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 cancer cells, tumor antigens, antigen presenting cells such as dendritic cells stimulated 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 alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogues topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesin, carzelesin and biszelesin); scutellarin (especially scutellarin 1 and scutellarin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, new nitrogen mustard, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, pyroglucose nitrosourea, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics antibiotics (e.g., calicheamicins, particularly calicheamicin gamma 1I and calicheamicin phi 1I, see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); danemycins, including danemycin A; bisphosphonates, such as clodronate; esperamicins; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin, authramycin, azaserine, bleomycin, actinomycin C, carabicin, carminomycin ( caminomycin), carcinoglossin, chromomycin, actinomycin D, daunomycin, detoxorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivetomycin, peplomycin, potfiromycin, puromycin, triferon, rhodorubicin, streptozocin, streptozocin, tuberculin, ubenimex, nepoxicam statin, doxycycline; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as dimethoate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, floxuridine; androgens such as caprotestosterone, drostanolone propionate, epitiostanol, melastane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid (frolinic acid); acid); acetoglucuronide; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate;defofamine; colcemid; diazocine; elformithine; elformithine; epothilone; etoglu; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansin and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitracrine; pentostatin; methamine mustard; pirarubicin; losoxantrone; podophyllic acid; 2-acetylhydrazine; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazoquinone; 2,2',2"-trichlorotriethylamine; crescent toxins (particularly T-2 toxin, verracurin A, A), bacitracin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; cytarabine ("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 inhibitor RFS2000; difluoromethylornithine (DMFO); vitamin A acids such as retinoic acid; capecitabine Tabin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are anti-hormonal agents that can be used to regulate or inhibit the effects of hormones on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, troxifene, keoxifene, LY117018, onapristone and toremifene (Falathon); aromatase inhibitors that inhibit aromatase that regulates estrogen production in the adrenal glands, such as, for example, 4 (5) -imidazole, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozole, letrozole and anastrozole; and anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. ;

[0212] In some embodiments, CD123 antibodies are used in combination with one or more other therapeutic agents targeting immune checkpoint regulators, such as, but not limited to, therapeutic agents targeting the following: 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, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1), OX40, OX40L, GITR, G ITRL, 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), SLAMF8(BLAME), SLAMF9(CD2F), CD28, CEACAM1(CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1-15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39–CD73–adenosine pathway (A2AR), BTKs, TIKs, CXCR2, CCR4, CCR8, CCR5, VEGF pathway, CSF-1, or innate immune response regulators.In some embodiments, the CD123 antibody is used in combination with, for example, an anti-PD-L1 antagonist antibody such as, for example, BMS-936559 (MDX-1105; CD123 antibodies such as, for example, nivolumab, pembrolizumab, and pidilizumab; anti-CTLA-4 antagonist antibodies such as, for example, ipilimumab; anti-LAG-3 antagonist antibodies such as BMS-986016 and IMP701; anti-TIM-3 antagonist antibodies; anti-B7-H3 antagonist antibodies such as, for example, MGA271; anti-VISTA antagonist antibodies; anti-TIGIT antagonist antibodies; anti-CD28 antagonist antibodies; anti-CD80 antibodies; anti-CD86 antibodies; anti-B7-H4 antagonist antibodies; anti-ICO S agonist antibody;Anti-CD28 agonist antibody;Innate immune response regulator (such as TLR, KIR, NKG2A) and IDO inhibitor. In some embodiments, CD123 antibody is used in combination with 4-1BB (CD137) agonist such as PF-05082566 or BMS-663513. In some embodiments, CD123 antibody is used in combination with OX40 agonist such as anti-OX-40 agonist antibody. In some embodiments, CD123 antibody is used in combination with GITR agonist such as anti-GITR agonist antibody such as, for example, but not limited to TRX518. In some embodiments, CD123 antibody is used in combination with IDO inhibitor. In some embodiments, CD123 antibody is used in combination with cytokine therapy such as, for example, but not limited to IL-15, CSF-1, MCSF-1, etc.

[0213] In some embodiments, CD123 antibodies are used in combination with one or more other therapeutic antibodies such as, for example, but not limited to, antibodies targeting CD19, CD22, CD40, CD52, or CCR4.

[0214] In some embodiments, CD123 antibody therapy can be carried out at intervals of several minutes to several weeks before or after other agents are treated. In the embodiment in which other agents and / or proteins or polynucleotides are administered separately, it is generally ensured that significant time will not be exceeded between each delivery, so that the medicament and composition of the present invention can still play a favorable combined effect on the object. In this case, it is considered that two schemes can be applied to each other in about 12-24h and more preferably in about 6-12h. However, in some cases, it may be required to significantly extend the application time, wherein between each application, through several days (2,3,4,5,6 or 7) to several weeks (1,2,3,4,5,6,7 or 8).

[0215] In some 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.

[0216] In some embodiments, the CD123 antibody composition is combined with a treatment regimen that further comprises a conventional therapy selected from surgery, radiotherapy, chemotherapy, targeted therapy, immunotherapy, hormone therapy, angiogenesis inhibition, and palliative therapy.

[0217] The composition used in the present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: The Science and practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. KE Hoover) in the form of a freeze-dried formulation or an aqueous solution. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosage and concentration, and may include buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol 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 residual yl) 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 dextran; 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 nonionic surfactants such as TWEEN TM 、PLURONICS TM Or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0218] Polynucleotides, vectors and host cells

[0219] The present invention also provides isolated polynucleotides encoding antibodies of the present invention, and vectors and host cells comprising the polynucleotides. On the other hand, the present invention provides compositions (such as pharmaceutical compositions) comprising any polynucleotide of the present invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any antibody described herein. On the other hand, the present invention provides methods for preparing any polynucleotide described herein.

[0220] Polynucleotides complementary to any such sequence are also encompassed in the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules and mRNA molecules, the HnRNA molecules contain introns and correspond to DNA molecules in a one-to-one manner, and the mRNA molecules do not contain introns. Other coding or non-coding sequences may (but not necessarily) be present in the polynucleotides of the present invention, and the polynucleotides may (but not necessarily) be connected to other molecules and / or support materials.

[0221] 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 the sequence. The polynucleotide variant comprises one or more substitutions, additions, deletions, and / or insertions so that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide is generally 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 preferably at least about 95% identity to a polynucleotide sequence encoding a native antibody or a portion thereof.

[0222] Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between two sequences are 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, typically 30 to about 75, or 40 to about 50 contiguous positions, in which a sequence can be compared to a reference sequence having the same number of contiguous positions after the two sequences have been optimally aligned.

[0223] Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Madison, WI) using default parameters. This program implements several alignment methods described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 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 Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80:726-730.

[0224] Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, usually 5% to 15%, or 10% to 12% additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated as follows: the number of positions where the same nucleic acid base or amino acid residue appears in the two sequences is determined to obtain the number of matching positions, the number of matching positions is divided by the total number of positions in the reference sequence (i.e., the window size), and the result is multiplied by 100 to obtain the percentage of sequence identity.

[0225] Variants may also or alternatively be substantially homologous to a native gene or a portion or complement thereof. The polynucleotide variants are capable of hybridizing to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence) under moderately stringent conditions.

[0226] Suitable "moderately stringent conditions" include pre-washing with 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) solution; hybridization overnight at 50°C-65°C, 5X SSC; followed by washing twice each for 20 minutes at 65°C with 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

[0227] As used herein, "high stringency conditions" or "high stringency conditions" refer to: (1) using low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent, such as formamide, for example, 50% (volume / volume) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinyl pyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, containing 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS and 10% dextran sulfate, and washed with 0.2×SSC (sodium chloride / sodium citrate) at 42° C. and 50% formamide at 55° C., followed by a high stringency wash with 0.1×SSC containing EDTA at 55° C. Those skilled in the art will know how to adjust conditions such as temperature, ionic strength, etc. to accommodate factors such as probe length when necessary.

[0228] It will be appreciated by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding the polypeptides described herein. Some of these polynucleotides have very low homology to the nucleotide sequence of any natural gene. However, the present invention specifically contemplates polynucleotides that vary due to differences in codon usage. In addition, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered due to one or more mutations such as deletions, additions and / or substitutions of nucleotides. The resulting mRNA and protein may, but not necessarily, have altered structures or functions. Alleles can be identified using standard techniques such as hybridization, amplification and / or database sequence comparisons.

[0229] The polynucleotides of the present invention can be obtained by chemical synthesis, recombinant methods or PCR. The methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail here. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequence.

[0230] To prepare polynucleotides using recombinant methods, polynucleotides containing the desired sequence can be inserted into an appropriate vector, which can then be introduced into an appropriate host cell for replication and amplification, as discussed further below. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, F-mating, or electroporation. Once introduced, the exogenous polynucleotides can be maintained in the cell in a non-integrating vector (such as a plasmid) or integrated into the host cell genome. The polynucleotides amplified in this way can be isolated from the host cell using methods well known in the art. See, for example, Sambrook et al., 1989.

[0231] In addition, PCR can replicate DNA sequences. PCR technology is well known in the art and is described in U.S. Patents 4,683,195, 4,800,159, 4,754,065 and 4,683,202 and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0232] RNA can be obtained by using DNA isolated in an appropriate vector and inserting it into an appropriate host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, such as described in Sambrook et al., 1989 (supra).

[0233] Suitable cloning vectors can be constructed according to standard techniques, or can be selected from the numerous cloning vectors available in the art. Although the selected cloning vector may vary depending on the intended host cell, suitable cloning vectors generally have the ability to self-replicate, may have a single target for a specific restriction endonuclease, and / or may carry a marker gene that can be used to select 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 can be purchased from commercial sellers such as BioRad, Strategene, and Invitrogen.

[0234] An expression vector is typically a replicable polynucleotide construct containing a polynucleotide of the present invention. This means that the expression vector must be able to be replicated in a host cell as an episome or as part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components typically include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons, are also typically required.

[0235] The vector containing the polynucleotide of interest can be introduced into the host cell by any appropriate means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; lipofection; and infection (for example, the vector is an infectious agent such as vaccinia virus). The choice of the introduction vector or polynucleotide will generally be determined by the characteristics of the host cell.

[0236] The present invention also provides a host cell comprising any polynucleotide described herein. Any host cell capable of overexpressing heterologous DNA can be used to separate the gene encoding an antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa and CHO cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli (E.coli) or Bacillus subtilis (B.subtillis)) and yeast (such as Saccharomyces cerevisiae (S.cerevisae), Schizosaccharomyces pombe (S.pombe); or Kluyveromyces lactis (K.lactis)). Preferably, the host cell expresses cDNA in an amount about 5 times, more preferably 10 times, and even more preferably 20 times higher than the corresponding endogenous antibody or protein of interest (if present in the host cell). Screening for host cells that specifically bind to CD123 is performed by immunoassay or FACS. Cells that overexpress an antibody or protein of interest can be identified.

[0237] Reagent test kit

[0238] The present invention also provides a kit for the method of the present invention. The kit of the present invention includes one or more containers comprising CD123 antibodies or CD123 ADCs described herein and instructions for use according to any method of the present invention described herein. Typically, these instructions include the use of CD123 antibodies or CD123 ADCs for the description of the above-mentioned therapeutic treatment.

[0239] Instructions for using the CD123 antibodies or CD123 ADCs described herein typically include information on the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, a large number of packages (e.g., multiple dose packages), or a sub-unit dose. The instructions provided in the kit of the present invention are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0240] The kit of the present invention is suitably packaged. Suitable packaging includes, but is not limited to, vials, bottles, cans, bendable packages (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packages used in combination with special devices, such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices such as mini-pumps. The kit may have a sterile interface (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). The container may also have a sterile interface (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is a CD123 antibody. The container may additionally include a second pharmaceutically active agent.

[0241] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container.

[0242] Biological Deposit

[0243] Representative materials of the present invention were deposited in American Type Culture Collection (ATCC) on June 29, 2017. The carrier with ATCC numbering PTA-124283 is a polynucleotide encoding a humanized CD123 antibody heavy chain sequence, and the carrier with ATCC numbering PTA-124284 is a polynucleotide encoding a humanized CD123 antibody light chain sequence. The preservation is carried out according to the Budapest Treaty (Budapest Treaty) and its regulations for the preservation of microorganisms for patent procedures recognized internationally. This ensures that the preserved culture of survival is maintained for 30 years from the preservation date. The deposit will be made available by ATCC under the terms of the Budapest Treaty and will be subject to an agreement between Pfizer, Inc. and ATCC which ensures that progeny of cultures of the deposit will be available to the public in perpetuity and without restriction upon the issuance of a related U.S. patent or publication of any U.S. or foreign patent application, whichever comes first, and will ensure that progeny will be available to persons entitled thereto as determined by the Director of the United States Patent and Trademark Office pursuant to 35 U.S.C. § 122 and the regulations thereunder (including 37 CFR § 1.14, with particular reference to 886 OG 638).

[0244] The assignee of this application agrees that if a culture of the deposited material dies or is lost or destroyed when grown under appropriate conditions, the material will be replaced immediately upon notification. The availability of the deposited material shall not be construed as a license to practice the present invention in violation of rights granted by any government agency under the patent laws of that country. Example

[0245] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, many modifications of the present invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0246] Example 1: In vitro cytotoxicity of CD123 antibody-drug conjugates

[0247] This example illustrates the cytotoxicity of various CD123 ADCs.

[0248] In this study, the cytotoxicity of various CD123ADCs was tested in the following AML cell lines using 2D in vitro cytotoxicity assays: MOLM13, MV411, JVM3, Granata519, OCI-AML3, NB4 and HL60. Table 4 shows the CD123 expression level of each of the individual cells used.

[0249] Table 4: Expression levels of CD123 in various AML cell lines

[0250]

[0251] The first group of ADCs tested were CD123 ADCs conjugated with CPI using an AcLysValCitPABC linker. These ADCs were: 18G3-CPI, 16D6-CPI, 3D1-CPI, and 20D7-CPI, all of which had a drug: antibody ratio (DAR) of about 2. The preparation of CD123 ADCs is described in detail in Example 5 below. A control IgG (IgG that does not bind CD123) ("Neg.8.8") with a CPI agent having a DAR of about 2 was used as a negative control.

[0252] For 2D in vitro cytotoxicity assays, AML cells were incubated for 96 hours with the following doses of ADC or control: 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. Cell viability was measured with CelltiterGlo (Promega, Madison, WI), and luminescence was determined using a Victor plate reader (Perkin Elmer, Waltham, MA). XLfit (IDBS, Boston, MA) 4-parameter curve fitting was used to generate 50% inhibition value (IC50) calculations. The results are summarized in Table 5.

[0253] Table 5: IC50 values ​​of CD123-CPI ADC in AML cells

[0254]

[0255]

[0256] The results show that CD123-CPI is cytotoxic to cells expressing CD123, and is more cytotoxic than non-specific CP1 (Neg8.8-CPI). For example, in MOLM13 cells, the IC50 of CD123 ADC 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. Relatively, the IC50 of Neg.8.8-CPI is> 88.43 ± 18.66 (Table 5, last column). Neg.8.8-CPI with DAR of about 2 is substantially less active at the highest test dose (Table 5, last column). The IC50 value of the CD123 ADC shown is well correlated with the CD123 expression level on the cell (Tables 4 and 5). For example, the IC50 of 18G3-CPI in MV411 cells expressing high levels of CD123 was 0.41±0.07. In contrast, the IC50 of 18G3-CPI in NB4 cells expressing low levels of CD123 was 63.92±36.08, and the IC50 of 18G3-CPI in HL60 cells not expressing CD123 was >84.4±33.30.

[0257] CD123-18G3 ADC conjugated with different agents iPr-calicheamicin or CTI (glucuronide) had similar cytotoxic effects. The results of the cytotoxicity assay of 18G3 conjugated with iPr-calicheamicin ("iPr") are summarized in Table 6. The results of the cytotoxicity assay of 18G3 conjugated with CTI (glucuronide) are summarized in Table 7.

[0258] Table 6: IC50 values ​​of CD123-iPr ADC in AML cells

[0259]

[0260] Table 7: IC50 values ​​of CD123-CTI ADC in AML cells

[0261]

[0262] These data show that CD123-ADCs with a DAR of about 2 are active and induce cell death in cancer cell lines expressing CD123, but not in cells that do not express CD 123. This shows the potency and specificity of these ADCs.

[0263] Example 2: Interleukin-3 (IL-3) signaling pathway and cytotoxicity of CD123 antibodies and ADCs

[0264] This example demonstrates the ability of CD123 antibodies to block IL-3 signaling and the cytotoxicity of CD123 ADC.

[0265] This study was conducted to determine whether any CD123 antibody clones competitively bind to IL-3 binding sites in TF-1 cells expressing CD123 / IL-3Rα. TF-1 cells expressing CD123 / IL-3Rα were incubated with 20ng / ml of IL-3 and the following CD123 antibodies: 3D1, 18G3 and 16D6, at doses of 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. CD123 antibody 7G3 was used as a benchmark antibody that has been shown to block the IL-3 signaling pathway and Neg 8.8 antibody was used as a negative control. In the CTG assay measuring cell survival, cells were treated at 37°C for four days. After the treatment period, cells were collected and protein was prepared. Western blot analysis was used to analyze STAT5, phosphorylated STAT5, and actin levels. Western blot results are shown in Figure 1 .

[0266] The presence of phosphorylated STAT5 ("phosphoStat5"), a downstream signaling protein, 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 phosphorylated Stat5 ( Figure 1 ). Treatment with CD123 antibody 7G3 blocked 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.

[0267] The cytotoxicity of CD123-18G3-CPI and CD123-16D6-CPI was maintained in the presence of IL-3 (Table 8).

[0268] Table 8

[0269]

[0270] Example 3: In vivo efficacy of CD123-ADC

[0271] This example demonstrates the in vivo efficacy of CD123 ADC.

[0272] The anti-tumor activity of CD123 ADC was tested in vivo using an acute myeloid leukemia (AML) cell line xenograft model. In each of the following models, the first dose was given on day 0. Tumors were measured at least once a week and their volumes were calculated as follows: Tumor volume (mm 3 )=0.5x(tumor width 2 ) (tumor length). The mean tumor volume (± SEM) for each treatment group was calculated from 10 animals included. All animal experiments were performed at an institution certified by the Association for Assessment of Laboratory Animal Care under the guidelines of the Institutional Animal Care and Use Committee and appropriate animal research approval. CD123 ADC showed high efficacy in a dose-dependent manner in cell lines with various genetic mutations or overexpressed genes and / or proteins.

[0273] AH1MOLM13 AML xenograft

[0274] The antitumor activity of CD123 ADC was evaluated in NOD / SCID immunodeficient mice against human tumors grown in vivo. In a subcutaneous (sc) AML model, 5×10 6 MOLM13 cells were implanted subcutaneously in the flank of female mice. 3 The average volume of 100 μg / kg was measured by animal staging to ensure the consistency of tumor size between different treatment groups. MOLM13 AML sc xenograft model was intravenously administered four times (Q4dx4) every four days: PBS vehicle, 0.3mg / kg or 1mg / kg of CD123-CPI (18G3, 16D6 or 3D1) or 0.3mg / kg or 1mg / kg of control Neg-8.8-CPI. Data are summarized in Table 9.

[0275] Table 9

[0276]

[0277]

[0278] CD123-CPI significantly inhibited 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, ten of ten animals in each group showed tumor regression. All of these mice remained tumor-free until at least day 60, when the study ended. At a CD123 ADC dose of 1 mg / kg, tumor regression occurred earlier (day 12).

[0279] These data show that CD123-CPI inhibits the growth of MOLM13 AML xenograft tumors.

[0280] To test the effect of CD123-ADC at lower dosage, the same MOLM13 model was used to perform in vivo efficacy studies. Animals were treated as described above. As shown in Table 10 below, 0.1mg / kg CD123-CPI was very effective and inhibited tumor growth in all mice in a dose-dependent manner (Table 10, middle column "18G3-CPI" and "16D6-CPI"). Relatively, Neg.8.8-CPI treated mice with continuous tumor growth (Table 10, right column "Neg 8.8-CPI").

[0281] Table 10

[0282]

[0283]

[0284] To test the efficacy of CD123-18G3-CTI, an in vivo efficacy study was performed using the same MOLM13 model. Animals were treated as described above. As shown in Table 11, CD123-18G3-CTI treatment inhibited tumor growth in mice in a dose-dependent manner. In contrast, tumors in Neg.8.8-CTI treated mice continued to grow.

[0285] Table 11

[0286]

[0287]

[0288] BH2MV4-11 AML xenograft

[0289] Immunodeficient mice were used to evaluate the effect of CD123 ADC on human tumor growth. In a subcutaneous (sc) AML model, 5×10 6 MV4-11 cells were implanted subcutaneously in the flank of female NOD-SCID mice. 3 Animals were staged to ensure consistency of tumor size between treatment groups when the mean volume of MV4-11 AML sc xenograft animals was dosed intravenously four times every four days (Q4dx4): PBS vehicle, CD123-CPI or 8.8-CPI at the following doses: 0.1, 0.3 and 0.6 mg / kg. The data are summarized in Table 12. CD123-18G3-H16-CPI was compared with Neg8.8-CPI and PBS vehicle.

[0290] 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, nine of ten mice showed tumor regression around day 25 and remained tumor-free until the end of the study at day 65. The data show that CD123-CPI inhibits the growth of MV4-11 xenograft tumors.

[0291] Table 12

[0292]

[0293] To test the efficacy of CD123-CTI, an in vivo efficacy study was performed using the same MV4-11 model. Animals were treated as described above. As shown in Table 13, CD123-CTI also inhibited mouse tumor growth in a dose-dependent manner, whereas tumors in mice treated with Neg.8.8-CTI continued to grow.

[0294] Table 13

[0295]

[0296]

[0297]

[0298] These results show that CD123 ADC is highly effective in treating tumors.

[0299] Example 4: In vivo efficacy of CD123-ADC

[0300] This example demonstrates the in vivo efficacy of CD123 ADC using AML patient-derived disseminated xenografts (AML PDX).

[0301] The efficacy of CD123-CPI on in vivo growth in a disseminated model established using patient bone marrow cells obtained according to appropriate consent procedures was evaluated in immunodeficient mice. Table 14 provides a summary of the patient samples used in this study.

[0302] Table 14

[0303]

[0304]

[0305] In the study, 1.0X 10 6Bone marrow cells from each patient were injected intravenously into the lateral tail vein of irradiated NSG mice. The study was staged and randomized based on the engraftment of human CD45+ / CD123+ / CD33+ cells (12-55% in peripheral blood as measured by flow cytometric staining). AML PDX mice were dosed intravenously twice every seven days (Q7dx2): PBS vehicle or 18G3-CPI. Peripheral blood and bone marrow were collected from mice sacrificed approximately 3-5 days after the second / last dose. Remaining tumor cells in the bone marrow and blood of sacrificed animals were analyzed by flow cytometry. The results are summarized in Figure 2 and Table 15. In Table 15, the numbers represent the percentages of remaining tumor cells in peripheral blood and bone marrow.

[0306] In each study, each group had approximately 6 to 10 mice. The data showed that CD123 ADC effectively reduced the number of tumor cells in peripheral blood and bone marrow in a dose-dependent manner ( Figure 2 and Table 15).

[0307] Table 15

[0308]

[0309]

[0310] To determine whether CD123-18G3-CPI can increase the overall survival of tumor-bearing mice, when peripheral blood shows a high degree of implantation (ie, an average of about 32%), PDX2407 is used to perform survival studies. Mice are intravenously administered twice every seven days (Q7dx2): PBS vehicle or CD123-CPI, and monitored every day. When mice show clinical signs, such as fatigue and weight loss (according to the Association for Assessment of Laboratory Animal Care, Institutional Animal Care and Use Committee guidelines), mice are euthanized.

[0311] The results are summarized in Figure 3 CD123-18G3-CPI treatment prolonged the overall survival of tumor-bearing animals in a dose-dependent manner. Specifically, treatment with a 0.1 mg / kg dose of CD123-18G3-CPI prolonged survival by approximately 25%, and treatment with a 0.3 mg / kg dose of CD123-18G3-CPI approximately doubled the length of survival ( Figure 3 ).

[0312] These results show that CD123-ADC treatment induced AML regression and inhibited progression, and prolonged survival.

[0313] Example 5: Preparation of CD123 Antibody Drug Conjugate (ADC)

[0314] This example illustrates 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").

[0315] 18G3-CPI is an ADC comprising CD123 humanized mAb 18G3 (see Tables 2.0 and 2.1, supra, "h18G3"), AcLysValCitPABC linker and cyclopropylpyrroloindoline (CPI) agent. K222R, E295L, Q295L, Y296Q, N297G mutations were introduced into the upper hinge and Fc to enable transglutaminase to catalyze site-specific conjugation of the CPI agent. The light chain and heavy chain amino acid sequences of CD123-18G3-H16-N60G-K222R humanized IgG1 antibody (referred to herein as "h18G3") are as follows:

[0316] Light chain:

[0317] DIQMTQSPSSSLSASVGDRVTITCKSSQSLLSSGTRKNYLAWYQQKPGKAPKLLIYWASTRQSGVPS

[0318] RFSGSGSGTDFTLTISSLQPEDFATYYCSQSYNLYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTA

[0319] SVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:30)

[0320] Heavy Chain:

[0321] EVQLVESGGGLVQPGGSLRLSCAASGFSLTSGDISWVRQAPGKGLEWVAVIWSGGGTNY G SRLMS

[0322] RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDWGNFYFDYWGQGTLVTVSSASTKGPSVFPLAP

[0323] SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT

[0324] YICNVNHKPSNTKVDKKVEPKSCDRTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV

[0325] DVSHEDPEVKFNWYVDGVEVHNAKTKPRELLQGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA

[0326] LPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO:27)

[0327] The H16 site-directed mutation sequence is shown above in bold italics and includes five H16 site-directed transglutaminase mutations K222R (in the hinge), E294L, Q295L, Y296Q and N297G. The LLQG sequence is the recognition tag for transglutaminase, and the linker-agent is conjugated at Q296. The G (underlined) in CDR-H2 is the N60G mutation.

[0328] The linker-agent (AcLysValCitPABC-DMAE-CO_CPI) was site-specifically conjugated at amino acid Q296. The AcLysValCitPABC-DMAE-CO_CPI-000638314 linker-agent structure is shown below.

[0329]

[0330] h18G3 was conjugated with AcLysValCitPABC-DMAE-CO_CPI-000638314 linker-agent by using bacterial transglutaminase (Sigma, 45U / mg protein). Specifically, the antibody was exchanged into a buffer containing 100mM KPO4, 200mM NaCl pH 7. The linker-agent was added in a 10-fold molar excess to the antibody in the presence of 7.5% (v / v) dimethyl sulfoxide (DMSO). The enzymatic reaction was initiated by adding 1U of bacterial transglutaminase per mg of antibody and incubated overnight at 25°C with constant mixing.

[0331] The reaction mixture was incubated at room temperature with 15% isopropanol for 30 min. Then diluted to 4 volumes of 1M KPO4 buffer and purified via hydrophobic interaction chromatography (HIC) using a butyl-agarose HP column (GE Lifesciences). The method utilizes 1M KPO4, 50mM Tris pH 7 to bind and 50mM Tris, pH 7 to elute the ADC over 10CV. The HIC-purified compound was dialyzed into a final buffer of 20mM histidine, 85mg / mL sucrose pH 5.8. The purity of the ADC was further characterized via SEC, and the drug-antibody ratio was calculated via reverse phase chromatography. The protein concentration was determined via a UV spectrophotometer.

[0332] The lead antibody had very good expression, evaluated in CHO pools up to 700 mg / L, 88% recovery yield and 99% purity after three purification steps. The conjugation of 18G3 was performed very well, as evaluated by achieving DAR 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) exhibited good thermal stability and molecular integrity.

[0333] Example 6: Method for conjugating antibodies to AcLysValCitPABC-DMAE-CO_CPI-000638314 using transglutaminase

[0334] This example illustrates the conjugation of an antibody to a linker-agent (AcLysValCitPABC-DMAE-CO_CPI-000638314, also referred to herein as AcLysPABC-CPI-8314).

[0335] Conjugation of CPI linker-cytotoxic agent. The conjugation of AcLysPABC-CPI-8314 (structure shown in Example 5 above) to the H16 site of the antibody was optimized by varying a variety of different parameters, such as molar concentration and salt composition, pH, enzyme concentration, time and temperature (Table 16). Briefly, the antibody was buffer exchanged into the appropriate buffer and pH for the various conditions shown in Table 16. A 10-fold molar excess of AcLysPABC-CPI-8314 linker-agent was added to the antibody and 5-10% dimethyl sulfoxide was added to dissolve the linker-agent in the reaction mixture. After the addition of bacterial transglutaminase (0.5-5 U / mg antibody), the reaction mixture was continuously mixed for the specified time and temperature. Unreacted linker-agent and transglutaminase were removed by size exclusion chromatography and / or hydrophobic interaction chromatography. The drug-antibody ratio was calculated by LCMS or RP-HPLC.

[0336] The conjugation efficiency measured by antibody-drug ratio (DAR) under various conditions is shown in Table 16. For example, when the antibody was exchanged to a buffer containing 30 mM KPO4, 150 mM NaCl pH 7, a DAR of 1.6 was achieved.

[0337] 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.

[0338] Table 16

[0339] Buffer ionic strength NaCl concentration, mM pH TG units / mg Ab Drug-Antibody Ratio (DAR) 20mM NaAc 75 5.8 1 0.3 30mM KPO4 150 6.5 1 1.3 30mM KPO4 150 7.0 0.5 1.6 30mM KPO4 150 7.0 2 1.6 30mM KPO4 150 7.0 5 1.4 30mM KPO4 150 7.5 1 1.5 30mM KPO4 150 8.0 1 0.6 25mM Tris 150 8.0 1 0.6

[0340] Although the teachings of the present disclosure have been described with reference to various applications, methods, kits and compositions, it should be understood that various changes and modifications may be made without departing from the teachings herein and the inventions claimed below. The foregoing examples are provided to more clearly illustrate the teachings of the disclosure and are not intended to limit the scope of the teachings herein. Although the teachings have been described by these exemplary embodiments, those skilled in the art will readily appreciate that many variations and modifications may be made to the exemplary embodiments without undue experimentation. All such variations and modifications are included within the scope of the teachings.

[0341] The references cited herein (including patents, patent applications, papers, textbooks, etc.) and the references cited in the references are hereby incorporated by reference in their entirety if not incorporated herein. If one or more of the incorporated references and similar materials differ or conflict with the present application, including but not limited to defined terms, term usage, described techniques, etc., the present application shall prevail.

[0342] The foregoing detailed description and examples describe in detail certain specific embodiments of the present invention and describe the best mode considered by the inventor. However, it should be understood that no matter how detailed the foregoing description is, the present invention can be implemented in many ways, and the present invention should be interpreted according to the appended claims and any equivalent scope thereof.

Claims

1. An isolated antibody that specifically binds to CD123, wherein the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region comprises three complementarity determining regions (CDRs) of a VH comprising an amino acid sequence of SEQ ID NO: 6, 24, 32, 44, 51 or 64, and the light chain variable region comprises three CDRs of a VL comprising an amino acid sequence of SEQ ID NO: 17, 28, 39, 48, 57 or 71.

2. The antibody of claim 1, wherein the VH comprises: (i) a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 7, 33, 52 or 65; (ii) a VH CDR2 comprising an amino acid sequence of SEQ ID NO: 8, 25, 34, 45, 53 or 66; and (iii) a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 9, 35, 46 or 67; and / or wherein the VL region comprises: (i) a VLCDR1 comprising an amino acid sequence of SEQ ID NO: 18, 40, 58 or 72; (ii) a VL CDR2 comprising an amino acid sequence of SEQ ID NO: 19, 42, 60 or 74; and (iii) a VL CDR3 comprising an amino acid sequence of SEQ ID NO: 20, 42, 60 or 74.

3. The antibody of claim 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 wherein 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.

4. The antibody of claim 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 in 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 in amino acids not within the CDRs.

5. The antibody of claim 3, wherein the antibody comprises a light chain and a heavy chain, the light chain comprises the sequence shown in SEQ ID NO: 30, and the heavy chain comprises the sequence shown in SEQ ID NO:

27.

6. An isolated antibody that specifically binds to CD123, wherein the antibody comprises a heavy chain variable region produced by an expression vector with ATCC Accession No. PTA-124283 and a light chain variable region produced by an expression vector with ATCC Accession No. PTA-124284.

7. An isolated antibody that specifically binds to CD123 and competes with the antibody of any one of claims 1 to 6 for binding to CD123.

8. The antibody of any one of claims 1-7, wherein the antibody comprises a tag containing the acyl donor glutamine engineered at a specific site.

9. The antibody of claim 8, wherein the tag containing the acyl donor glutamine comprises an amino acid sequence 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), 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). 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 NO:106), LLQGAP (SEQ ID NO:107) and LLQLQG (SEQ ID NO:108).

10. The antibody of claim 9, wherein the tag containing the acyl donor glutamine is LLQG (SEQ ID NO: 78).

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