Anti-CD123 antibodies, and conjugates and derivatives thereof

Abstract

To provide immunoconjugates that bind to CD123 antigen (the a chain of the interleukin 3 receptor, or IL-3Ra) for diagnosing and treating diseases, such as B-cell malignancies.SOLUTION: The invention provides an antibody or antigen-binding fragment thereof configured to: (a) bind an epitope within amino acids 101 to 346 of human CD123 / IL3-Rα antigen; and (b) inhibit IL3-dependent proliferation in antigen-positive TF-1 cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119 of the filing dates of U.S. Provisional Application No. 62 / 186,161, filed June 29, 2015, U.S. Provisional Application No. 62 / 338,203, filed May 18, 2016, and U.S. Provisional Application No. 62 / 346,730, filed June 7, 2016. The entire contents of each of the aforementioned related applications are incorporated herein by reference.

[0002] The present invention relates generally to antibodies, antigen-binding fragments thereof, polypeptides, and immunoconjugates that bind to the CD123 antigen (the alpha chain of the interleukin-3 receptor or IL-3Rα). The present invention also relates to methods of using such CD123-binding molecules to diagnose and treat diseases such as B-cell malignancies. [Background technology]

[0003] CD123 (interleukin-3 receptor alpha, IL-3Rα) is a 40 kDa molecule that is part of the interleukin-3 receptor (IL-3R) complex. The cytokine interleukin-3 (IL-3) induces early differentiation of pluripotent stem cells into erythroid, myeloid, and lymphoid progenitor cells. CD123 is associated with CD34 + Expressed on committed progenitor cells, but not on conventional CD34 + / CD38 - It is not expressed by hematopoietic stem cells (HSCs). CD123 is expressed by basophils, mast cells, and plasmacytoid dendritic cells, with some expression by monocytes, macrophages, and eosinophils, and is poorly expressed or absent by neutrophils and megakaryocytes. Some nonhematopoietic tissues (e.g., placenta, Leydig cells of the testes, certain brain cellular components, and some endothelial cells) also express CD123. However, its expression there is mostly cytoplasmic.

[0004] CD123 has been reported to be expressed by leukemic blast cells ("leukemic blasts") and leukemic stem cells (LSCs) (Jordan et al., Leukemia 14:1777-1784, 2000; Jin et al., Blood 113:6603-6610, 2009). In normal human progenitor cell populations, CD123 is expressed by a subset of hematopoietic progenitor cells (HPCs), but not by normal HSCs. CD123 is also reportedly expressed by plasmacytoid dendritic cells (pDCs) and basophils, and to a lesser extent by monocytes and eosinophils.

[0005] CD123 has been reported to be overexpressed on malignant cells in a wide range of hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Munoz et al., Haematologica 86(12):1261-1269, 2001). Overexpression of CD123 is associated with poor prognosis in AML (Tettamanti et al., Br. J. Haematol. 161:389-401, 2013). AML and MDS are thought to arise from and be perpetuated by a small population of leukemic stem cells (LSCs), which are typically dormant (i.e., not rapidly differentiating cells) and therefore resistant to cell death (apoptosis) and conventional chemotherapeutic agents. LSCs are characterized by overexpression of CD123, whereas CD123 is absent from the corresponding normal hematopoietic stem cell population in normal human bone marrow (Jin et al., Blood 113:6603-6610, 2009; Jordan et al., Leukemia 14:1777-1784, 2000). CD123 expression is also found in multiple other malignancies / premalignancies, including chronic myeloid leukemia (CML) progenitor cells (including blast crisis CML), Hodgkin's Reed-Sternberg (RS) cells, transformed non-Hodgkin's lymphoma (NHL), and Some chronic lymphocytic leukemia (CLL) (CD11c + ), a subset of acute T-lymphoblastic leukemia (T-ALL) (16%, mostly immature, most often adult), plasmacytoid dendritic cell (pDC) (DC2) malignancies, and CD34+ / CD38 - It is also associated with myelodysplastic syndrome (MDS), a myeloid cell malignancy.

[0006] AML is a clonal disease characterized by the proliferation and accumulation of transformed myeloid progenitor cells in the bone marrow, which ultimately leads to hematopoietic failure. The incidence of AML increases with age, and older patients generally have a worse treatment outcome than younger patients (Robak et al., 2004). (Et al., Clin. Ther. 2:2349-2370, 2009). Unfortunately, most adults currently afflicted with AML will die from the disease.

[0007] Treatment of AML initially focuses on inducing remission (induction therapy). Once remission is achieved, treatment shifts to focus on ensuring this type of remission (postremission or consolidation therapy) and, in some cases, maintenance therapy. The standard remission induction strategy for AML is chemotherapy with an anthracycline / cytarabine combination followed by consolidation chemotherapy, usually with either high doses of the same agents used during induction or human stem cell transplant, depending on the patient's ability to tolerate intensive treatment and the likelihood of cure with chemotherapy alone (see Roboz, Curr. Opin. Oncol. 24:711-719, 2012).

[0008] Drugs often used in induction therapy include cytarabine and anthracyclines. Cytarabine, also known as AraC, kills cancer cells and other rapidly differentiating normal cells by inhibiting DNA synthesis. Side effects associated with AraC treatment include decreased resistance to infection due to decreased white blood cell production, bleeding due to decreased platelet production, and anemia due to a potential decrease in red blood cells. Other side effects include nausea and vomiting. Anthracyclines (e.g., daunorubicin, doxorubicin, and idarubicin) have several modes of action, including inhibition of DNA and RNA synthesis, disruption of DNA conformation, and generation of cytotoxic oxygen free radicals. The most serious side effect of anthracyclines is cardiac toxicity, which limits the lifetime dose administered and, to some extent, significantly limits their usefulness.

[0009] Unfortunately, despite substantial advances in the treatment of newly diagnosed AML, 20%–40% of patients do not achieve remission with standard induction chemotherapy, and 50%–70% of patients who enter first complete remission are expected to relapse within 3 years. The optimal approach for patients with relapsed or refractory disease remains unclear. Stem cell transplantation has been established as the most effective form of anti-leukemia treatment in AML patients in first or next remission (Roboz, 2012).

[0010] Antibody-drug conjugates (ADCs) and other cell-binding agent-drug conjugates have emerged as a powerful class of antitumor agents with efficacy against a variety of cancers. Cell-binding agent-drug conjugates (e.g., ADCs) generally consist of three distinct components: a cell-binding agent (e.g., an antibody), a linker, and a cytotoxic moiety. Traditionally, the cytotoxic drug moiety is covalently attached to a lysine or cysteine ​​residue on the antibody by reduction of an interchain disulfide bond, resulting in ADCs that are heterogeneous mixtures carrying a varying number of drugs attached to different positions on the antibody molecule. Summary of the Invention

[0011] The present invention is based on the surprising discovery that the conjugates of the present invention are highly potent against a variety of CD123-expressing cancer cells, particularly leukemias with at least one negative prognostic factor.

[0012] One aspect of the present invention is (a) amino acids 101 to 34 of the human CD123 / IL3-Rα antigen. 6, and (b) inhibits IL3-dependent proliferation of antigen-positive TF-1 cells.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within amino acids 101 to 204 of the human CD123 antigen. In other embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within amino acids 205 to 346 of the human CD123 antigen.

[0014] A related aspect of the invention is a compound that (a) binds an epitope within amino acids 1-100 of human CD123, and (b) has an IC of 0.1 nM or less (e.g., 0.08 nM, 0.05 nM, 0.03 nM). 50 The present invention provides antibodies or antigen-binding fragments thereof that inhibit IL3-dependent proliferation of antigen-positive TF-1 cells, which have therapeutic value.

[0015] In certain embodiments, the antibody or antigen-binding fragment thereof inhibits proliferation of leukemic stem cells or leukemic blast cells but not hematopoietic stem cells.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof has a dissociation constant (K) of 0.3 nM or less, e.g., 0.01 nM to 0.3 nM, 0.01 nM to 0.2 nM, 0.01 nM to 0.19 nM, 0.01 nM to 0.18 nM, 0.01 nM to 0.15 nM, or 0.01 nM to 0.1 nM. d ) and binds to human CD123 antigen-positive cells.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey CD123. In certain embodiments, the antibody or antigen-binding fragment thereof has a K of 0.05 nM to 0.3 nM, 0.05 nM to 0.2 nM, 0.05 nM to 0.19 nM, 0.05 nM to 0.18 nM, 0.05 nM to 0.15 nM, or 0.05 nM to 0.1 nM. d In certain embodiments, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus CD123 with substantially similar binding affinity. For example, the antibody or antigen-binding fragment thereof may have a K of 0.05 nM to 0.3 nM, 0.05 nM to 0.2 nM, or 0.05 nM to 0.1 nM. d and can bind to human and cynomolgus monkey CD123. d can be measured by flow cytometry, surface plasmon resonance, or radioimmunoassay.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof inhibits at least 50% of IL3-dependent proliferation of antigen-positive TF-1 cells at a concentration of 0.5 nM or less.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a) at least one heavy chain variable region or fragment thereof comprising three contiguous complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 1, 5, and 12; CDR2 is selected from the group consisting of SEQ ID NOs: 2, 3, 6-10, 13, and 14; and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 4, 11, 15, and 70; or and b) at least one light chain variable region or fragment thereof comprising three contiguous complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 16, 19, 20, 23, and 72; CDR2 is selected from the group consisting of SEQ ID NOs: 17, 21, 24, and 71; and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 18, 22, and 25.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a) at least one overlapping CDR comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, respectively; and b) at least one heavy chain variable region or fragment thereof, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 1, 5, and 12, CDR2 is selected from the group consisting of SEQ ID NOs: 2, 3, 6-10, 13, and 14, and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 4, 11, and 15; and b) at least one light chain variable region or fragment thereof, comprising three contiguous complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 16, 19, 20, and 23, CDR2 is selected from the group consisting of SEQ ID NOs: 17, 21, and 24, and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 18, 22, and 25.

[0021] In certain embodiments, conservative amino acid substitutions comprise substitution of at least one Lys in a CDR with Arg.

[0022] In certain embodiments, the antibody is a CDR-grafted humanized antibody comprising murine CDR regions, wherein one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) heavy and / or light chain framework region Vernier zone residues of the antibody are of murine origin.

[0023] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa, the second residue from the N-terminus of SEQ ID NO: 34, is Phe. In other embodiments, Xaa is Val.

[0025] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise: a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40, except that the N-terminal residue is Ser; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise: a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41, except that the N-terminal residue is Ser.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the N-terminal residue is Ser and except that the residue corresponding to the fifth-to-last residue of SEQ ID NO: 54 is Cys (i.e., Cys at position 442 according to the EU / OU numbering system); and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the residue corresponding to the fifth to last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41, except that the N-terminal residue is Ser.

[0029] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35.

[0030] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:56, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO:35.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:54, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO:37.

[0033] In certain embodiments, Xaa, the second residue from the N-terminus of SEQ ID NOs: 38, 34, 56 and 54, is Phe. In other embodiments, Xaa is Val.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the residue corresponding to the fifth to last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof can comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO:54, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO:35.

[0036] In certain embodiments, Xaa, the second residue from the N-terminus of SEQ ID NO: 54 or 56, is Phe. In other embodiments, Xaa is Val.

[0037] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise: a) an immunoglobulin heavy chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 or 3, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 4; and b) an immunoglobulin light chain variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 16, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 18.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof may comprise: a) an immunoglobulin heavy chain variable region comprising: CDR1 having the amino acid sequence set forth in SEQ ID NO: 5; CDR2 having the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 9, or 10; and CDR3 having the amino acid sequence set forth in SEQ ID NO: 11; and b) an immunoglobulin light chain variable region comprising: CDR1 having the amino acid sequence set forth in SEQ ID NO: 19 or 20; CDR2 having the amino acid sequence set forth in SEQ ID NO: 21; and CDR3 having the amino acid sequence set forth in SEQ ID NO: 22.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain variable region comprising: a) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 13 or 14, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 15; and b) a CDR1 having the amino acid sequence set forth in SEQ ID NO: 23, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 24, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 25. The immunoglobulin light chain variable region may comprise an immunoglobulin light chain variable region comprising a CDR2 having the amino acid sequence set forth in SEQ ID NO: 24 and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 25.

[0040] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) a reference V selected from the group consisting of SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably 26, 28, 30, 32, 34, and 38). H V with sequences at least 95% identical Hand / or b) a reference V selected from the group consisting of SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (preferably 27, 29, 31, 35, and 37). L V with sequences at least 95% identical L In certain embodiments, V H The sequence is at least 99% identical to one of SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably 26, 28, 30, 32, 34, and 38), and / or V L The sequence is at least 99% identical to one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (preferably 27, 29, 31, 35, and 37). In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: a) SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably 26, 28, 30, 32, 34, and 38). H and / or b) V selected from the group consisting of SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (preferably 27, 29, 31, 35, and 37). L In certain embodiments, the antibody or antigen-binding fragment thereof may comprise the sequence V of SEQ ID NO: 26. H Sequence and V of SEQ ID NO: 27 L V of sequence SEQ ID NO: 28 H Sequence and V of SEQ ID NO: 29 L V of sequence SEQ ID NO: 30 H Sequence and V of SEQ ID NO: 31 L or V of SEQ ID NO: 34 H Sequence and V of SEQ ID NO: 35 L It can contain an array.

[0041] In certain embodiments, the antibody is a murine, non-human mammalian, chimeric, humanized, or human antibody. For example, a humanized antibody can be a CDR-grafted antibody or a resurfaced antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, the antigen-binding fragment thereof includes Fab, Fab', F(ab'), F d, single chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, small antibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0042] Another aspect of the invention is the V of any of the subject antibodies or antigen-binding fragments thereof. H and V L The polypeptide may be a fusion with a protein that is not a Pseudomonas toxin.

[0043] Another aspect of the present invention provides cells that produce the antibody or antigen-binding fragment thereof, or the polypeptide of the present invention.

[0044] Another aspect of the invention provides a method for producing an antibody or antigen-binding fragment thereof of the invention, or a polypeptide of the invention, said method comprising (a) culturing a cell of the invention, and (b) isolating the antibody, antigen-binding fragment thereof, or polypeptide from the cultured cell. In certain embodiments, the cell is a eukaryotic cell.

[0045] Another aspect of the present invention is a compound of the formula: [ka] an immunoconjugate comprising During the ceremony, CBA is Cy L1 The antibody of the present invention or its antibody is covalently bound by a heterodimer residue. a polypeptide of the present invention, W L is an integer between 1 and 20, Cy L1 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4)alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; W' is -NR e ' and R e ' is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x3 is (C1-C6) alkyl, L' has the following formula: -NR5-PC(=O)-(CR a R b ) m -C(=O)- (B1'), or -NR5-PC(=O)-(CR a R b ) m -SZ s1 - (B3'), represented by R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R a and R b are each independently, in each occurrence, —H, (C1-C3) alkyl, or a charged substituent or ionic group, Q; m is an integer from 1 to 6, Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is H + or a cation.

[0046] In certain embodiments, R a and R b are both H and R5 is H or Me.

[0047] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues, for example, P can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- P may be selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P is selected from Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. It is a.

[0048] In certain embodiments, Q is -SO3M.

[0049] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10, and the double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0050] A related aspect is a compound of the formula: [ka] an immunoconjugate comprising During the ceremony, CBA is a heterodimer of Cy L2an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to W L is an integer between 1 and 20, Cy L2 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4) alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; R x1 and R x2 are each (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e ' and R e ' is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is -H + or a cation.

[0051] In certain embodiments, R eis H or Me, and R x1 and R x2 are independently -(CH2) p -(CR f R g )-, wherein R f and R g are each independently —H or (C1-C4) alkyl; and p is 0, 1, 2, or 3.

[0052] In certain embodiments, R f and R g are the same or different and are selected from -H and -Me.

[0053] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10, and the double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0054] In certain embodiments, the doublet between N and C [ka] represents a double bond, X is absent, and Y is -H. In certain embodiments, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M. In certain embodiments, M is H + , Na + or K + is.

[0055] Another related aspect of the invention is a compound of the formula: [ka] providing an immunoconjugate having During the ceremony, CBA is a Cysteine-linked molecule that is linked to a Lys residue. L3 an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to In the formula, W L is an integer between 1 and 20, Cy L3 is the following formula: [ka] is represented by m' is 1 or 2, R1 and R2 are each independently H or (C1-C3) alkyl; Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is H + or a cation.

[0056] In certain embodiments, m' is 1 and R1 and R2 are both H. In certain embodiments, m' is 2 and R1 and R2 are both Me.

[0057] In certain embodiments, the immunoconjugate has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10.

[0058] In certain embodiments, M is H + , Na + or K + is.

[0059] Another aspect of the present invention is a compound of the formula: [ka] an immunoconjugate comprising During the ceremony, CBA is a CB an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to a ' group, Ws is 1, 2, 3, or 4; J CB ' is an aldehyde group resulting from oxidation of a 2-hydroxyethylamine moiety (the 2-hydroxyethylamine moiety can be part of a serine, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residue) at the N-terminus of the antibody or antigen-binding fragment thereof of the present invention or the polypeptide of the present invention, and Cy s1 and J CB ' is the following formula: [ka] is expressed as where s1 is the site covalently attached to CBA and s2 is the site of Cy s1 is a moiety covalently bonded to Cy s1 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; Z d1 is absent, -C(=O)-NR9-, or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; R a and R b is, in each occurrence, independently —H, (C1-C3) alkyl, or a charged substituent or ionic group, Q; r and r' are independently integers of 1 to 6; W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x3 is (C1-C6) alkyl, L is -NR9-(CR a R b ) r” or absent, r″ is an integer of 0 to 6.

[0060] For simplicity's sake, when each example below lists Ser as the N-terminal residue, it should be understood that other 2-hydroxyethylamine moieties as part of serine, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residues, where applicable, are specifically contemplated with respect to Thr.

[0061] In certain embodiments, R a and R b are both H, and R5 and R9 are both H or Me.

[0062] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues, for example, P can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu -Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg , Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. In certain embodiments, Q is -SO3M.

[0063] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] [ka] [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0064] Another aspect of the present invention is a compound of the formula: [ka] an immunoconjugate comprising During the ceremony, CBA is a CB an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to a ' group, J CB ' is an aldehyde group obtained by oxidation of the 2-hydroxyethylamine moiety at the N-terminus of the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide of the present invention, and Cy s2 is a moiety formed by reacting the aldehyde reactive group of CB ' is the following formula: [ka] is expressed as where s1 is the site covalently attached to CBA and s2 is the site of Cy s2 is a moiety covalently bonded to Cy s2 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4)alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; M is H + or a cation, R x1 is (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x2 is (C1-C6) alkyl, L1 is the following formula: [ka] is represented by During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s2 is the moiety covalently bonded to the -S- group of Z a2 is absent, -C(=O)-NR9- or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; Q is H, a charged substituent, or an ionic group; R a1 , R a2 , R a3 , R a4 is, in each occurrence, independently H or (C1-C3) alkyl; q1 and r1 are each independently an integer from 0 to 10, provided that q1 and r1 are not both 0.

[0065] In certain embodiments, -L1- has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein R is H or —SO3M.

[0066] In certain embodiments, R e is H or Me, and R x1 is -(CH2) p -(CR f R g )- and R x2 is -(CH2) p -(CR f R g )- and R f and R g are each independently —H or (C1-C4) alkyl, and p is 0, 1, 2 or is 3. In certain embodiments, R f and R g are the same or different and are selected from -H and -Me.

[0067] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0068] In certain embodiments, the doublet between N and C [ka] represents a double bond, X is absent, and Y is -H.

[0069] In certain embodiments, the doublet between N and C [ka] represents a single bond, X is -H, and Y is -SO3M. In certain embodiments, M is H + , Na + or K + is.

[0070] Another aspect of the present invention is a compound of the formula: [ka] providing an immunoconjugate having During the ceremony, CBA is a CB an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to a ' group, J CB ' is an aldehyde group obtained by oxidation of the 2-hydroxyethylamine moiety at the N-terminus of the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide of the present invention, and Cy s3 and J CB ' is the following formula: [ka] is expressed as where s1 is the site covalently attached to CBA and s2 is the site of Cy s3 is a moiety covalently bonded to Cy s3 is the following formula: [ka] is expressed as During the ceremony, m' is 1 or 2, R1 and R2 are each independently H or (C1-C3) alkyl; L1 is the following formula: [ka] is expressed as During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s3 is the moiety covalently bonded to the -S- group of Z a2 is absent, -C(=O)-NR9- or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; Q is H, a charged substituent, or an ionic group; R a1 , R a2 , R a3 , R a4 is, in each occurrence, independently H or (C1-C3) alkyl; q1 and r1 are each independently an integer from 0 to 10, provided that q1 and r1 are not both 0.

[0071] In certain embodiments, m' is 1 and R1 and R2 are both H. In certain embodiments, m' is 2 and R1 and R2 are both Me.

[0072] In certain embodiments, -L1- has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein R is H or —SO3M and M is H + or a cation.

[0073] In certain embodiments, the immunoconjugate has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM is represented by the following formula: [ka] It is expressed as:

[0074] Another aspect of the present invention is a compound of the formula: [ka] providing an immunoconjugate having During the ceremony, CBA is a CB an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to a ' group, J CB ' is an aldehyde group obtained by oxidation of the 2-hydroxyethylamine moiety at the N-terminus of the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide of the present invention, and Cy s4 and J CB ' is the following formula: [ka] is expressed as where s1 is the site covalently attached to CBA and s2 is the site of Cy s4 is a moiety covalently bonded to Cy s4 is the following formula: [ka] is expressed as L1' is of the following formula: [ka] is expressed as During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s4 is the moiety covalently bonded to the -NMe- group of Z b1 and Z b2 are both absent, or Z b1 and Z b2 one of which is absent and the other is -CH2-O- or -O-CH2-; Z b1 ' and Z b2each ' is independently absent, -CH2-O-, -O-CH2-, -NR9-C(=O)-CH2-, or -CH2-C(=O)-NR9-; R9 is H or (C1-C3) alkyl; n1 and m1 each independently represent an integer of 1 to 6, one of E1 and E2 is -C(=O)- and the other is -NR9-, or one of E1 and E2 is -C(=O)- or -NR9- and the other is absent; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R b1 , R b2 , R b3 , R b4 , R b5 and R b6 is each independently H or (C1-C3) alkyl in each occurrence.

[0075] In certain embodiments, R b1 , R b2 , R b3 , R b4 , R b5 , and R b6 are all H. In certain embodiments, R9 is H.

[0076] In certain embodiments, Z b1 ' and Z b2 ' are both absent or Z b1 ' is -CH2-O-, and Z b2 ' is absent or Z b1 ' is -CH2-C(=O)-NR9-, and Z b2 ' is O-CH2- or absent.

[0077] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues, for example, P can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D -Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P is Gly-Gly-Gly, Ala-Val, A Ia-Ala, Ala-D-Ala, D-Ala-Ala, and D-Ala-D-Ala.

[0078] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM is represented by the following structural formula: [ka] It is expressed as:

[0079] Another aspect of the present invention is a compound of the formula: [ka] An immune complex represented by the formula: During the ceremony, CBA is a cysteine-linked C1 an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to Wc is 1 or 2, Cy C1 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R a and R b is, in each occurrence, independently -H, (C1-C3) alkyl, or a charged is a substituent or ionic group Q, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, Rx3 is (C1-C6) alkyl, Lc is [ka] where s1 is the site covalently attached to CBA and s2 is the site of Cy C1 is a moiety covalently bonded to the —C(═O)— group of the formula R 19 and R 20 is, in each occurrence, independently —H or (C1-C3) alkyl; m" is an integer from 1 to 10, R h is —H or (C1-C3) alkyl.

[0080] In certain embodiments, R a and R b are both H and R5 is H or Me.

[0081] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues, for example, P can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- P may be selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala. In certain embodiments, Q is -SO3M.

[0082] In certain embodiments, R 19 and R 20 are both H, and m″ is an integer from 1 to 6.

[0083] In certain embodiments, -L C - is the following formula: [ka] It is expressed as:

[0084] In certain embodiments, the immunoconjugate has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0085] Another aspect of the present invention is a compound of the formula: [ka] An immune complex represented by the formula: During the ceremony, CBA is a cysteine-linked C2 an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to Wc is 1 or 2, Cy C2 is the following formula: [ka] is expressed as An immunoconjugate or a pharmaceutically acceptable salt thereof is provided, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R x1 is (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R kis -H or -Me, R x2 is (C1-C6) alkyl, L C ' is the following formula: [ka] is expressed as During the ceremony, s1 is the site covalently attached to CBA, and s2 is the site of Cy C2 is the moiety covalently bonded to the -S- group of Z is -C(=O)-NR9- or -NR9-C(=O)-; Q is -H, a charged substituent, or an ionic group; R9, R 10 , R 11 , R 12 , R 13 , R 19 , R 20 , R 21 and R 22 is, in each occurrence, independently —H or (C1-C3) alkyl; q and r, in each occurrence, are independently an integer from 1 to 10; m and n each independently represent an integer of 0 to 10; R h is —H or (C1-C3) alkyl, P' is an amino acid residue or a peptide containing 2 to 20 amino acid residues.

[0086] In certain embodiments, P' is a peptide containing 2 to 5 amino acid residues, for example, P' can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- P' may be selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P' is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0087] In certain embodiments, immunoconjugate-L C '- is the formula: [ka] It is expressed as:

[0088] In certain embodiments, R e is H or Me, and R x1 is -(CH2) p -(CR f R g )- and R x2 is -(CH2) p -(CR f R g )-, wherein R f and R g are each independently —H or (C1-C4) alkyl, and p is 0, 1, 2, or 3. In certain embodiments, R f and R g are the same or different and are selected from -H and -Me.

[0089] In certain embodiments, the immunoconjugate has the following formula: [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M.

[0090] In certain embodiments, the doublet between N and C [ka] represents a double bond, X is absent, and Y is -H.

[0091] In certain embodiments, the doublet between N and C [ka] represents a single bond, X is -H, and Y is -SO3M. In certain embodiments, M is H + , Na + or K + is.

[0092] Another aspect of the present invention is a compound of the formula: [ka] providing an immunoconjugate having During the ceremony, CBA is a cysteine-linked C3 an antibody or antigen-binding fragment thereof, or a polypeptide of the invention, covalently linked to Wc is 1 or 2, Cy C3 is the following formula: [ka] is expressed as During the ceremony, m' is 1 or 2, R1 and R2 are each independently —H or (C1-C3) alkyl; L C ' is the following formula: [ka] is expressed as During the ceremony, s1 is the site covalently attached to CBA, and s2 is the site of Cy C3 is the moiety covalently bonded to the -S- group of Z is -C(=O)-NR9- or -NR9-C(=O)-; Q is H, a charged substituent, or an ionic group; R9, R 10 , R 11 , R 12 , R 13 , R 19 , R 20 , R 21 and R 22 is, in each occurrence, independently —H or (C1-C3) alkyl; q and r, in each occurrence, are independently an integer from 1 to 10; m and n each independently represent an integer of 0 to 10; R h is —H or (C1-C3) alkyl, P' is an amino acid residue or a peptide containing 2 to 20 amino acid residues.

[0093] In certain embodiments, P' is a peptide containing 2 to 5 amino acid residues, for example, P' can be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Leu-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- and Met-Ala. In certain embodiments, P' is selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In certain embodiments, P' is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0094] In certain embodiments, -L C '- is the formula: [ka] is expressed as In the formula, M is H + or a cation.

[0095] In certain embodiments, m' is 1 and R1 and R2 are both H. In certain embodiments, m' is 2 and R1 and R2 are both Me.

[0096] In certain embodiments, the immunoconjugate has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM is represented by the following formula: [ka] is the drug moiety, represented by

[0097] Another aspect of the present invention provides a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding fragment thereof, or the polypeptide of the present invention, or the immunoconjugate of the present invention, and a pharmaceutically acceptable carrier.

[0098] Another aspect of the present invention provides a method for inhibiting the proliferation of CD123-expressing cells, said method comprising contacting the cells with an antibody or antigen-binding fragment thereof of the present invention, or a polypeptide of the present invention, or an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention.

[0099] In certain embodiments, the cell is a tumor cell, hi certain embodiments, the cell is a leukemia cell or a lymphoma cell.

[0100] Another aspect of the present invention provides a method for treating a subject suffering from cancer, wherein cells of the cancer express CD123, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a polypeptide of the present invention, or an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention.

[0101] In certain embodiments, the cancer or cell proliferative disorder is leukemia or lymphoma. In certain embodiments, the cancer or cell proliferative disorder is acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), including B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (H-ALL), or lymphoma. In certain embodiments, the cancer is selected from the group consisting of acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), myelodysplastic syndrome, blastic plasmacytoid dendritic cell neoplasm (BPDCN) leukemia, non-Hodgkin's lymphoma (NHL), including mantle cell lymphoma, and Hodgkin's leukemia (HL). In certain embodiments, the cancer is acute myeloid leukemia (AML). In certain embodiments, the cancer is B-cell acute lymphoblastic leukemia (B-ALL).

[0102] Another aspect of the present invention provides a method of treating a cell proliferative disorder in a subject, wherein cells of the cell proliferative disorder express CD123, the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention, or a polypeptide of the present invention, or an immunoconjugate of the present invention, or a pharmaceutical composition of the present invention, in an amount sufficient to treat the cell proliferative disorder.

[0103] It is contemplated that any one embodiment described herein, including those described as only one aspect of the invention (but not described elsewhere or repeated elsewhere) and those described by example only, can be combined with any one or more other embodiments of the invention, unless expressly stated otherwise or inappropriate. [Brief explanation of the drawings]

[0104] [Figure 1] 1 shows the inhibition of IL-3-dependent proliferation of TF-1 cells by chimeric CD123-6 antibody (chCD123-6) and its CDR-grafted huCD123-6 antibodies (huCD123-6Gv4.6 and huCD123-6Gv4.7). [Figure 2A] Figure 1 shows that three murine anti-CD123 antibodies (muCD123-3, -6, and -14) inhibit IL-3-dependent proliferation of TF-1 cells at least as well as 7G3. Figure 2 shows the inhibition of TF-1 cells cultured in the presence of IL-3 (1 ng / mL) by various anti-CD123 antibodies, including the CD123-binding control antibodies 7G3, 6H6, and 9F5. [Figure 2B]Figure 1 shows that three murine anti-CD123 antibodies (muCD123-3, -6, and -14) inhibit IL-3-dependent proliferation of TF-1 cells at least as well as 7G3. Figure 2 shows the inhibition of TF-1 cells cultured in the presence of GM-CSF (2 ng / mL) by the same anti-CD123 antibodies. [Figure 3] This shows that the murine anti-CD123 antibodies muCD123-3, -6, and -14 inhibit the IL-3 (1 ng / mL)-dependent proliferation of TF-1 cells in a dose-dependent manner to a greater extent than the 7G3 antibody. muCD123-16 is a negative control anti-CD123 antibody that binds to CD123 but does not inhibit the IL-3-dependent proliferation of TF-1 cells. [Figure 4A] 1 shows that the murine muCD123-3, -6, and -14 antibodies have higher binding affinity to CD123-positive AML cells than the 7G3 antibody to CD123-expressing TF-1 cells. [Figure 4B] 1 shows that mouse muCD123-3, -6, and -14 antibodies have higher binding affinity to CD123-positive AML cells than the 7G3 antibody to CD123-expressing HNT-34 cells. [Figure 5A] This shows that the chimeric anti-CD123 antibodies chCD123-3, -6, and -14 retain the high binding affinity of their murine counterparts using HNT-34 cells. The chimeric antibody chKTI, which does not bind to CD123, was included as a negative control. [Figure 5B] We demonstrate that the chimeric anti-CD123 antibodies chCD123-3, -6, and -14 retain the high binding affinity of their murine counterparts using the CD123-positive acute myeloid leukemia (AML) cell line MOLM-13. The chimeric antibody chKTI, which does not bind to CD123, was included as a negative control. [Figure 6] This shows that the chimeric chCD123-3, -6, and -14 anti-CD123 antibodies retain the functional activity of their murine counterparts, as evidenced by their ability to inhibit IL-3-dependent proliferation of TF-1 cells. A non-functional chimeric anti-CD123 antibody (chCD123-18), which binds to CD123 but does not inhibit IL-3-dependent proliferation of TF-1 cells, was included as a negative control. [Figure 7A] Figure 7A shows that the murine (muCD123-6), chimeric (chCD123-6), and CDR-grafted huCD123-6 antibodies (huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3) all have higher affinity for CD123-expressing HNT-34 cells than 7G3. The chimeric antibody chKTI, which does not bind to CD123, was included as a negative control. [Figure 7B] Figures 7B and 7C show that covalent attachment of the huCD123-6Gv4.7S3 or -Gv4.7 antibody to the D1 or D2 compound via a Lys-, Ser-, or Cys-linkage only moderately affects the binding affinity of these ADC complexes, i.e., Ser-linked huCD123-6Gv4.7S3-SeriMab-sD1 (see structures in Figure 17) and huCD123-6Gv4.7S3-SeriMab-D8 in Figure 7B, and Lys-linked huCD123-6Gv4.7S3-sSPDB-D1 and huCD123-6Gv4.7S3-D2, and Cys-linked huCD123-6Gv4.7-CysMab-D4 and huCD123-6Gv4.7-CysMab-D5 in Figure 7C. In Figure 7C, the unconjugated huCD123-6Gv4.7 antibody has the heavy chain sequence of SEQ ID NO: 54, where Xaa is Val. Conjugates with "S3-SeriMab" have a cytotoxin (in this case, an indolinobenzodiazepine or "IGN" compound hereafter) attached through an oxidized N-terminal Ser in the light chain. Conjugates with "CysMab" have a cytotoxin (in this case, an IGN compound) attached through an engineered Cys in the heavy chain (i.e., the Cys corresponding to the penultimate Cys in SEQ ID NO: 54). [Figure 7C]Figures 7B and 7C show that covalent attachment of the huCD123-6Gv4.7S3 or -Gv4.7 antibody to the D1 or D2 compound via a Lys-, Ser-, or Cys-linkage only moderately affects the binding affinity of these ADC complexes, i.e., Ser-linked huCD123-6Gv4.7S3-SeriMab-sD1 (see structures in Figure 17) and huCD123-6Gv4.7S3-SeriMab-D8 in Figure 7B, and Lys-linked huCD123-6Gv4.7S3-sSPDB-D1 and huCD123-6Gv4.7S3-D2, and Cys-linked huCD123-6Gv4.7-CysMab-D4 and huCD123-6Gv4.7-CysMab-D5 in Figure 7C. In Figure 7C, the unconjugated huCD123-6Gv4.7 antibody has the heavy chain sequence of SEQ ID NO: 54, where Xaa is Val. Conjugates with "S3-SeriMab" have a cytotoxin (in this case, an indolinobenzodiazepine or "IGN" compound hereafter) attached through an oxidized N-terminal Ser in the light chain. Conjugates with "CysMab" have a cytotoxin (in this case, an IGN compound) attached through an engineered Cys in the heavy chain (i.e., the Cys corresponding to the penultimate Cys in SEQ ID NO: 54). [Figure 8A] 1 shows that chimeric (chCD123-6) and CDR-grafted (huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3) huCD123-6 antibodies inhibit the IL-3 dependent proliferation of TF-1 cells better than the 7G3 antibody. [Figure 8B] These antibodies had no inhibitory effect when cells were grown in the presence of GM-CSF, indicating that inhibition was IL-3 dependent. [Figure 9A] Expression constructs for the IL-3Rα (CD123) extracellular domain and a chimeric receptor protein containing the IL-3Rα (gray) and GMRα domains (white) are shown. [Figure 9B] This shows that the CD123-6 antibody binds primarily to the CRM domain of IL-3Rα (residues 101 to 306). [Figure 9C]This shows that the CD123-3 antibody binds primarily to the CRM domain of IL-3Rα (residues 101 to 306). [Figure 9D] This shows that the CD123-14 antibody binds only to the N-terminal domain of IL-3Rα (residues 1 to 100). [Figure 9E] This shows that the 7G3 antibody binds only to the N-terminal domain of IL-3Rα (residues 1 to 100). [Figure 9F] This shows that the 6H6 antibody binds only to the N-terminal domain of IL-3Rα (residues 1-100). [Figure 9G] This shows that the 9F5 antibody binds only to the N-terminal domain of IL-3Rα (residues 1 to 100). [Figure 10] We show that the resurfaced huCD123-6Rv1.1 antibody, huCD123-6Rv1.1-CX1-1-DM1, conjugated with the maytansinoid DM1, exhibits dose-dependent cytotoxicity on the growth factor-independent CD123-expressing AML cell line OCI-AML4. Cytotoxicity is CD123-dependent, as evidenced by the ability of excess unconjugated huCD123-6 antibody (500 nM) to inhibit cytotoxicity. [Figure 11A] 1 shows the in vitro cytotoxicity of various resurfaced lysine-linked huCD123-6Rv1.1-IGN complexes on multiple CD123-positive malignant cell lines of different origins. [Figure 11B] Figure 1 shows the in vitro cytotoxicity of various lysine- or cysteine-linked huCD123-6-IGN conjugates on multiple CD123-positive B-ALL cell lines. A non-binding KTI antibody-based conjugate is included as a negative control. [Figure 11C] Figure 1 shows that various Lys- or Cys-linked IGN compounds are highly active in the P-gp (P-glycoprotein)-positive AML cell lines Kasumi-3 and MOLM-1. Control curves, white data points, are generated in the presence of excess unconjugated matched huCD123 antibody. [Figure 11D]We show that almost all of the various Lys- or Cys-linked CD123-IGN conjugates of the present invention kill 90% of AML progenitor cells from nine AML patient samples at nM or sub-nM concentrations. [Figure 11E] We show that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugate kills normal blood cells at concentrations over 100-fold higher than those required to kill AML progenitor cells, whereas Mylotarg does not show such preferential killing effect. [Figure 12A] Figure 1 shows the in vitro cytotoxicity of various lysine-linked huCD123-6Rv1.1-IGN conjugates on primary cells from an AML patient. Results from a conventional CFU assay on one primary patient sample are shown. [Figure 12B] Figure 1 shows the in vitro cytotoxicity of various lysine-linked huCD123-6Rv1.1-IGN conjugates on primary cells from AML patients. IC90 values ​​are shown for all AML patient samples treated with the conjugates. [Figure 13A] The IGN conjugate of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Gv4.6-D2, filled circles) against the AML cell line EOL-1 (Figure 13A), the B-ALL cell line KOPN-8 (Figure 13B), and the CML cell line MOLM-1 (Figure 13C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Gv4.6-CysMab-D5 and open squares for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated chCD123-6 antibody. [Figure 13B]The IGN conjugate of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Gv4.6-D2, filled circles) against the AML cell line EOL-1 (Figure 13A), the B-ALL cell line KOPN-8 (Figure 13B), and the CML cell line MOLM-1 (Figure 13C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Gv4.6-CysMab-D5 and open squares for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated chCD123-6 antibody. [Figure 13C] The IGN conjugate of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Gv4.6-D2, filled circles) against the AML cell line EOL-1 (Figure 13A), the B-ALL cell line KOPN-8 (Figure 13B), and the CML cell line MOLM-1 (Figure 13C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Gv4.6-CysMab-D5 and open squares for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated chCD123-6 antibody. [Figure 14A] The figures show that the huCD123-6 SeriMab (huCD123-6Rv1.1S2-SeriMab-D8, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Rv1.1-D2, filled triangles) in the AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B) and the CML cell line MOLM-1 (Figure 14C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Rv1.1S2-SeriMab-D8 and open triangles for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated huCD123-6 antibody. [Figure 14B]The figures show that the huCD123-6 SeriMab (huCD123-6Rv1.1S2-SeriMab-D8, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Rv1.1-D2, filled triangles) in the AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B) and the CML cell line MOLM-1 (Figure 14C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Rv1.1S2-SeriMab-D8 and open triangles for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated huCD123-6 antibody. [Figure 14C] The figures show that the huCD123-6 SeriMab (huCD123-6Rv1.1S2-SeriMab-D8, filled circles) is at least as active as a lysine-linked conjugate of the same antibody (huCD123-6Rv1.1-D2, filled triangles) in the AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B) and the CML cell line MOLM-1 (Figure 14C). The dotted curves connecting the open data points in each figure represent the activity of the respective conjugates (i.e., open circles for huCD123-6Rv1.1S2-SeriMab-D8 and open triangles for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of unconjugated huCD123-6 antibody. [Figure 15] A schematic diagram is provided showing the general steps that can be used to synthesize Ser-binding complexes of the invention. [Figure 16] A schematic diagram is provided showing the general steps that can be used to synthesize Ser-binding complexes of the invention. [Figure 17] A schematic diagram is provided showing the general steps that can be used to synthesize Ser-binding complexes of the invention. [Figure 18] Figure 1 shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugate has higher activity than gemtuzumab ozogamicin (GO) (also known as Mylotarg) in unselected AML patient samples. [Figure 19]We show that the Cys-linked huCD123-6Gv4.7-CysMab-D5 conjugate kills normal progenitor cells at concentrations over 100-fold higher than those required to kill AML progenitor cells, whereas Mylotarg and huCD123-6G4.7-CysMab-D5' (ADC of the DNA cross-linker D5') do not show such preferential killing. [Figure 20] 1 shows the in vivo efficacy of CD123-IGN complexes in the MV4-11 AML subcutaneous model. [Figure 21] This shows that the Cys-linked huCD123-6Gv4.7-CysMab-D5 complex is highly active against various CD123-positive AML cells with poor prognostic factors. [Figure 22] Figure 1 shows in vivo bioluminescence imaging of mice treated with the huCD123-6Gv4.7-CysMab-D5 conjugate compared to vehicle and control treated mice on day 26. Treatment with the conjugate significantly reduces tumor burden in mice. [Figure 23] FIG. 1 shows that treatment with the huCD123-6Gv4.7-CysMab-D5 conjugate prolongs survival of 6 / 6 mice compared to vehicle and control treated mice. [Figure 24] Figure 1 shows that incubation of MV4-11 cells with huCD123-6Gv4.7-CysMab-D5 complexes causes DNA damage, S-phase arrest of the cell cycle, and apoptosis-mediated cell death. [Figure 25] 1 shows the in vivo efficacy of CD123-IGN complexes in the Molm-13 AML disseminated model. [Figure 26] 1 shows the in vivo efficacy of CD123-IGN complexes in an EOL-1 subcutaneous model. [Figure 27] Figure 1 shows the in vivo efficacy of huCD123-CysMab-D5 conjugate at various doses in the EOL-1 subcutaneous model. [Figure 28]1 shows the in vivo efficacy of huCD123-CysMab-D5 conjugates compared to the corresponding free drug forms of payload (FGN849 or D5), naked antibody, control, cytarabine, and azacitidine in the EOL-1 subcutaneous model. [Figure 29] 1 shows the in vivo efficacy of CD123-IGN complexes in the MV4-11 AML disseminated model. [Figure 30] 1 shows the in vivo efficacy of CD123-IGN complexes in the MV4-11 AML subcutaneous model. [Figure 31] 1 shows that treatment with huCD123-CysMab-D5 conjugates prolongs survival of mice compared to vehicle and control treated mice. [Figure 32] Figure 1 shows the in vivo tolerability of huCD123-CysMab-D5 and huCD123-SeriMab-sD1 in mice. [Figure 33] 1 shows the in vivo tolerability of huCD123-lysine-linked-D2 conjugates in mice. DETAILED DESCRIPTION OF THE INVENTION

[0105] 1.Definition To facilitate the understanding of the present invention, several terms and phrases are defined below.

[0106] The terms "(human) IL-3Rα," "interleukin-3 receptor α," or "CD123," when used interchangeably herein, refer to any native (human) IL-3Rα or CD123, unless otherwise indicated. The CD123 protein is the interleukin-3-specific subunit of the heterodimeric cytokine receptor (IL-3 receptor or IL-3R). IL-3R consists of a ligand-specific α subunit and a signaling common β subunit (also called CD131) that is common to the receptors for interleukin 3 (IL3), colony-stimulating factor 2 (CSF2 / GM-CSF), and interleukin 5 (IL5). Binding of CD123 / IL-3Rα to IL3 depends on the β subunit, which is activated by ligand binding and is required for the biological activity of IL3.

[0107] All such above-mentioned terms relating to CD123 refer to either the protein or nucleic acid sequence set forth herein. The term "CD123 / IL-3Rα" includes "full-length," unprocessed CD123 / IL-3Rα, and any form of CD123 / IL-3Rα resulting from intracellular processing. The term also encompasses naturally occurring variants of CD123 / IL-3Rα protein or nucleic acid (e.g., splice variants, allelic variants, and isoforms). The CD123 / IL-3Rα polypeptides and polynucleotides described herein can be isolated from various sources (e.g., from human tissue types or from another source) or prepared by recombinant or synthetic methods. CD12 Examples of IL-3Rα sequences include, but are not limited to, NCBI reference numbers NP_002174 and NM_002183 (protein and nucleic acid sequences of human CD123 variant 1), and NP_001254642 and NM_001267713 (protein and nucleic acid sequences of human CD123 variant 2).

[0108] The term "antibody" refers to an immunoglobulin molecule that specifically recognizes and binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) at at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, and Fv fragments), single-chain Fv (scFv) variants, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing an antigen-recognizing portion of an antibody, and any other modified immunoglobulin molecule containing an antigen recognition site, provided that the antibody exhibits the desired biological activity. Antibodies can be of any of the five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, based on the identity of their heavy chain constant domains, called α, δ, ε, γ, and μ, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). The different classes of immunoglobulins have different, well-known subunit structures and three-dimensional shapes. Antibodies can be naked or conjugated to other molecules (e.g., toxins, radioisotopes, etc.).

[0109] In some embodiments, the antibody is a non-naturally occurring antibody. In some embodiments, the antibody is purified from natural components. In some embodiments, the antibody is recombinantly produced. In some embodiments, the antibody is produced by a hybridoma.

[0110] A "blocking" or "antagonist" antibody is one that inhibits or reduces the biological activity of the antigen (e.g., CD123 / IL-3Rα) to which it binds. In certain embodiments, a blocking or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Desirably, biological activity is reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 100%.

[0111] The terms "anti-CD123 antibody," "anti-IL-3Rα antibody," or "antibody that (specifically) binds to CD123 / IL-3Rα" refer to an antibody that can bind to CD123 / IL-3Rα with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting CD123 / IL-3Rα. Unless otherwise indicated, the extent of binding of an anti-CD123 / IL-3Rα antibody to an unrelated non-CD123 / IL-3Rα protein is less than about 10% of the binding of the antibody to CD123 / IL-3Rα, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to CD123 / IL-3Rα has a dissociation constant (K d In one embodiment, the anti-CD123 / IL-3Rα antibody does not bind to the common β chain CD131. In one embodiment, the anti-CD123 / IL-3Rα antibody is a known commercially available CD123 antibody (e.g., 7G3 (mouse IgG 2a ), but does not bind to the same epitope on CD123 bound by 6H6 (mouse IgG1) and 9F5 (mouse IgG1) (Sun et al., Blood 87(1):83-92, 1996).

[0112] The sequences of the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof of the present invention are shown in Tables 1 to 6 below. The nomenclature for the various antibodies and immunoconjugates of the present invention is provided separately below.

[0113] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab'), and F. v Antibody fragments include, but are not limited to, antibody fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. The term "antigen-binding fragment of an antibody" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by certain fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include (i) Fab fragments, V L, V H , C L , and C H1 (ii) a monovalent fragment consisting of a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (e.g., an antibody digested with papain yields three fragments: two antigen-binding Fab fragments and one Fc fragment that does not bind to antigen), and its associated F(ab')2 monovalent unit; (iii) a V H and C H1 F consisting of domains d (iv) a V fragment of a single arm of an antibody (i.e., a portion of the heavy chain contained in Fab); L and V H F consisting of domains v fragments, as well as the associated disulfide bonds F v , (v) V H domains, including (but not limited to) dAb (domain antibody) or sdAb (single domain antibody) fragments (Ward et al., Nature 341:544-546, 1989), and (vi) isolated complementarity determining regions (CDRs).

[0114] "Monoclonal antibody" refers to a homogeneous antibody population that is responsible for the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. As used herein, the term "monoclonal antibody" refers to both intact and full-length monoclonal antibodies and antibody fragments (e.g., Fab, Fab', F(ab')2 and F). v ), single chain (scFv) variants, fusion proteins containing antibody portions, and other modified immunoglobulin molecules containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies produced by any number of methods, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0115] The term "humanized antibody" refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequence. Generally, humanized antibodies are human immunoglobulins in which residues from the complementarity-determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534-1536, 1988).

[0116] In some cases, human immunoglobulin F v Framework region (FR) residues are replaced by corresponding residues in an antibody from a non-human species having the desired specificity, affinity, and capacity. v Further modification can be made by substitution of additional residues in the framework regions and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or potency of the antibody. Generally, a humanized antibody will comprise substantially all of at least one, and usually two or three, variable domains containing all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are those of a human immunoglobulin sequence. Humanized antibodies also comprise immunoglobulin constant regions or domains (F C ), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Patent Nos. 5,225,539 and 5,639,641, Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994, and Roguska et al., Proc. Natl. Acad. Sci. USA 91(3):969-973, 1994. ska et al., Protein Eng. 9(10):895-904, 1996 (all incorporated herein by reference). In some embodiments, a "humanized antibody" includes a resurfaced antibody. In some embodiments, a "humanized antibody" is a CDR-grafted antibody.

[0117] 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, either alone or in combination. The heavy and light chain variable regions each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs of each chain are held in close proximity by the FRs, along with the CDRs from the other chain, and contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, Vol. 1, No. 1, pp. 111-114, 2002); (1) an approach based on crystallographic studies of antigen-antibody complexes (Al-Lazikani et al., J. Molec. Biol., 273:927-948, 1997). Furthermore, a combination of these two approaches is sometimes used in the art to determine CDRs.

[0118] The Kabat numbering system is commonly used when referring to variable domain residues (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0119] Kabat-like numbering of amino acid positions refers to the numbering system used for the heavy or light chain variable domains of the antibody compilation of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991) (incorporated herein by reference). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or CDR of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by sequence comparison in the regions of sequence homology of the antibody with the "standard" Kabat numbered sequence. Chothia instead relates to the position of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987). The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop. This is because the Kabat numbering system places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. [Table 1]

[0120] The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human, made using any technique known in the art. In certain embodiments, a human antibody is free of non-human sequences. This definition of a human antibody includes intact or full-length antibodies, or antigen-binding fragments thereof.

[0121] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecules are derived from two or more species. Generally, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies from one mammalian species (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in antibodies from another species (usually human) to avoid or reduce the possibility of eliciting an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody can comprise an antibody or antigen-binding fragment thereof comprising at least one human heavy and / or light chain polypeptide (e.g., an antibody comprising a murine light chain and a human heavy chain polypeptide).

[0122] The terms "epitope" and "antigenic determinant" are used interchangeably herein to refer to a portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and from noncontiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed from contiguous amino acids are typically retained when the protein is denatured, whereas epitopes formed by tertiary folding are typically lost when the protein is denatured. Epitopes typically contain at least three, more commonly at least five or 8-10 amino acids in a unique spatial arrangement.

[0123] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity," as used herein, refers to the intrinsic binding affinity, which indicates a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for a partner Y is generally determined by the dissociation constant (K d ) or median effective concentration (EC 50 ) Affinity can be measured by common methods well known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate quickly, whereas high-affinity antibodies generally bind antigen quickly and tend to remain bound longer. Various methods for measuring binding affinity are well known in the art, any of which can be used in the present invention. Certain exemplary embodiments are described herein.

[0124] As used herein, "or higher" refers to binding affinity, which refers to stronger binding between a molecule and its binding partner. As used herein, "or higher" refers to a smaller numerical value of K d For example, an antibody having an affinity for an antigen of "0.3 nM or greater" has an affinity for the antigen of 0.3 nM or less, e.g., 0.29 nM, 0.28 nM, 0.27 nM, etc., or any value less than or equal to 0.3 nM. In one embodiment, the K d The affinity of the antibody, as determined by -3 ~about 10 -12 M, about 10 -6 ~about 10 -11 M, about 10 -6 ~about 10 -10 M, about 10 -6 ~about 10 -9 M, about 10 -6 ~about 10 -8 M, or about 10 -6 ~about 10 -7 I am M.

[0125] "Specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain, and that this binding requires some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope if it binds to the epitope via its antigen-binding domain more readily than it binds to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" can be considered to have higher specificity for a given epitope than antibody "B," or antibody "A" can be said to bind to epitope "C" with higher specificity than it has for related epitope "D."

[0126] In certain embodiments, an antibody or antigen-binding fragment of the invention "specifically binds" to the CD123 antigen in that it has higher binding specificity for the CD123 antigen (from any species) than it has binding specificity for a non-CD123 antigen. In certain embodiments, an antibody or antigen-binding fragment of the invention "specifically binds" to the human CD123 antigen in that it has higher binding specificity for the human CD123 antigen than it has binding specificity for a non-human CD123 antigen (e.g., mouse or rat CD123).

[0127] "Preferentially binds" means that an antibody specifically binds to an epitope more readily than it binds to a related, similar, homologous, or analogous epitope. Thus, an antibody that "preferentially binds" to a given epitope is more likely to bind to the given epitope than to a related epitope, although such an antibody may cross-react with the related epitope. For example, in certain embodiments, an antibody or antigen-binding fragment of the invention "preferentially binds" to the human CD123 antigen on mouse CD123.

[0128] If an antibody preferentially binds to an epitope to such an extent that it inhibits the binding of the reference antibody to the epitope to some extent, the antibody is said to "competitively inhibit" the binding of the reference antibody to the given epitope. Competitive inhibition can be measured by any method well known in the art, for example, by competitive ELISA assay. The antibody can be said to competitively inhibit the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0129] As used herein, the phrases "substantially similar" or "substantially identical" mean that one of ordinary skill in the art would understand that the difference between two values ​​is within the range of said values ​​(e.g., K d "value" refers to a sufficiently high degree of similarity between two numerical values ​​(typically one associated with an antibody of the invention and the other associated with a reference / comparator antibody) that there is little or no biological and / or statistical significance within the context of the biological property measured by the value of the reference / comparator antibody, where the difference between the two values ​​is less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%, depending on the value of the reference / comparator antibody.

[0130] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are not in a form found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure.

[0131] As used herein, "substantially pure" means that the material is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0132] As used herein, the terms "immunoconjugate," "conjugate," or "ADC" refer to a compound or derivative thereof linked to a cell-binding agent (i.e., an anti-CD123 / IL-3Rα antibody or fragment thereof) and are defined by the general formula: ALC, where C = cytotoxin, L = linker, and A = cell-binding agent (CBA) (e.g., an anti-CD123 / IL-3Rα antibody or antibody fragment). An immunoconjugate can also be defined in reverse order by the general formula: CLA.

[0133] A "linker" is any chemical moiety capable of attaching a compound, usually a drug, such as a cytotoxic agent described herein (e.g., a maytansinoid or IGN (indolinobenzodiazepine) compound), to a cell-binding agent (e.g., an anti-CD123 / IL-3Rα antibody or fragment thereof) in a stable, covalent manner. The linker can be sensitive or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions under which the compound or antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-labile groups, photolabile groups, peptidase-labile groups, and esterase-labile groups. Linkers also include charged linkers and hydrophilic forms thereof, as described herein and known in the art.

[0134] The terms "increased" CD123 / IL-3Rα, "increased expression" of CD123 / IL-3Rα, and "overexpression" of CD123 / IL-3Rα refer to a sample containing high levels of CD123 expression. CD123 may be elevated, increased, or overexpressed compared to a control value (e.g., expression levels in a biological sample, tissue, or cell from a cancer-free subject, a sample or cancer known to not express or to have low levels of CD123 / IL-3Rα, a normal sample or cancer that does not have elevated CD123 / IL-3Rα values). For example, a sample with increased expression (e.g., from a blood cancer such as leukemia or lymphoma) can include at least a 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, or at least a 50-fold increase compared to a control / normal value.

[0135] A "reference sample" can be used to correlate and compare the results obtained by the methods of the present invention with test samples. A reference sample can be cells (e.g., cell lines, cell pellets) or tissues. The CD123 / IL-3Rα level of a "reference sample" can be an absolute or relative amount, a range of amounts, a minimum and / or maximum amount, an average amount, and / or a median amount of CD123 / IL-3Rα. A "reference sample" can also serve as a baseline of CD123 / IL-3Rα expression to which a test sample is compared. A "reference sample" can include a previous or baseline sample from the same patient, a normal reference with a known level of CD123 / IL-3Rα expression, or a reference from a relevant patient population with a known level of CD123 / IL-3Rα expression. CD123 / IL-3Rα levels can also be expressed as values ​​from a standard curve. A standard curve is a quantitative test method in which assay data is plotted to determine the CD123 / IL-3Rα concentration of a sample. In one embodiment, the reference sample is purified CD123 / IL-3Rα. The diagnostic method of the present invention can include comparing the expression level of CD123 / IL-3Rα in a test sample with a "reference value." In some embodiments, the reference value is the expression level of CD123 / IL-3Rα in a reference sample. The reference value can be a predetermined value or can be measured from a reference sample (e.g., a control biological sample or a reference sample) tested together with the test sample. The reference value can be a single cutoff value, such as a median or mean value, or a range of values, such as a confidence interval. Reference values ​​can be set for various individual subgroups.

[0136] As used herein, the term "primary antibody" refers to an antibody that specifically binds to a target protein antigen in a sample. A primary antibody is generally the first antibody used in an ELISA assay or IHC procedure. In one embodiment, the primary antibody is the only antibody used in an IHC procedure.

[0137] As used herein, the term "secondary antibody" refers to an antibody that specifically binds to a primary antibody, thereby forming a bridge or bond, if any, between the primary antibody and subsequent reagents. A secondary antibody is generally a second antibody used in an immunohistochemistry procedure.

[0138] A "sample" or "biological sample" of the present invention is derived from a living organism, e.g., from a eukaryotic organism, in certain embodiments. In some embodiments, the sample is a human sample, although animal samples can also be used. Non-limiting sources of samples for use in the present invention include, for example, solid tissue, biopsy aspirate, ascites, fluid extract, blood, plasma, serum, cerebrospinal fluid, lymph, skin, external sections of the respiratory, intestinal, and genitourinary tracts, tears, saliva, breast milk, tumors, organs, cell cultures, and / or cell culture components. A "cancerous sample" is a sample containing cancer cells. The methods can be used to examine aspects of CD123 / IL-3Rα expression or the state of a sample, including, but not limited to, comparing different types of cells or tissues, comparing different stages of development, and detecting or determining the presence and / or type of disease or abnormality.

[0139] As used herein, the term "capture reagent" refers to a reagent that can bind and capture a target molecule of a sample such that the capture reagent-target molecule complex can be separated from the remainder of the sample under appropriate conditions. In one embodiment, the capture reagent is immobilized. In one embodiment, the capture reagent in a sandwich immunoassay is an antibody or a mixture of different antibodies against the target antigen.

[0140] As used herein, the term "detectable antibody" refers to an antibody that can be detected directly by a label that is amplified by a detection means, or indirectly, for example, by another labeled antibody. In direct labeling, the antibody is usually conjugated to a moiety that is detectable by some means. In one embodiment, the detectable antibody is a biotinylated antibody.

[0141] As used herein, the term "detection means" refers to a moiety or technique used to detect the presence of a detectable antibody, and includes detection agents that amplify immobilized labels (e.g., labels captured on a microtiter plate). In one embodiment, the detection means is a fluorometric detection agent (e.g., avidin or streptavidin).

[0142] Generally, a "sandwich ELISA" uses the following steps: (1) coating a microtiter plate with a capture antibody, (2) adding the sample and allowing any antigen present to bind to the capture antibody, (3) adding a detection antibody and allowing it to bind to the antigen, (4) adding an enzyme-linked secondary antibody and allowing it to bind to the detection antibody, and (5) adding a substrate which is converted by the enzyme into a detectable form.

[0143] As used herein, the term "label" refers to a detectable compound or composition that is conjugated directly or indirectly to an antibody to produce a "labeled" antibody. They may themselves be detectable (eg, radioisotope-labeled or fluorescently labeled) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that is detectable.

[0144] "Correlate" or "correlating" refers to comparing the performance and / or results of a first assay with the performance and / or results of a second assay, in any case. For example, the results of the first assay can be used in performing the second assay, and / or can be used to determine whether the second assay should be performed, and / or the results of the first assay can be compared with the results of the second assay. In one embodiment, increased expression of CD123 / IL-3Rα correlates with an increased likelihood that a CD123 / IL-3Rα-targeted therapy will be effective.

[0145] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth. "Tumor" and "neoplasm" refer to one or more cells resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0146] Examples of cancers include lymphomas and leukemias. Examples of cancers or tumorigenic diseases that can be treated and / or prevented by the methods and reagents of the present invention (e.g., anti-CD123 antibodies, antigen-binding fragments thereof, or immunoconjugates thereof) include AML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndromes, blastic plasmacytoid DC neoplasm (BPDCN) leukemia, non-Hodgkin's lymphoma (NHL), precursor B-cell lymphoblastic leukemia / lymphoma, and mature B-cell neoplasms (e.g., B-cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocy ... These include B-cell lymphomas including lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, mantle cell lymphoma (MCL), follicular lymphoma (FL) including low-, intermediate-, and high-grade FL, cutaneous follicle center lymphoma, marginal zone B-cell lymphoma (MALT type, nodal, and splenic marginal zone), hairy cell leukemia (HCL), diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasma cell myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, anaplastic large cell lymphoma (ALCL), and Hodgkin's leukemia (HL).

[0147] Cancers also include cancers containing cells with increased levels of CD123 / IL-3Rα expression. Such cancers with increased CD123 / IL-3Rα include, but are not limited to, AML, CML, ALL (e.g., B-ALL), and CLL.

[0148] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the entire population of cells derived from a tumor or precancerous lesion, including non-tumorigenic cells (which include many tumor cell populations) and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" is modified by the term "non-tumorigenic" when it simply refers to tumor cells that lack the ability to reproduce and differentiate, thus distinguishing tumor cells from cancer stem cells.

[0149] The term "subject" refers to any animal (e.g., mammal), which is the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, etc. Generally, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0150] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0151] The term "pharmaceutical formulation" refers to a formulation that is in a form that provides the biological activity of the active ingredient and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Such formulations may be sterile.

[0152] An "effective amount" of an antibody or immunoconjugate disclosed herein is an amount sufficient to carry out the particular purpose described. An "effective amount" can be determined empirically and routinely for the stated purpose.

[0153] The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, a therapeutically effective amount of a drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent, and in certain embodiments, stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent, and in certain embodiments, stop) tumor metastasis; inhibit tumor growth to some extent; alleviate to some extent one or more symptoms associated with cancer, and / or produce a favorable response (e.g., increased progression-free survival (PFS), disease-free survival (DFS) or overall survival (OS), complete response (CR), partial response (PR), or in some cases stable disease (SD), reduced progressive disease (PD), reduced time to progression (TTP), or any combination thereof). See the definition of "treat" herein. To the extent the drug can prevent growth and / or kill existing cancer cells, it can be cytostatic and / or cytotoxic.

[0154] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0155] The term "favorably responding" generally refers to the occurrence of a favorable state in a subject. In the context of cancer treatment, the term refers to providing a therapeutic effect to a subject. Positive cancer therapeutic effects can be measured in various ways (see W.A. Weber, J. Nucl. Med. 50:1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging diagnostic techniques can all be used to evaluate the therapeutic efficacy of anti-cancer treatments. Regarding tumor growth inhibition, according to the NCI standard, T / C<42% is the minimum level of anti-tumor activity. T / C(%)=median treated tumor volume / median control tumor volume×100, and T / C<10% is considered a high level of anti-tumor activity. A favorable response may be assessed, for example, by progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), or in some cases, an increase in stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof.

[0156] PFS, DFS, and OS can be measured by the criteria established by the National Cancer Institute and the Food and Drug Administration for new drug approval. See Johnson et al., (2003) J. Clin. Oncol. 21(7):1404-1411.

[0157] "Progression-free survival" (PFS) refers to the time from enrollment to disease progression or death. PFS is typically measured using the Kaplan-Meier method and Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Progression-free survival generally refers to the duration of a patient's life without further progression of their cancer.

[0158] "Complete response" or "complete remission" or "CR" refers to the disappearance of all signs of tumor or cancer in response to treatment. It does not necessarily mean that the cancer has been cured.

[0159] "Partial response" or "PR" refers to a reduction in the size or volume of one or more tumors or lesions or the extent of cancer in the body in response to treatment.

[0160] "Stable disease" means disease without progression or recurrence. In stable disease, there is neither sufficient tumor shrinkage to qualify as a partial response nor sufficient tumor growth to be considered progressive disease.

[0161] "Progressive disease" means the appearance of another new lesion or tumor and / or a clear progression of existing non-target lesions. Progressive disease can also refer to tumor growth of more than 20% since the start of treatment, either due to an increase in tumor weight or an increase in tumor spread.

[0162] "Disease-free survival" (DFS) means the period during and after treatment during which a patient is free of disease.

[0163] "Overall survival" (OS) refers to the period from patient enrollment to death or censored at the last known date of survival. OS includes an increase in life expectancy compared to a naive or untreated individual or patient. Overall survival refers to the situation where a patient survives for a certain period of time (e.g., 1 year, 5 years, etc.) from, for example, the time of diagnosis or treatment.

[0164] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. The terms "treat" or "treatment" or "treating" or "alleviate" or "alleviating" refer to therapeutic measures that cure, slow, alleviate and / or stop the progression of a diagnosed condition or disorder. Thus, those in need of treatment include those already diagnosed with the disease, and can also include those with minimal residual disease, resistant disease, or replacement disease. In certain embodiments, if a patient exhibits one or more of the following: a reduction in the number or complete absence of cancer cells; a reduction in tumor size; inhibition or absence of cancer cell invasion into peripheral organs, including, for example, spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; a reduction in one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in the tumorigenicity, tumorigenic frequency, or tumorigenic potential of the tumor; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumor-forming cells to a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), or overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), a decrease in progressive disease (PD), a decrease in time to progression (TTP), or any combination thereof, the subject has been successfully "treated" for cancer by the methods of the present invention.

[0165] Prophylactic or preventative measures refer to measures that prevent and / or delay the onset of the target condition or disease. Thus, those in need of prophylactic or preventative measures include those susceptible to the disease as well as those in whom the disease is to be prevented.

[0166] Prophylactic or preventative measures refer to therapeutic measures that prevent and / or delay the onset of the target condition or disease. Thus, those in need of prophylactic or preventative measures include those susceptible to the disease as well as those in whom the disease is to be prevented.

[0167] As used herein, the term "healthcare practitioner" refers to an individual or entity that works directly with and provides care to living subjects (e.g., human patients). Non-limiting examples of healthcare practitioners include doctors, nurses, technicians, therapists, pharmacists, counselors, alternative medicine practitioners, medical facilities, doctors' offices, hospitals, emergency rooms, clinics, urgent care centers, alternative medicine clinics / facilities, and any other entity that provides general and / or specialty treatment, evaluation, maintenance, therapy, medication, and / or advice for all or any part of a patient's condition, including, but not limited to, general medical, specialty medical, surgical, and / or any other type of treatment, evaluation, maintenance, therapy, medication, and / or advice.

[0168] In some embodiments, a healthcare professional may supervise or direct another healthcare professional administering a therapy to treat cancer. As used herein, "administering" a therapy includes prescribing the therapy to a subject, as well as administering, applying, or giving the therapy to a subject. A healthcare professional may perform, or have another healthcare professional or patient perform or instruct the following actions: obtaining a sample, processing a sample, submitting a sample, receiving a sample, transporting a sample, analyzing or measuring a sample, quantifying a sample, providing results obtained after analyzing / measuring / quantifying a sample, receiving results obtained after analyzing / measuring / quantifying a sample, comparing / scoring results obtained after analyzing / measuring / quantifying one or more samples, providing a comparison / score from one or more samples, obtaining a comparison / score from one or more samples, applying a treatment or therapeutic agent (e.g., a CD123 / IL-3Rα binding agent), initiating administration of a treatment, stopping administration of a treatment, continuing administration of a treatment, temporarily interrupting administration of a treatment, increasing the dosage of a treatment agent, decreasing the dosage of a treatment agent, continuing administration of the amount of a treatment agent, increasing the frequency of administration of a treatment agent, decreasing the frequency of administration of a treatment agent, maintaining the same dosage frequency of a treatment agent, replacing a treatment or therapeutic agent with at least another treatment or therapeutic agent, or combining a treatment or therapeutic agent with at least another treatment or therapeutic agent. These procedures can be performed automatically by medical personnel using computer-implemented methods (eg, via a web service or a stand-alone computer system).

[0169] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymer of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, substitution "caps" with analogs of one or more naturally occurring nucleotides, internucleotide modifications (e.g., modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L lysine, etc.), modifications with intercalating agents (e.g., acridine, psoralens, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylating agents, modifications with modified linkages (e.g., α-anomeric nucleic acids, etc.), and unmodified forms of polynucleotide(s). Additionally, any of the hydroxyl groups normally present on the sugar can be substituted, for example, with phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to provide additional linkages to additional nucleotides, or covalently attached to a solid support. The 5' and 3' terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars (e.g., arabinose, xylose, or lyxose), pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs (e.g., methyl riboside). One or more phosphodiester linkages can be replaced with alternative linking groups. These alternative linking groups include, but are not limited to, phosphate-substituted P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate ... P(S)S ("dithioate"), P(S)S (O)R, P(O)OR * , CO, or CH2 ("formacetal"), where each R and R is independently H, or a substituted or unsubstituted alkyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl, optionally containing an ether (-O-) linkage. Not all linkages in a polynucleotide need be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

[0170] The term "vector" refers to a construct that is capable of delivering and expressing one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid 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 (e.g., production cells).

[0171] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. A polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also include amino acid polymers that are modified, either naturally or by intervening modifications, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., covalent conjugation with a labeling component). Also included within the definition are polypeptides containing, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. Because the polypeptides of the present invention are based on antibodies, it is understood that in certain embodiments, the polypeptide can occur as single chains or associated chains. In some embodiments, the polypeptide, peptide, or protein is non-naturally occurring. In some embodiments, the polypeptide, peptide, or protein is purified from other naturally occurring components. In some embodiments, the polypeptide, peptide, or protein is recombinantly produced.

[0172] "Identical" or "percent identity," with respect to two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence (introducing gaps, if necessary), are identical or have a specified percentage of identical nucleotide or amino acid residues, without considering any conservative amino acid substitutions as part of the sequence identity. Percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software that can be used to align amino acid or nucleotide sequences are well known in the art. One such non-limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. 87:2264-2268, 1990, modified in Karlin et al., Proc. Natl. Acad. Sci. 90:5873-5877, 1993, and incorporated into the NBLAST and XBLAST programs (Altschul et al., Nucleic Acids Res. 25:3389-3402, 1991). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; BLAST-2, WU-BLAST-2 (Altschul et al., Methods in Enzymology 266:460-480, 1996); ALIGN, ALIGN-2 (Genentech, South San Francisco, California); or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences can be determined using the GAP program in GCG software (e.g., NWSgapdna.CM). P matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6. In certain alternative embodiments, the GAP program of the GCG software package (J. Mol. Biol. (48):444-453, 1970), which incorporates the Needleman and Wunsch algorithm, can be used to measure percent identity between two amino acid sequences (e.g., using either a Blossum62 matrix or a PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, or 5). Alternatively, in certain embodiments, percent identity between nucleotide or amino acid sequences is measured using the Myers and Miller algorithm (CABIOS, 4:11-17, 1989). For example, percent identity can be measured using the ALIGN program (version 2.0) and using a residue table and PAM120 with a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximum alignment with a particular alignment software can be determined by one of skill in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percent identity "X" of a first amino acid sequence to a second amino acid sequence is calculated as 100 x (Y / Z), where Y is the number of amino acid residues scored as perfect matches in the alignment of the first and second sequences (aligned by visual inspection or a particular sequence alignment program), and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will exceed the percent identity of the second sequence to the first sequence.

[0173] As a non-limiting example, whether a particular polynucleotide has a particular percent sequence identity (e.g., at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence in accordance with the present invention, parameters are set so that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and so that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0174] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning that they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as determined using a sequence comparison algorithm or by visual inspection. In particular embodiments, identity exists over a range of sequences that is at least about 10, about 20, about 40-60 residues in length, or any integer value therebetween, or over a region longer than 60-80 residues, at least about 90-100 residues, or the sequences are substantially identical over the entire length of the sequences being compared (e.g., the coding region of a nucleotide sequence).

[0175] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. A group of amino acid residues having similar side chains is As defined in the art, these include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate binding of the polypeptide or antibody containing the amino acid sequence to the antigen(s), i.e., to CD123 / IL-3Rα to which the polypeptide or antibody binds. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187, 1993; Kobayashi et al., Protein Eng. 12(10):879-884, 1999; and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417, 1997).

[0176] As used herein, "P-glycoprotein 1," also known as "permeability glycoprotein," "P-gp or Pgp," "multidrug resistance protein 1 (MDR1)," "ATP-binding cassette subfamily B member 1 (ABCB1)," or "cluster of differentiation antigen 243 (CD243)," is an ABC transporter of the MDR / TAP subfamily that transports a wide variety of substrates across extracellular and intracellular membranes. It is an ATP-dependent efflux pump with broad substrate specificity. P-gp is widely distributed and expressed in intestinal epithelium, where it transports xenobiotics (e.g., toxins or drugs) back into the intestinal lumen, in hepatocytes, where it transports them into the bile duct, in cells of the proximal tubules of the kidney, where it transports them into the urinary duct, and in capillary endothelial cells, including those of the blood-brain barrier and blood-testis barrier, where it transports them back into the capillaries. Some cancer cells also express large amounts of P-gp, which confers multidrug resistance to these cancers.

[0177] As used herein, "alkyl" refers to a saturated, straight- or branched-chain monovalent hydrocarbon group of 1 to 20 carbon atoms. Examples of alkyl include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CHCH(CH), 2-butyl, 2-methyl-2-propyl, 1-pentyl, 2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl, 1-heptyl, 1-octyl, and the like. Preferably, alkyl has 1 to 10 carbon atoms. More preferably, the alkyl has 1 to 4 carbon atoms.

[0178] The number of carbon atoms in the group is indicated by the prefix "C x-xx ", where x and xx are integers. For example, "C 1-4 "Alkyl" is an alkyl group having 1 to 4 carbon atoms.

[0179] The terms "compound" or "cytotoxic compound" are used interchangeably. They are intended to include compounds whose structures or formulas, or any derivatives thereof, are disclosed in the present invention or whose structures or formulas, or derivatives thereof, are incorporated by reference. The terms also include stereoisomers, geometric isomers, tautomers, solvates, metabolites, and salts (e.g., pharmaceutically acceptable salts) of compounds of all formulas disclosed in the present invention. The terms also include any solvates, hydrates, and polymorphs of any of the foregoing. The specific recitation of "stereoisomers," "geometric isomers," "tautomers," "solvates," "metabolites," "salts," "complexes," "hydrates," or "polymorphs" of particular embodiments of the invention listed herein should not be construed as an intentional omission of these forms of other embodiments of the invention where the term "compound" is used without a recitation of these other forms.

[0180] The term "chiral" refers to a molecule that possesses the property of being non-superimposable on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0181] The term "stereoisomers" refers to compounds that have identical chemical constitution and bonding, but differ in the arrangement of their atoms in space, which are not interconvertible by rotation about a single bond.

[0182] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high-resolution analytical techniques such as crystallization, electrophoresis, and chromatography.

[0183] "Enantiomers" refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0184] Stereochemical definitions and conventions used herein generally follow those of S.P. Parker (ed.), McGraw-Hill, Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York, and Eliel, E. and Wilen, S., Stereochemistry of Organic Compounds, John Wiley & Sons, Inc., New York, 1994. The compounds of the present invention may contain asymmetric or chiral centers and therefore may exist in various stereoisomeric forms. All stereoisomers of the compounds of the present invention, including but not limited to diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, including racemic mixtures, are intended to form part of the present invention. Many organic compounds exist in optically active forms; that is, they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and l, or (+) and (-), are used to indicate the sign of rotation of plane-polarized light by a compound; (-) or l means the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Specific stereoisomers are sometimes called enantiomers, and mixtures of such isomers are often called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur when there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species that lacks optical activity.

[0185] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton (e.g., keto-enol and imine-enamine isomerizations). Valence tautomers include interconversions via reorganization of some of the bonding electrons.

[0186] The term "imine-reactive reagent" refers to a reagent capable of reacting with an imine group. Examples of imine-reactive reagents include sulfite (H2SO3, H2SO2, or HSO3 formed with the cation) - , SO3 2- or HSO2 - salts), metabisulfite (H2S2O5, or S2O5 formed with cations 2- salts), mono-, di-, tri- and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3, and PO3S formed with cations 3- , PO2S2 3- ,POS3 3- Or PS4 3- salts), thiophosphate esters (R i O)2PS(OR i ), R i S.H., R. i S.O.H., R. i SO2H, R i SO3H, various amines (hydroxylamine (e.g., NH2OH), hydrazine (e.g., NH2NH2), NH2O-R i , R i ', NH-R i , NH2-R i ), NH2-CO-NH2, NH2-C(=S)-NH2' thiosulfate (H2S2O3, or S2O3 formed with cation 2- salts), dithionites (H2S2O4, or S2O4 formed with cations 2- salts), phosphorodithioates (P(=S)(OR k )(SH)(OH), or salts formed with cations), hydroxamic acids (R kC(=O)NHOH, or salts formed with cations), hydrazides (R k CONHNH2), formaldehyde sulfoxylate (HOCH2SO2H, or HOCH2SO2 - Na + Formed with cations such as HOCH2SO2 - salts thereof), glycosylated nucleotides (e.g., GDP-mannose), fludarabine, or mixtures thereof, wherein R i and R i ' are each independently a straight or branched chain alkyl having 1 to 10 carbon atoms, -N(R J )2, -CO2H, -SO3H, and -PO3H; R i and R i’ may be further optionally substituted with alkyl substituents as described herein; R j is a straight or branched chain alkyl having 1 to 6 carbon atoms; R k is a linear, branched or cyclic alkyl, alkenyl or alkynyl, aryl, heterocyclyl or heteroaryl having 1 to 10 carbon atoms (preferably R k is a straight or branched chain alkyl having 1 to 4 carbon atoms, more preferably R k is methyl, ethyl or propyl). Preferably the cation is Na + or K + Preferably, the imine-reactive reagent is selected from sulfite, hydroxylamine, urea, and hydrazine. More preferably, the imine-reactive reagent is NaHSO or KHSO.

[0187] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present invention. Examples of salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentianate, fumarate, gluconate, glucuronate, sucrose, formate, benzoate, glutamate, methanesulfonate, "mesylate," ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion may be an organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have two or more charged atoms in its structure. Examples in which multiple charged atoms are part of a pharmaceutically acceptable salt may have multiple counterions. Thus, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0188] When the compound of the present invention is a base, the desired pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, by reaction with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, etc.). acid, methanesulfonic acid, phosphoric acid, etc.), or an organic acid (e.g., acetic acid, maleic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid), a pyranosidyl acid (e.g., glucuronic acid or galacturonic acid), an alpha hydroxy acid (e.g., citric acid or tartaric acid), an amino acid (e.g., aspartic acid or glutamic acid), an aromatic acid (e.g., benzoic acid or cinnamic acid), a sulfonic acid (e.g., p-toluenesulfonic acid or ethanesulfonic acid), and the like.

[0189] When the compound of the present invention is an acid, the desired pharmaceutically acceptable salt can be prepared by any suitable method, for example, treatment of the free acid with an inorganic or organic base, such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide, or an alkaline earth metal hydroxide. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids (glycine and arginine), ammonia, primary, secondary, and tertiary amines and cyclic amines (e.g., piperidine, morpholine, and piperazine), and inorganic salts derived from sodium, calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

[0190] As used herein, the term "solvate" refers to a compound that further includes a stoichiometric or non-stoichiometric amount of a solvent (e.g., water, isopropanol, acetone, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine dichloromethane, 2-propanol, etc.) bound by non-covalent intermolecular forces. Solvates or hydrates of a compound are readily prepared by adding at least one molar equivalent of a hydroxylic solvent (e.g., methanol, ethanol, 1-propanol, 2-propanol, or water) to the compound, resulting in solvation or hydration of the imine moiety.

[0191] A "metabolite" or "catabolic product" is a product produced by metabolism or catabolism in the body of a particular compound, its derivative, or conjugate, or its salt. Metabolites of a compound, its derivative, or conjugate can be identified using routine techniques known in the art, and their effects can be measured using tests such as those described herein. Such products can result, for example, from oxidation, hydroxylation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of an administered compound. Accordingly, the present invention includes metabolites of a compound, its derivative, or conjugate of the present invention, including compounds, derivatives, or conjugates thereof, produced by a method comprising contacting a compound, derivative, or conjugate of the present invention with a mammal for a period of time sufficient to yield the metabolic product.

[0192] The term "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients comprising the formulation and / or the mammal being treated therewith.

[0193] The term "protecting group" or "protecting moiety" refers to a substituent that is commonly used to block or protect a particular functional group while reacting with other functional groups on a compound, its derivatives, or complexes thereof. For example, an "amine protecting group" or "amino protecting moiety" is a substituent attached to an amino group that blocks or protects the amino functionality of the compound. Such groups are well known in the art (see, for example, P. Wuts and T. Greene, 2007, Protective Groups in Organic Synthesis, Chapter 7, J. Wiley & Sons, NJ), and examples include carbamates (e.g., methyl and ethyl carbamates, FMOC, substituted ethyl carbamates, carbamates that are cleaved by 1,6-β elimination (also known as "self-immolative"), urea, amides, peptides, alkyl and aryl derivatives). Suitable amino protecting groups include: Groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see P.G.M. Buts & T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.

[0194] The term "amino acid" refers to a naturally occurring amino acid or a non-naturally occurring amino acid. In one embodiment, an amino acid is an amino acid having the formula NH2-C(R aa’ R aa )-C(=O)OH, where R aa and R aa’ are each independently H, an optionally substituted linear, branched, or cyclic alkyl, alkenyl, or alkynyl having 1 to 10 carbon atoms, aryl, heteroaryl, or heterocyclyl, or R aa and the N-terminal nitrogen atom can together form a heterocycle (e.g., in proline). The term "amino acid residue" refers to a residue in which one hydrogen atom is attached to an amino acid (e.g., -NHC(R aa 'R aa When removed from the amine and / or carboxy terminus of —C(═O)O—, it refers to the corresponding residue.

[0195] The term "cation" refers to a positively charged ion. Cations are monovalent (e.g., Na + , K. + , NH4 + etc.), divalent (e.g., Ca 2+ , Mg 2+ ), or multivalent (e.g., Al 3+ etc.) Preferably, the cation is monovalent.

[0196] The term "reactive ester group" refers to an ester group that can readily react with an amine group to form an amide bond. Representative reactive ester groups include, but are not limited to, N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxysulfosuccinimide esters, para-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, and derivatives thereof, which facilitate amide bond formation. In certain embodiments, the reactive ester group is an N-hydroxysuccinimide ester or an N-hydroxysulfosuccinimide ester.

[0197] The term "amine-reactive group" refers to a group that can react with an amine group to form a covalent bond. Representative amine-reactive groups include, but are not limited to, a reactive ester group, an acyl halide, a sulfonyl halide, an imide ester, or a reactive thioester group. In certain embodiments, the amine-reactive group is a reactive ester group. In one embodiment, the amine-reactive group is an N-hydroxysuccinimide ester or an N-hydroxysulfo-succinimide ester.

[0198] The term "thiol-reactive group" refers to a group that can react with a thiol (-SH) group to form a covalent bond. Exemplary thiol-reactive groups include, but are not limited to, maleimide, haloacetyl, haloacetamide, vinylsulfone, vinylsulfonamide, or vinylpyridine. In one embodiment, the thiol-reactive group is maleimide.

[0199] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the content clearly dictates otherwise.

[0200] Whenever an embodiment is described herein with the word "comprising," it is understood that other similar embodiments described with the words "consisting of" and / or "consisting essentially of" are also provided.

[0201] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0202] Names of antibodies, compounds, and immune complexes As used herein, the names used for anti-CD123 antibodies, cytotoxic compounds, and their immunoconjugates generally adopt the following meanings.

[0203] CD123-3, -6, and -14 (or CD123Mu-3, -6, and -14; or muCD123-3, -6, and 14) are three murine anti-CD123 monoclonal antibodies. The heavy and light chain CDR1-3 sequences (VH-CDR1-3 and VL-CDR1-3) are provided in Tables 1 and 2 with associated SEQ ID NOS: 1-25. The heavy chain variable region (HCVR) sequences are provided in Table 3A with associated SEQ ID NOS: 26, 28, and 30. The light chain variable region (LCVR) sequences are provided in Table 4A with associated SEQ ID NOS: 27, 29, and 31. The full-length heavy chain (HC) sequences of the murine antibodies are provided in Table 5 (SEQ ID NOS: 42, 44, and 46), and the full-length light chain (LC) sequences of the murine antibodies are provided in Table 6 (SEQ ID NOS: 43, 45, and 47).

[0204] chCD123-3, -6, and -14 are corresponding mouse-human chimeric antibodies with mouse heavy and light chain variable regions and human constant region sequences. For example, chimeric antibody chCD123-6 consists of the mouse HCVR and LCVR of SEQ ID NOs: 28 and 29, along with human IgG1 and κ constant sequences in the heavy and light chains, respectively. See Example 3.

[0205] huCD123-3, -6, and -14 are the corresponding humanized antibodies. When humanization is by CDR-grafting of the six corresponding murine CDR regions (HC and LC CDR1-3), the letter "G" immediately follows the clone designation, followed by a version number indicating the origin of the human light and heavy chain variable region sequences. Thus, huCD123-6Gv4.6 refers to a humanized CD123 antibody based on grafting ("G") the six CDR regions from the corresponding muCDR123-6 antibody onto the human light chain variable region Gv4 and heavy chain variable region Gv6. Similarly, -Gv4.7 contains the human light chain variable region Gv4 and heavy chain variable region Gv7, and -Gv1.1 contains the human light chain variable region Gv1 and heavy chain variable region Gv1.

[0206] Three HCVR sequences, huCD123-6Gv1, -Gv6, and -Gv7, are provided in Table 3A (SEQ ID NOs:32 and 34, where SEQ ID NO:34 differs only at the second residue, Xaa, and therefore represents -Gv6 and -Gv7), and their DNA coding sequences are provided in Table 3B (SEQ ID NOs:62, 64, and 66). Three full-length HC sequences, huCD123-6Gv1, -Gv6, and -Gv7, are provided in Table 5 (SEQ ID NOs:48 and 50, where SEQ ID NO:50 differs only at the second residue, Xaa, and therefore represents full-length -Gv6 and -Gv7).

[0207] Two LCVR sequences, huCD123-6Gv1 and -Gv4, are provided in Table 4A (SEQ ID NOs: 33 and 35), and their DNA coding sequences are provided in Table 4B (SEQ ID NOs: 63 and 65). Two full-length LC sequences (huCD123-6Gv1 and -Gv4) are provided in Table 6 (SEQ ID NOs: 49 and 51).

[0208] When humanization is by resurfacing, the resurfaced heavy chain sequence is designated by "rh" immediately following the mouse CD123 antibody clone number and is further designated by one of two versions of the resurfaced sequence, v1.0 or v1.1. Thus huCD123-6rhv1.0 and -rhv1.1 are resurfaced heavy chain sequences with CDR regions corresponding to the muCD123-6 antibody, with the version designations 1.0 and 1.1, respectively. See HCVR SEQ ID NOS: 39 and 40 in Table 3A, and SEQ ID NOS: 68 and 69 in Table 3B. See also full-length HC SEQ ID NOS: 59 and 60 in Table 5.

[0209] Similarly, the only version of the resurfaced light chain sequence, huCD123-6rlv1.0, has LCVR SEQ ID NO:41 in Table 4A and full-length LC SEQ ID NO:61 in Table 6.

[0210] The resurfaced antibody with huCD123-6rhv1.0 and huCD123-6rlv1.0 is huCD123-6Rv1.0, and the resurfaced antibody with huCD123-6rhv1.1 and huCD123-6rlv1.0 is huCD123-6Rv1.1.

[0211] NTS2, or abbreviated "S2," refers to an antibody with Ser engineered into the N-terminus of the heavy chain. The S2 variant of huCD123-6Gv6 / 7 has the HCVR sequence of Table 3A, SEQ ID NO:38, and the full-length HC protein sequence of Table 5, SEQ ID NO:53.

[0212] Similarly, NTS3, or abbreviated "S3," refers to an antibody with an engineered Ser at the N-terminus of the light chain. The S3 variant of huCD123-6Gv4 has the LCVR sequence of Table 4A, SEQ ID NO:37, and the full-length LC protein sequence of Table 6, SEQ ID NO:58.

[0213] Antibodies containing an engineered N-terminal Ser (S2 or S3) can be covalently conjugated to a cytotoxic drug / agent via an oxidized N-terminal Ser or via a "traditional" Lys linkage. When the drug is conjugated via an oxidized N-terminal Ser, the name of the conjugate includes the "SeriMab" designation. When the drug is conjugated via Lys, the name of the conjugate does not include SeriMab (despite the presence of an S2 or S3 designation indicating the presence of an engineered Ser at the N-terminus). The specific type of conjugation is also apparent based on the cytotoxic reactive group. For example, huCD123-6Gv4.7S3-SeriMab-D8 refers to a conjugate between D8 and the humanized CD123 antibody huCD123-6Gv4.7S3 via an oxidized N-terminal Ser of the light chain. The humanized CD123 antibody has grafted mouse CD123-6 CDR regions, human LC Gv4, and heavy chain Gv7, with the N-terminus of the light chain containing an engineered Ser (S3). In contrast, huCD123-6Gv4.7S3-sSPDB-D1 refers to the conjugate between D1 and the same humanized CD123 antibody huCD123-6Gv4.7S3 by Lys linkage via a sulfonated SPDB linker.

[0214] In certain embodiments, when both the light and heavy chain N-termini contain an engineered Ser, "S2S3" or "S2S3-SeriMab" may appear in the antibody name.

[0215] Certain antibodies of the invention have an engineered Cys in the heavy chain CH3 domain at a position corresponding to the same Kabat position as the penultimate Cys in SEQ ID NO: 54. Such HCs, or antibodies containing such HCs, are designated CysMab. Thus, huCD123-6Gv4.6-CysMab is a humanized CD123 antibody grafted with muCD123-6 CDR regions and based on the human light chain Gv4 and heavy chain Gv6 sequences, in which an engineered Cys is located in the HC CH3 domain at a position corresponding to the penultimate Cys in SEQ ID NO: 54. Similarly, its heavy chain sequence is huCD123-6Gv6-CysMab. Additionally, huCD123-6Gv4.6S2-CysMab is otherwise identical but has an engineered Ser at the N-terminus of the heavy chain, and its heavy chain sequence is huCD123-6Gv6S2-CysMab.

[0216] The resurfaced antibodies described above can be further engineered to contain an N-terminal Ser in either the light chain (S3 variant of the resurfaced antibody) or the heavy chain (S2 variant of the resurfaced antibody), or both (see below). Alternatively or additionally, the resurfaced antibody has an engineered Cys in the heavy chain CH3 domain at a position corresponding to the same Kabat position as the penultimate Cys in SEQ ID NO: 54 (CysMab version of the resurfaced antibody). The resurfaced antibody can have both an engineered Cys and an N-terminal Ser.

[0217] However, in such a conjugate formed between a CysMab and a cytotoxin, at least theoretically, the cytotoxin can be bound to the CysMab via Cys or via the conventional Lys. However, as used herein, unless otherwise specified, a conjugate with a CysMab designation also refers to a conjugate between a CysMab and a cytotoxin as a Cys bond (not a Lys bond). The specific type of bond is also clear based on the cytotoxin reactive group.

[0218] Other variations or combinations of the above general nomenclature are contemplated and will be readily apparent to those skilled in the art. For example, huCD123-6Rv1.1-CysMab is a resurfaced version of huCD123-6(v1.1) with an engineered Cys located in the HC CH3 region at a position corresponding to the penultimate Cys of SEQ ID NO:54.

[0219] The antibodies or antigen-binding fragments thereof of the present invention can be conjugated to specific cytotoxic agents via either a Lys side chain amino group, a Cys side chain thiol group, or an oxidized N-terminal Ser / Thr. Certain representative (non-limiting) cytotoxic agents described herein (including in the Examples) are listed below for illustrative purposes. Note that most compounds (e.g., D1, D2, D4, DGN462, D3, D6, etc.) can be sulfonated with one of the indolinobenzodiazepine monomers of certain Examples (not shown here, but see compound sD1 in Figure 17, compound sDGN462 in Figure 15, and compound sD8 in Figure 16). In compound D5', both indolinobenzodiazepine monomers can be sulfonated. [Table 2-1] [Table 2-2]

[0220] Note that due to the different linkage chemistries required to attach the cytotoxin to different antibody side chains (i.e., Lys-linked, Cys-linked, oxidized N-terminal Ser-linked), the agents are only slightly different. Nevertheless, these related cytotoxins are given different "D" designations. See D1 and D4, and D2, D5, and D8.

[0221] Conjugates of the subject antibodies and cytotoxic agents generally follow the names of the antibody and cytotoxic agent as described above.

[0222] For example, huCD123-6Gv4.6-sulfo-SPDB-D1 is a conjugate of the huCD123-6Gv4.6 antibody to compound D1 via an SPDB linker sulfonated at one or more Lys residues of the antibody. is a conjugate of the huCD123-6 antibody conjugated at one or more Lys residues of the antibody with the cytotoxic agent DM1 via a triglycyl linker termed the "CX1-1 linker." See Example 9e.

[0223] One notable exception is the conjugate huCD123-6-SeriMab-sD1 shown in Figure 7B and Figure 17, where the short linker sequence between huCD123-6-SeriMab and the sD1 cytotoxin is not explicitly indicated in the conjugate name. Similarly, the conjugate huCD123-6-SeriMab-sDGN462 in Figure 15 is also an exception to the general rule described above.

[0224] 2. CD123-binding agents In a first aspect, the present invention provides agents that specifically bind to CD123 / IL-3Rα (e.g., human CD123 / IL-3Rα). These agents are generally referred to herein as "CD123 / IL-3Rα binding agents." In certain embodiments, the CD123 / IL-3Rα binding agent is an antibody or antigen-binding fragment thereof (or simply "antibody"), an immunoconjugate thereof, or a polypeptide thereof.

[0225] The amino acid and nucleotide sequences of CD123 / IL-3Rα in humans and other species are well known in the art. For example, as shown in NCBI RefSeq NP_002174, the human CD123 / IL-3Rα splice variant 1 protein sequence is reproduced as follows: [ka]

[0226] The above sequence represents the CD123 / IL-3Rα precursor chain protein, which is composed of 378 amino acids, including an extracellular domain (residues 1-306, including an 18-residue N-terminal signal peptide), a 20-amino acid transmembrane domain, and a short 52-amino acid cytoplasmic tail.

[0227] As shown in NCBI RefSeq NM_002183, the human CD123 / IL-3Rα splice variant 1 nucleic acid sequence is reproduced as follows: [ka] [ka]

[0228] Protein and nucleic acid sequences of CD123 / IL-3Rα from other non-human species can be readily retrieved from public databases such as GenBank using sequence search tools well known in the art (e.g., NCBI BLASTp or BLASTn) and the above-mentioned protein and nucleic acid sequences, respectively, as query sequences.

[0229] Such sequences from non-human species can be aligned with human sequences using any of a number of art-recognized sequence alignment tools (e.g., those described herein and above), so that any amino acid residue or nucleotide that "corresponds" to any given human sequence or region of a sequence can be readily obtained.

[0230] Accordingly, one aspect of the present invention provides an antibody or antigen-binding fragment thereof that (a) binds an epitope within amino acids 101 to 346 of the human CD123 antigen, and (b) inhibits IL3-dependent proliferation of antigen-positive TF-1 cells.

[0231] In some embodiments, the anti-CD123 / IL-3Rα antibody or antigen-binding fragment thereof can specifically bind to an epitope of SEQ ID NO: 36. In certain embodiments, the epitope is within a region corresponding to residues 101-346 of human CD123 / IL-3Rα. In certain embodiments, the epitope is within a region corresponding to residues 101-204 of SEQ ID NO: 36. In certain other embodiments, the epitope is within a region corresponding to residues 205-346 of SEQ ID NO: 36. In certain embodiments, the epitope is not within a region corresponding to residues 1-100 of human CD123 / IL-3Rα.

[0232] In certain embodiments, the CD123 / IL-3Rα binding agents (e.g., antibodies) inhibit IL-3-dependent signaling (e.g., IL-3-dependent proliferation of CD123-positive TF-1 cells). Without being bound by any particular theory, the CD123 / IL-3Rα binding agents (e.g., antibodies) of the present invention bind to CD123, such as within a region of CD123 corresponding to residues 101-346 (e.g., residues 101-204, or 205-346) of human CD123 / IL-3Rα, and inhibit productive binding and / or signaling between CD123 and an IL-3 ligand. prevents, reduces, attenuates, or inhibits productive binding between CD123 and the common beta chain CD131, resulting in reduced or abolished IL-3-dependent signaling.

[0233] In a related aspect, the invention provides a compound that (a) binds an epitope within amino acids 1-100 of the human CD123 antigen, and (b) has an IC of 0.1 nM or less (e.g., 0.08 nM, 0.05 nM, 0.03 nM). 50 The present invention provides an antibody or antigen-binding fragment thereof that inhibits IL3-dependent proliferation of antigen-positive TF-1 cells, the antibody or antigen-binding fragment having therapeutic value.

[0234] In certain embodiments, binding by a CD123 / IL-3Rα binding agent (e.g., an antibody) of the present invention inhibits (e.g., preferentially inhibits) proliferation of leukemic stem cells (LSCs), leukemic progenitor cells (LPs), or leukemic blasts, but does not substantially inhibit proliferation of normal hematopoietic stem cells (HSCs).

[0235] Inhibition of cell proliferation can be performed using any standard assay known in the art, including, but not limited to, flow cytometry. For example, normal HSCs, LSCs, LPs, and leukemic blasts can be separated using flow cytometry based on differential expression of cell surface markers, and the relative numbers of surviving or remaining cells can be quantitatively determined and compared after incubation with a test agent.

[0236] Inhibition of cell proliferation of LSCs, LPs, or leukemic blasts relative to normal HSCs can also be measured using in vitro potency assays of primary cancer cells (e.g., primary AML cells). For example, AML cells (or normal human bone marrow samples containing normal HSCs) can be exposed to various concentrations of a target anti-CD123 antibody, its antigen-binding fragment, its immunoconjugate, or a polypeptide containing the antibody or antigen-binding fragment for 24 hours. A non-targeting (isotype-matched) antibody or immunoconjugate (ADC) control can also be used in the assay. Samples can be split into short-term liquid culture (STLC) assays to measure cytotoxicity against LSCs, LPs, or leukemic blasts, and into long-term liquid culture (LTLC) assays to measure effects on LSCs and normal HSCs. STLC can be used to measure colony-forming units (CFUs) for 10–14 days after plating cells, for example, in semi-solid MethoCult H4230 medium (Stemcell Technologies). LTLC assays can also be performed with growth factors added during long-term culture for 5–7 weeks. In both assays, colonies can be counted to measure colony-forming units per number of cells initially plated. LTLC colonies were further analyzed for the presence of molecular markers of cancer (e.g., AML) using PCR or FISH or both.

[0237] In certain embodiments, the antibody or antigen-binding fragment thereof has a dissociation constant (K dIn certain embodiments, the antibody or antigen-binding fragment thereof binds to human CD123 antigen-positive cells with a K of 0.05 nM to 0.3 nM, or 0.05 nM to 0.2 nM, or 0.05 nM to 0.1 nM, or 0.01 nM to 0.3 nM, or 0.01 nM to 0.2 nM, or 0.01 nM to 0.1 nM. d and binds to human CD123.

[0238] In certain embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus monkey CD123. In certain embodiments, the antibody or antigen-binding fragment thereof has a K of 0.05 nM to 0.3 nM, or 0.05 nM to 0.2 nM, or 0.05 nM to 0.1 nM. d and binds to cynomolgus monkey CD123.

[0239] In certain embodiments, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus CD123 with substantially similar binding affinity. In certain embodiments, the antibody or antigen-binding fragment thereof has a K of 0.05 nM to 0.3 nM, or 0.05 nM to 0.2 nM, or 0.05 nM to 0.1 nM. d and binds to both human and cynomolgus CD123.

[0240] In certain embodiments, K d Values ​​are based on cell-based binding assays. In certain embodiments, K d The K value is measured by flow cytometry. d The K value is measured by surface plasmon resonance (e.g., using a BIOCORE™ surface plasmon resonance system). d The value is measured by radioimmunoassay (RIA). In certain embodiments, K d is measured by any other art-recognized method.

[0241] In certain embodiments, the antibody or antigen-binding fragment thereof inhibits at least 50% of IL3-dependent proliferation of antigen-positive TF-1 cells at a concentration of 0.5 nM or less.

[0242] In certain embodiments, the CD123 / IL-3Rα binding agent is a CD123 / IL-3Ra antibody or antigen-binding fragment thereof, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), each comprising three CDR regions (e.g., CDR1-CDR3 in the HCVR and CDR1-CDR3 in the LCVR), and the complex CDRs in the HCVR and LCVR have any of the sequences shown in Tables 1 and 2 below. [Table 3] [Table 4]

[0243] In certain embodiments, anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) at least one heavy chain variable region or fragment thereof comprising three contiguous complementarity-determining regions (CDRs), CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 1, 5, and 12; CDR2 is selected from the group consisting of SEQ ID NOs: 2-3, 6-10, and 13-14; and optionally, CDR3 is selected from the group consisting of SEQ ID NOs: 4, 11, 15, and 70. and b) at least one light chain variable region or fragment thereof, each comprising three contiguous complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 16, 19-20, 23, and 72; CDR2 is selected from the group consisting of SEQ ID NOs: 17, 21, 24, and 71; and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 18, 22, and 25.

[0244] In certain embodiments, anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) at least one heavy chain variable region or fragment thereof comprising three contiguous complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 1, 5, and 12; CDR2 is selected from the group consisting of SEQ ID NOs: 2-3, 6-10, and 13-14; and optionally, CDR3 is selected from the group consisting of SEQ ID NOs: 4, 11, and 15. or a fragment thereof; and b) at least one light chain variable region or a fragment thereof comprising three contiguous complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, respectively, wherein, except for one, two, or three conservative amino acid substitutions, CDR1 is selected from the group consisting of SEQ ID NOs: 16, 19-20, and 23; CDR2 is selected from the group consisting of SEQ ID NOs: 17, 21, and 24; and optionally CDR3 is selected from the group consisting of SEQ ID NOs: 18, 22, and 25.

[0245] In certain embodiments, the conservative amino acid substitution comprises a substitution of at least one Lys in a CDR with Arg (such as the Lys to Arg substitutions in SEQ ID NOs: 6 and 7, 8 and 9, and 19 and 20). In certain embodiments, the antibody comprises a CDR-grafted humanized antibody comprising murine CDR regions, and one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) heavy chains and / or a CDR of the antibody. Alternatively, the light chain framework region Vernier zone residues are of murine origin.

[0246] In certain embodiments, anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2 or 3, and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO: 4; and 2) an immunoglobulin light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 16, CDR2 having the amino acid sequence set forth in SEQ ID NO: 17, and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 2. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 3.

[0247] In certain embodiments, anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain variable region comprising: CDR1 having the amino acid sequence set forth in SEQ ID NO:5; CDR2 having the amino acid sequence set forth in SEQ ID NO:6, 7, 8, 9, or 10; and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO:11; and 2) an immunoglobulin light chain variable region comprising: CDR1 having the amino acid sequence set forth in SEQ ID NO:19 or 20; CDR2 having the amino acid sequence set forth in SEQ ID NO:21; and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO:22. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO:6, and CDR1 of the light chain variable region is SEQ ID NO:19. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO:7, and CDR1 of the light chain variable region is SEQ ID NO:19. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO:8, and CDR1 of the light chain variable region is SEQ ID NO:19. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 9 and CDR1 of the light chain variable region is SEQ ID NO: 19. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 10 and CDR1 of the light chain variable region is SEQ ID NO: 19. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 6 and CDR1 of the light chain variable region is SEQ ID NO: 20. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 7 and CDR1 of the light chain variable region is SEQ ID NO: 20. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 8 and CDR1 of the light chain variable region is SEQ ID NO: 20. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 9 and CDR1 of the light chain variable region is SEQ ID NO: 20. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 10 and CDR1 of the light chain variable region is SEQ ID NO: 20. In each of the above-mentioned combinations of heavy chain variable region CDR2 and light chain variable region CDR1, the heavy chain variable region CDR1 and CDR3 are SEQ ID NOs: 5 and 11, respectively, and the light chain variable region CDR2 and CDR3 are SEQ ID NOs: 21 and 22, respectively.

[0248] In certain embodiments, anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 12, CDR2 having the amino acid sequence set forth in SEQ ID NO: 13 or 14, and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO: 15; and 2) an immunoglobulin light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 23, CDR2 having the amino acid sequence set forth in SEQ ID NO: 24, and optionally, CDR3 having the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 13. In certain embodiments, CDR2 of the heavy chain variable region is SEQ ID NO: 14.

[0249] In certain embodiments, CDR1 sequences from the light and heavy chains of one antibody (e.g., SEQ ID NOs: 5 and 19) can be combined with CDR2 sequences from the light and heavy chains of another antibody (e.g., SEQ ID NOs: 2 and 17), and optionally with CDR3 sequences from the light and heavy chains of the same (e.g., SEQ ID NOs: 4 and 18, or 11 and 22) or even another antibody (e.g., SEQ ID NOs: 15 and 25). Based on SEQ ID NOs: 1 to 25 in Table 1, all possible combinations, particularly those relating to the same antibody number (e.g., all six light and heavy chain CDRs), are possible. is derived from CD123-3, or CD123-6, or CD123-14) are contemplated herein without exhaustively listing all specific combinations.

[0250] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof conserve amino acid substitutions at one, two, or three consecutive residues in any one or more of the above CDR sequences. That is, in some embodiments, the subject antibodies and antigen-binding fragments thereof may conserve amino acid substitutions at one, two, or three consecutive residues in any one or more of SEQ ID NOs: 1-25.

[0251] In certain embodiments, the CD123 / IL-3Rα binding agent is a CD123 / IL-3Ra antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), where the HCVR and LCVR are any of the sequences set forth in Tables 3A and 4A below. Selected corresponding nucleic acid sequences encoding the HCVRs and LCVRs are in Tables 3B and 4B. [Table 5] [Table 6-1] [Table 6-2] [Table 7] [Table 8]

[0252] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of (a) reference V and VIII antibodies having the amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38). H V with sequences at least 95% identical H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L V with sequences at least 95% identical L Contains arrays.

[0253] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of: (a) the amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38). Reference V Hsequences and V that are at least 96% identical H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L sequences and V that are at least 96% identical L Contains arrays.

[0254] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of (a) reference V and VIII antibodies having the amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38). H sequences and V that are at least 97% identical H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L sequences and V that are at least 97% identical L Contains arrays.

[0255] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of (a) reference V and VIII antibodies having the amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38). H sequences and V that are at least 98% identical H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L sequences and V that are at least 98% identical L Contains arrays.

[0256] In certain embodiments, the anti-CD123 antibodies and antigen-binding fragments thereof are selected from the group consisting of (a) a reference V antibody having an amino acid sequence represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38).H sequence and V that are at least 99% identical H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L sequence and V that are at least 99% identical L Contains arrays.

[0257] In certain embodiments, CD123 / IL-3Rα antibodies / antigen-binding fragments thereof having a particular percentage of sequence identity to SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (preferably SEQ ID NOs: 26, 28, 30, 32, 34, and 38) and / or SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) differ from SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38) and / or SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) only by conservative amino acid substitutions (e.g., 1, 2, or 3 conservative amino acid substitutions). In certain embodiments, the conservative amino acid substitutions are one, two, or three conservative amino acid substitutions in one or more CDR regions of the heavy and / or light chain.

[0258] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of (a) reference V and VIII antibodies having the amino acid sequences represented by SEQ ID NOs: 26, 28, 30, 32, 34, 38, 39, and 40 (or SEQ ID NOs: 26, 28, 30, 32, 34, and 38). H V identical to the array H and / or (b) a reference V selected from the group having the amino acid sequences represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37). L V identical to the array L Contains arrays.

[0259] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise the V H sequence, and / or V as set forth in SEQ ID NO: 27 L Contains arrays.

[0260] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise the V H sequence, and / or V as set forth in SEQ ID NO: 29 L Contains arrays.

[0261] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise the V H and / or V as set forth in SEQ ID NO: 31 L Contains arrays.

[0262] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise the V H and / or V as set forth in SEQ ID NO: 35 L Contains arrays.

[0263] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof are selected from the group consisting of: V, ... H Sequence and V L Contains arrays.

[0264] For example, in one embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41. H The sequence is set forth in SEQ ID NO: 39, and VL The sequence is set forth in SEQ ID NO: 41. In certain embodiments, V H The sequence is set forth in SEQ ID NO: 40, and V L The sequence is set forth in SEQ ID NO:41.

[0265] In a related aspect, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa in SEQ ID NO: 34 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 34 is Val (V).

[0266] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40, except that the first residue is substituted with Ser (S); and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0267] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 39 or 40; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41, except that the first residue is substituted with Ser (S).

[0268] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the N-terminal residue is Ser and except that the residue corresponding to the fifth-to-last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0269] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the residue corresponding to the fifth to last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 41, except that the N-terminal residue is Ser. It contains the globulin light chain variable region.

[0270] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 38, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa in SEQ ID NO: 38 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 38 is Val (V).

[0271] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, Xaa in SEQ ID NO: 34 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 34 is Val (V).

[0272] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 56, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa in SEQ ID NO: 56 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 56 is Val (V).

[0273] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, Xaa in SEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54 is Val (V).

[0274] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise: a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 59 or 60, except that the residue corresponding to the fifth-to-last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 41.

[0275] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa in SEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54 is Val (V).

[0276] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 56, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, Xaa in SEQ ID NO: 56 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 56 is Val (V).

[0277] In another embodiment, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof comprise a) an immunoglobulin heavy chain region having the amino acid sequence set forth in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, Xaa in SEQ ID NO: 54 is Phe (F). In certain embodiments, Xaa in SEQ ID NO: 54 is Val (V).

[0278] In certain embodiments, the anti-CD123 / IL-3Rα antibodies and antigen-binding fragments thereof bind to CD1 In certain embodiments, the CD123 / IL-3Rα antibody or antigen-binding fragment thereof is a murine, chimeric, humanized, or non-human antibody or antigen-binding fragment thereof that specifically binds to CD123 / IL-3Rα. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is a CDR-grafted or resurfaced antibody or antigen-binding fragment thereof.

[0279] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a full-length antibody. A full-length antibody can include any of the above-mentioned antibodies defined by one to four CDRs (e.g., CDR1 and CDR2 of the heavy chain, CDR1 and CDR2 of the heavy and light chains), one to six CDR sequences (e.g., CDR1-CDR3 of the heavy chain, CDR1-CDR3 of the heavy and light chains), or any of the above-mentioned antibodies defined by an LCVR and / or HCVR, or any of the full-length antibodies having a heavy chain sequence in Table 5, or any of the full-length antibodies having a light chain sequence in Table 6, or any of the full-length antibodies having a heavy chain sequence in Table 5 and a light chain sequence in Table 6. [Table 9-1] [Table 9-2] [Table 9-3] [Table 10]

[0280] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a full-length antibody comprising (a) a heavy chain having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the full-length heavy chain sequences described above (e.g., any of the full-length heavy chain sequences in Table 5), and / or (b) a light chain having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the full-length light chain sequences described above (e.g., any of the full-length light chain sequences in Table 6). In certain embodiments, the anti-CD123 / IL-3Rα antibody is selected from the group consisting of SEQ ID NOs: 42 / 43, 44 / 45, 46 / 47, 48 / 49, 50 / 49, 53 / 4 and 60 / 61, or antibodies having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to either the full-length heavy chain sequence and / or the full-length light chain sequence selected from the group consisting of: 9, 54 / 49, 56 / 49, 59 / 49, 60 / 49, 48 / 51, 50 / 51, 53 / 51, 54 / 51, 56 / 51, 59 / 51, 60 / 51, 48 / 58, 50 / 58, 53 / 58, 54 / 58, 56 / 58, 59 / 58, 60 / 58, 59 / 49, 59 / 51, 59 / 58, 59 / 61, 60 / 49, 60 / 51, 60 / 58, and 60 / 61.

[0281] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a full-length antibody comprising a combination of full-length heavy chain and full-length light chain sequences selected from the group consisting of SEQ ID NOs: 59 / 61 and 60 / 61, or an antibody having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the full-length heavy chain and / or light chain sequences. Such antibodies may further comprise an engineered N-terminal Ser / Thr in the light chain, the heavy chain, or both. Such antibodies may further comprise an engineered Cys in the heavy chain CH3 domain at a position corresponding to the penultimate Cys in SEQ ID NO: 54.

[0282] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a murine, chimeric, humanized, or human antibody that specifically binds to CD123 / IL-3Rα. In certain embodiments, an anti-CD123 / IL-3Rα antibody having a particular percentage of sequence identity to any of the full-length SEQ ID NOs differs from such SEQ ID NOs by conservative amino acid substitutions only (e.g., by 1, 2, 3, 4, or 5 consecutive conservative amino acid substitutions). In certain embodiments, the conservative amino acid substitutions are outside the CDRs.

[0283] In certain embodiments, the antigen-binding fragment is or comprises a Fab, Fd, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, small antibody, F(ab')3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc of any of the aforementioned antibodies.

[0284] In a related aspect, the present invention provides an antibody or antigen-binding fragment thereof, H and / or V L Also provided are polypeptides comprising any of the sequences, any of the HCVRs and / or LCVRs described above, or any of the CDR sequence(s) of the HCVRs and / or LCVRs described above. The polypeptides may be fused, for example, with a non-antibody protein or domain. In certain embodiments, the fusion protein is not a fusion with a Pseudomonas toxin.

[0285] The affinity or avidity of an antibody for an antigen can be experimentally measured using any suitable method known in the art (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., BIACORE™ analysis). Direct binding assay and competitive binding assay formats are readily available. See, for example, Berzofsky et al., "Antibody-Antigen Interactions," in Fundamental Immunology, Paul, W.E., Ed., Raven Press: New York, NY (1984); Kuby, Janis, Immunology, W.H. Freeman and Company: New York, NY (1992), and the methods described herein.

[0286] The measured affinity of a particular antibody-antigen interaction may differ when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, affinity and other antigen binding parameters may differ. (For example, K D , or K d , K. on , K. off Measurements of ) are performed using standardized solutions of antibody and antigen, and standardized buffers known in the art, such as those described herein.

[0287] In one embodiment, binding assays can be performed using flow cytometry of cells expressing the CD123 / IL-3Rα antigen on their surface. For example, CD123 / IL-3Rα positive cells are cultured at 1×10 per sample in 100 μL of FACS buffer (e.g., RPMI-1640 medium supplemented with 2% normal goat serum). 5Cells can be incubated with various concentrations of anti-CD123 / IL-3Rα antibodies. They can then be pelleted, washed, and incubated with 100 μL of FITC-conjugated goat anti-mouse or goat anti-human IgG antibody (e.g., 6 μg / mL in FACS buffer from Jackson Laboratory) for 1 hour. They are then pelleted again, washed with FACS buffer, and resuspended in 200 μL of PBS containing 1% formaldehyde. Samples can be acquired using, for example, a FACSCalibur flow cytometer equipped with an HTS multiwell sampler and analyzed using CellQuest Pro (all from BD Biosciences, San Diego, US). For each sample, the mean fluorescence intensity (FL1) (MFI) can be exported and plotted against antibody concentration on a semilogarithmic plot to generate a binding curve. A sigmoidal dose-response curve can be fitted to the binding curve, and the EC 50 Values ​​are calculated using a program such as GraphPad Prism v4 (GraphPad software, San Diego, CA) with default parameters. 50 The value is the apparent dissociation constant, K d " or "K D " can be used as a criterion for

[0288] Monoclonal antibodies can be prepared using the hybridoma method (e.g., as described in Kohler and Milstein (1975) Nature 256:495). Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized to induce the production by lymphocytes of antibodies that specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line, e.g., using polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas producing monoclonal antibodies specifically directed against a selected antigen, as determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)), can then be propagated in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), or in vivo as ascites tumors in animals. Monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies.

[0289] Alternatively, monoclonal antibodies can be produced using recombinant DNA methods, as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, for example, by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the antibody heavy and light chains, and their sequences are determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors and transfected into host cells (e.g., E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins), whereupon the monoclonal antibody is produced by the host cells. Furthermore, recombinant monoclonal antibodies of the desired species, or fragments thereof, can be isolated from phage display libraries expressing the CDRs of the desired species, as described (McCafferty et al., 2002). t al., Nature348:552-554, 1990, Clackson et al. al., Nature, 352:624-628, 1991, and Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0290] The polynucleotide(s) encoding a monoclonal antibody can be further modified in many different ways using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a murine monoclonal antibody can be replaced with 1) those regions of a human antibody, e.g., to generate a chimeric antibody, or 2) non-immunoglobulin polypeptides to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate desired antibody fragments of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0291] In some embodiments, the monoclonal antibody against human CD123 / IL-3Rα is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject.

[0292] Methods for engineering, humanizing, or resurfacing non-human or human antibodies are also available and well known in the art. Humanized, resurfaced, or similarly engineered antibodies can have one or more amino acid residues from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammal. These non-human amino acid residues are often replaced by residues referred to as "import" residues, which are usually taken from an "import" variable, constant, or other domain of a known human sequence.

[0293] Such imported sequences can be used to reduce immunogenicity, or to reduce, enhance, or alter binding affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property, as is well known in the art. Generally, the CDR residues are directly and most substantially involved in influencing CD123 / IL-3Rα binding. Thus, some or all of the non-human or human CDR sequences can be maintained, while the non-human sequences of the variable and constant regions can be substituted with human or other amino acids.

[0294] Antibodies can also be humanized, resurfaced, engineered, or humanized, optionally with retention of high affinity for the antigen CD123 / IL-3Rα and other favorable biological properties. To this end, humanized (or human) or engineered anti-CD123 / IL-3Rα antibodies and resurfaced antibodies can be prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the role of residues that may affect the function of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen (e.g., CD123 / IL-3Rα). In this way, framework (FR) residues from the consensus and import sequences can be selected and combined so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved.

[0295] Humanization, resurfacing or engineering of the antibodies of the present invention can be accomplished using methods such as, but not limited to, Winter (Jones et al.), Nature 321:522, 1986; Riechmann et al.,Nature332:323,1988, Verhoeyen et al. al.,Science239:1534,1988,Sims et al.,J.Immunol.151:2296,1993,Chothia and Lesk,J.Mol.Biol.196:901,1987,Carteret al.,Proc.Natl.Acad.Sci.USA89:4285,1992,Presta et al. al., J. Immunol. 151:2623, 1993, Raguska et al. al., Proc.Natl.Acad.Sci.USA91(3):969-973,1994, U.S. Patent No. 5,639,641, U.S. Patent No. 5,723, No. 323, No. 5,976,862, No. 5,824,514, No. 5,817,483, No. 5,814,476, No. 5,763,192 , No. 5,723,323, No. 5,766,886, No. 5,714,352, No. 6,204,023, No. 6,180,370, No. 5 , No. 693,762, No. 5,530,101, No. 5,585,089, No. 5,225,539, No. 4,816,567, PCT / US9 This can be done using well-known methods such as those described in PCT Publication Nos. WO 90 / 14443, WO 90 / 14424, WO 90 / 14430, EP 229246, EP 7,557,189, EP 7,538,195, and EP 7,342,110, each of which, including the references therein, is incorporated by reference in its entirety.

[0296] In certain alternative embodiments, the antibody against CD123 / IL-3Rα is a human antibody. Human antibodies can be prepared directly using various techniques known in the art. Immortalized human B lymphocytes can be generated by in vitro immunization or by isolation from immunized individuals that produce antibodies against a target antigen (see, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147(1):86-95; and U.S. Pat. No. 5,750,313). (See, e.g., Vaughan et al., Nat. Biotech. 14:309-314, 1996; Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162, 1998; Hoogenboom et al., J. Immunol. 1999, 11:117-118, 1999). Additionally, human antibodies can be selected from phage libraries, as described, e.g., in Vaughan et al., Nat. Biotech. 14:309-314, 1996; Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162, 1998; Hoogenboom et al., J. Immunol. 1999, 11:117-118, 1999). and Winter, J. Mol. Biol. 227:381, 1991 and Marks et al., J. Mol. Biol. 222:581, 1991. Techniques for the generation and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915, 6,593,081, 6,300,064, 6,653,068, 6,706,484, and 7,264,963, as well as Rothe et al., J. Mol. Bio. doi:10.1016 / j.jmb.2007.12.018, 2007 (each of which is incorporated by reference in its entirety). Affinity maturation and chain shuffling methods (Marks et al., Bio / Technology 10:779-783, 1992, incorporated by reference in its entirety) are well known in the art and can be used to generate high-affinity human antibodies.

[0297] Humanized antibodies can also be produced in transgenic mice containing human immunoglobulin loci that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production, as described in U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016.

[0298] Certain embodiments provide antibody fragments that, for example, increase tumor invasion. Various techniques for producing antibody fragments are well known. Traditionally, these fragments were obtained via proteolysis of intact antibodies (e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117, 1993; Brennan et al., Science 229:81, 1985). In certain embodiments, antibody fragments are recombinantly produced. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing for the production of large amounts of these fragments. Such antibody fragments can also be isolated from antibody phage libraries. Antibody fragments can also be linear antibodies, e.g., as described in U.S. Pat. No. 5,641,870, and can be monospecific or bispecific. Other techniques for producing antibody fragments will be apparent to those skilled in the art.

[0299] It should be understood that the modified antibodies of the present invention can comprise any type of variable region that provides for the association of the antibody with the human CD123 / IL-3Rα polypeptide. In this regard, the variable region can comprise or be derived from any type of mammal that can be elicited to mount a humoral response and produce immunoglobulins against a desired tumor-associated antigen. Thus, the variable region of the modified antibody can be derived, for example, from a human, a mouse, a non-human primate (e.g., cynomolgus monkey, macaque, etc.), or a lupin. In some embodiments, the variable and constant regions of the engineered immunoglobulin are human. In other embodiments, the variable region of a compatible antibody (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding characteristics of the molecule or reduce its immunogenicity. In this regard, variable regions useful in the present invention can be humanized or otherwise altered by the inclusion of imported amino acid sequences.

[0300] In certain embodiments, both the heavy and light chain variable domains are altered by at least partial replacement of one or more CDRs, and optionally by partial framework region replacement and sequence exchange. The CDRs can be derived from antibodies of the same class or subclass as the antibody from which the framework regions are derived, although it is contemplated that the CDRs will be obtained from antibodies of a different class, and in certain embodiments, from antibodies derived from a different species. To transfer the antigen-binding capacity of one variable domain to another, it may not be necessary to replace all CDRs with the complete CDRs from the donor variable region. Rather, it may be necessary to transfer only those residues necessary to maintain the activity of the antigen-binding site. Given the descriptions in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, it is well within the capabilities of one skilled in the art to obtain functional antibodies with reduced immunogenicity, either by routine experimentation or trial-and-error testing.

[0301] Notwithstanding modifications to the variable region, one of skill in the art will understand that modified antibodies of the present invention include antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least one or more fractions of the constant region domains have been deleted or otherwise altered to provide desirable biochemical properties, such as increased tumor localization or decreased serum half-life, when compared to an antibody of substantially the same immunogenicity containing a native or unchanged constant region. In some embodiments, the constant region of the modified antibody comprises a human constant region. Modifications to the constant region compatible with the present invention include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, modified antibodies disclosed herein can include alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some embodiments, modified constant regions in which one or more domains are partially or completely deleted are contemplated. In some embodiments, modified antibodies include domain-deleted constructs or mutants in which the entire CH2 region has been removed. (ACH2 construct). In some embodiments, the omitted constant region domain is replaced with a short amino acid spacer (e.g., 10 residues) that provides some of the molecular flexibility normally conferred by the absent constant region.

[0302] It should be noted that in certain embodiments, modified antibodies can be engineered to fuse the CH3 domain directly to the hinge region of each modified antibody. In other constructs, it may be desirable to provide a peptide spacer between the hinge region and the modified CH2 and / or CH3 domain. For example, a compatible construct can be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added, for example, to ensure that constant domain regulatory elements remain free and accessible or to keep the hinge region flexible. However, it should be noted that amino acid spacers can in some cases be immunogenic and may prove to elicit unwanted immune responses against the construct. Therefore, in certain embodiments, any spacers added to the construct are relatively non-immunogenic or omitted altogether in order to maintain the desired biochemical properties of the modified antibody.

[0303] It should be understood that, in addition to the deletion of entire constant region domains, antibodies of the present invention can be provided by the partial deletion or substitution of several or single amino acids. For example, the mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it may be desirable to simply delete portions of one or more constant region domains that control modulation of effector functions (e.g., complementing C1Q binding). Such partial deletion of a constant region can improve selected characteristics of the antibody (e.g., serum half-life) while leaving intact other desirable functions associated with the constant region domain of interest. Furthermore, as noted above, the constant regions of the disclosed antibodies can be modified, for example, by mutation or substitution of one or more amino acids, which can enhance the properties of the resulting construct. In this regard, it may be possible to inhibit the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the structural and immunogenic properties of the modified antibody. Certain embodiments can include the addition of one or more amino acids to the constant region to enhance a desirable characteristic (e.g., decrease or increase effector function) or to provide more cytotoxicity or carbohydrate attachment. In such embodiments, it may be desirable to insert or duplicate specific sequences from a selected constant region domain.

[0304] The present invention further encompasses variants and equivalents that are substantially homologous to the chimeric, humanized, and human antibodies, or antibody fragments thereof, described herein. These can include, for example, conservative substitution mutations, i.e., the substitution of one or more amino acids with similar amino acids. For example, a conservative substitution refers to the substitution of one amino acid for another within the same general class (e.g., one acidic amino acid for another, one basic amino acid for another, or one neutral amino acid for another). What is intended by conservative amino acid substitution is well known in the art (e.g., as defined herein above).

[0305] The polypeptides of the present invention may be recombinant, natural, or synthetic polypeptides, including antibodies or fragments thereof against human CD123 / IL-3Rα. It is recognized in the art that some amino acid sequences of the present invention can be altered without significantly affecting the structure or function of the protein. Thus, the present invention further includes variants of polypeptides that exhibit substantial activity against the CD123 antigen (e.g., human CD123) or that contain regions of antibodies or fragments thereof. Such variants include deletions, insertions, inversions, repeats, and type substitutions.

[0306] Polypeptides and analogs can be further modified to contain additional chemical moieties not normally part of the protein. Such derivatized moieties can improve the solubility, biological half-life, or absorption of the protein. The moieties can also reduce or eliminate any undesirable side effects of the protein. A summary of such moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed., Mack Publishing Co., Easton, PA (2000).

[0307] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis to the construction of a DNA sequence that encodes the isolated polypeptide sequence and expresses that sequence in a suitable transformed host. In some embodiments, the DNA sequence is constructed using recombinant techniques by isolating or synthesizing a DNA sequence encoding the wild-type protein of interest. Optionally, the sequence can be mutagenized by site-directed mutagenesis to provide a functional analog thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066, 1984 and U.S. Pat. No. 4,588,585.

[0308] In some embodiments, a DNA sequence encoding a polypeptide of interest (e.g., an antibody, antigen-binding fragment, or polypeptide of the present invention) is constructed, in whole or in part, by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide, selecting codons that are preferred in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Furthermore, DNA oligomers containing nucleotide sequences encoding particular isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0309] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), the polynucleotide sequence encoding the particular isolated polypeptide of interest is inserted into an expression vector and operably linked to expression control sequences suitable for protein expression in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of a transfected gene in a host, the gene is operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0310] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies against human CD123 / IL-3Rα or fragments thereof. A recombinant expression vector is a replicable DNA construct that carries a synthetic or cDNA-derived DNA fragment encoding the polypeptide chain of an anti-CD123 / IL-3Rα antibody or fragment thereof, operably linked to appropriate transcriptional or translational control elements derived from mammalian, microbial, viral, or insect genes. A transcription unit generally comprises (1) a collection of genetic elements (e.g., a transcriptional promoter or enhancer) that have a regulatory role in gene expression, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational initiation and termination sequences. Such control elements can contain an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated into the recombinant vector. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, when the recombinant protein is expressed without a leader or transport sequence, it can contain an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0311] The choice of expression control sequence and expression vector will depend on the choice of host. A wide variety of expression host / vector combinations can be used. Expression vectors useful in eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful in bacterial hosts include well-known bacterial plasmids (e.g., plasmids from Escherichia coli), including pCR1, pBR322, pMB9, and their derivatives, broad-host-range plasmids (M13 and filamentous single-stranded DNA phages).

[0312] Suitable host cells for expressing CD123 / IL-3Rα-binding polypeptides or antibodies (or CD123 / IL-3Rα proteins used as antigens) include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of a suitable promoter. Prokaryotes include gram-negative or gram-positive organisms (e.g., E. coli or bacilli). Higher eukaryotic cells include established cell lines of mammalian origin (e.g., CHO cells). Cell-free translation systems can also be used. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985), the relevant disclosure of which is incorporated herein by reference. Additional information regarding methods of protein production, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is incorporated by reference herein in its entirety.

[0313] Various mammalian or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include HEK-293T, the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), and other cell lines, including, for example, L cells, CI27, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors can include nontranscribed elements (e.g., an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed), and other 5'- or 3'-flanking nontranscribed sequences, as well as 5'- or 3'-untranslated sequences (e.g., essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites), and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed in Luckow and Summers, Bio / Technology 6:47, 1988.

[0314] Thus, one aspect of the present invention also provides a cell that produces any one of the subject antibodies or antigen-binding fragments thereof, or any one of the subject polypeptides. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a HEK-293 or HEK-2 93T cells, COS-7 cells, L cells, CI27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, or BHK cells. In a specific embodiment, the cells are CHO cells.

[0315] Proteins produced by transformed hosts can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags (e.g., hexahistidine, maltose binding domain, influenza coat sequence, and glutathione S-transferase) can be attached to the protein to allow for simple purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0316] For example, supernatants from systems secreting recombinant protein into the medium can be first concentrated using commercially available protein concentration filters (e.g., Amicon or Millipopore Pellicon ultrafiltration devices). After the concentration step, the concentrate can be applied to an appropriate purification matrix. Alternatively, an anion exchange resin (e.g., a matrix or substrate with pendant diethylaminoethyl (DEAE) groups) can be used. This matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with pendant methyl or other aliphatic groups) can be used to further purify the CD123 / IL-3Rα binding agent. Some or all of the aforementioned purification steps, in various combinations, can also be used to form a homogeneous recombinant protein.

[0317] Recombinant proteins produced in bacterial cultures can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used in final purification steps. Microbial cells used in recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0318] Methods well known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publication Nos. 2008 / 0312425, 2008 / 0177048, and 2009 / 0187005, each of which is incorporated herein by reference in its entirety.

[0319] In certain embodiments, the CD123 / IL-3Rα binding agents of the present invention have an N-terminal serine, which can be oxidized with an oxidizing agent to form an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde group.

[0320] Any suitable oxidizing agent can be used in step (a) of the above method. In certain embodiments, the oxidizing agent is a periodate. More specifically, the oxidizing agent is sodium periodate.

[0321] An excess of molar equivalents of oxidizing agent relative to the CD123 / IL-3Rα binding agent can be used. In certain embodiments, about 2-100, 5-80, 10-50, 1-10, or 5-10 molar equivalents of oxidizing agent can be used. In certain embodiments, about 10 or Approximately 50 equivalents can be used. When a large amount of oxidizing agent is used, a short reaction time is used to avoid over-oxidation. For example, when 50 equivalents of oxidizing agent are used, the oxidation reaction is carried out for about 5 to about 60 minutes. Alternatively, when 10 equivalents of oxidizing agent are used, the reaction is carried out for about 30 minutes to about 24 hours. In one embodiment, 5 to 10 molar equivalents of oxidizing agent are used, and the oxidation reaction is carried out for about 5 to about 60 minutes (e.g., about 10 to about 30 minutes, about 20 to about 30 minutes).

[0322] In certain embodiments, the oxidation reaction does not result in significant non-target oxidation, e.g., there is no significant (e.g., less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, or less than 1%) oxidation of methionine and / or glycans during the N-terminal serine oxidation step to produce an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde group.

[0323] In certain embodiments, the CD123 / IL-3Rα binding agents of the present invention have a recombinantly engineered Cys residue (e.g., the Cys residue corresponding to the fifth-to-last position in SEQ ID NO: 54 or 56 (i.e., the Cys residue at position 442 according to the EU / OU numbering system). Accordingly, the term "cysteine ​​engineered antibody" includes antibodies with at least one Cys that is not normally present at a given residue in the antibody light or heavy chain. Such a Cys, also referred to as an "engineered Cys," can be engineered using any conventional molecular biology or recombinant DNA technique (e.g., by replacing the coding sequence for a non-Cys residue with the coding sequence for Cys at the target residue). For example, if the original residue is Ser with a coding sequence of 5'-UGU-3', the coding sequence can be mutated (e.g., by site-directed mutagenesis) to 5'-UGU-3', which encodes Cys. In certain embodiments, the Cys-engineered antibodies of the present invention have an engineered Cys in the heavy chain. In certain embodiments, the engineered Cys is at or near the CH3 domain of the heavy chain. In certain embodiments, the engineered Cys is, for example, the Cys corresponding to the penultimate position of SEQ ID NO: 54 or 56. The engineered antibody heavy (or light) chain sequence can be inserted into a suitable recombinant expression vector to produce an engineered antibody with an engineered Cys residue in place of the original Ser residue.

[0324] 3. Immune complex In a second aspect, the present invention also provides an immunoconjugate comprising a CD123 / IL-3Rα binding agent as described herein covalently linked to one or more molecules of a cytotoxic agent as described herein.

[0325] In a first embodiment, the immunoconjugate of the present invention comprises a CD123 / IL-3Rα-binding agent (including an antibody, an antigen-binding fragment thereof, or a polypeptide comprising the antibody or antigen-binding fragment thereof) covalently attached to a cytotoxic agent described herein via the ε-amino group of one or more lysine residues present in the CD123 / IL-3Rα-binding agent.

[0326] In a first particular embodiment of the first embodiment, the immunoconjugate of the invention has the following formula: [ka] is expressed as During the ceremony, CBA is Cy L1 a CD123 / IL-3Rα binding agent (e.g., a subject antibody or antigen-binding fragment thereof, as described above, or a subject polypeptide thereof, as described above) covalently linked by a heterodimer residue; W L is an integer between 1 and 20, Cy L1 is the following formula: [ka] a cytotoxic compound represented by or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4)alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x3 is (C1-C6) alkyl, L' has the following formula: -NR5-PC(=O)-(CR a R b ) m -C(=O)- (B1'), or -NR5-PC(=O)-(CR a R b ) m -SZ s1 - (B2'), represented by R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R a and R b are each independently, in each occurrence, —H, (C1-C3) alkyl, or a charged substituent or ionic group, Q; m is an integer from 1 to 6, Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is H + or a cation.

[0327] In a second particular embodiment, for the conjugate of formula (L1), Cy L1 is represented by formula (L1a) or (L1a1), and the remaining variables are as described above in the first specific embodiment.

[0328] In a third particular embodiment, for the conjugate of formula (L1), Cy L1 is represented by formula (L1b) or (L1b1), and the remaining variables are as described above in the first specific embodiment. More specifically, R x3 is (C2-C4) alkyl.

[0329] In a fourth particular embodiment, for the conjugate of formula (L1), Cy L1 is expressed by formula (L1a), and R a and R b are both H, R5 is H or Me, and the remaining variables are as described above in the first specific embodiment.

[0330] In a fifth specific embodiment, P is a peptide containing 2 to 5 amino acid residues, with the remaining variables being as described above in the first, second, or fourth specific embodiment. In a further specific embodiment, P is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala- Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D- selected from the group consisting of Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. More specifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0331] In a sixth embodiment, Q is -SO3M and the remaining variables are as described above in the first, second, fourth, or fifth specific embodiment, or as any further specific embodiment described herein.

[0332] In a seventh particular embodiment, the immunoconjugate of the first embodiment has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10, and N and C are The double line between [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the double line between N and C [ka] represents a double bond, X is absent and Y is -H. In another further particular embodiment, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M.

[0333] In an eighth particular embodiment, the immunoconjugate of the first embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is Cy L2 a CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, as described above), covalently linked by a helixidine residue; W L is an integer between 1 and 20, Cy L2 is the following formula: [ka] a cytotoxic compound represented by or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4)alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; R x1 and R x2 are each (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is -H + or a cation.

[0334] In a ninth particular embodiment, for the immunoconjugate of formula (L2), Cy L2 is represented by formula (L2a) or (L2a1), and the remaining variables are as described above in the eighth specific embodiment.

[0335] In a tenth particular embodiment, for the immunoconjugate of formula (L2), Cy L2 is represented by formula (L2b) or (L2b1), and the remaining variables are as described above in the eighth specific embodiment.

[0336] In an eleventh particular embodiment, for the immunoconjugate of formula (L2), R e is H or Me, and R x1 and Rx2 are independently -(CH2) p -(CR f R g )- and R f and R g are each independently —H or (C1-C4) alkyl, p is 0, 1, 2, or 3, and the remaining variables are as described above in the eighth, ninth, or tenth specific embodiments. More specifically, R f and R g are the same or different and are selected from -H and -Me.

[0337] In a twelfth particular embodiment, the immunoconjugate of the first embodiment has the following formula: [ka] [ka] or [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10, and the double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the double line between N and C [ka] represents a double bond. In another further particular embodiment, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M.

[0338] In a thirteenth particular embodiment, the immunoconjugate of the first embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is Cy L3 a CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject antibody or antigen-binding fragment thereof as described above, or a subject polypeptide as described above), covalently linked via a Lys residue to W L is an integer between 1 and 20, Cy L3 is the following formula: [ka] is expressed as m' is 1 or 2, R1 and R2 are each independently H or (C1-C3) alkyl; Z s1 is the following formula: [ka] is selected from one of During the ceremony, q is an integer from 1 to 5, M is H + or a cation.

[0339] In a fourteenth particular embodiment, for the immunoconjugate of formula (L3), m' is 1, R1 and R2 are both H, and the remaining variables are as described above in the thirteenth particular embodiment.

[0340] In a fifteenth particular embodiment, for the immunoconjugate of formula (L3), m' is 2, R1 and R2 are both Me, and the remaining variables are as described above in the thirteenth particular embodiment.

[0341] In a sixteenth particular embodiment, the immunoconjugate of the first embodiment has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein W L is an integer between 1 and 10.

[0342] In a seventeenth particular embodiment, for the immunoconjugate of the first embodiment, M is H + , Na + or K + and the remaining variables are as described above in any one of the first through sixteenth specific embodiments, or as any further specific embodiments described herein.

[0343] In any of the first to seventeenth specific embodiments described above, the subject antibody or its antigen binding The antibody fragments may have one or more (e.g., substantially all or 100%) of the Lys residues in any of the six light and heavy chain CDR regions (if any) substituted with Arg. A subject antibody or antigen-binding fragment thereof may comprise an immunoglobulin heavy chain variable region (HCVR) having the amino acid sequence set forth in SEQ ID NO: 39 or 40, and an immunoglobulin light chain variable region (LCVR) having the amino acid sequence set forth in SEQ ID NO: 41. A subject antibody or antigen-binding fragment thereof may also comprise an Ig HCVR having the amino acid sequence set forth in SEQ ID NO: 34, and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 35. A subject antibody or antigen-binding fragment thereof may also comprise an Ig HCVR having the amino acid sequence set forth in SEQ ID NO: 32, 34, 38, 39, or 40, and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, or 41. In certain embodiments, the penultimate residue to the N-terminus of SEQ ID NO: 34 is Phe, and in certain other embodiments, the penultimate residue to the N-terminus of SEQ ID NO: 34 is Val.

[0344] The immunoconjugates described in the first embodiment or in any particular embodiment thereof can be prepared according to any method known in the art. See, for example, International Patent Nos. WO2012 / 128868 and WO2012 / 112687, which are incorporated herein by reference.

[0345] In certain embodiments, the immunoconjugate of the first embodiment can be prepared by a first method comprising reacting a CBA with a cytotoxic agent having an amine-reactive group.

[0346] In one embodiment, for the first method described above, the reaction is carried out in the presence of an imine-reactive reagent such as NaHSO3.

[0347] In one embodiment, for the first method described above, the cytotoxic agent having an amine-reactive reagent has the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L c ' is the following formula: -NR5-PC(=O)-(CR a R b ) m -C(=O)E (B1) or -NR5-PC(=O)-(CR a R b ) m -S-Zs (B2) C(═O)E is a reactive ester group, such as an N-hydroxysuccinimide ester, an N-hydroxysulfosuccinimide ester, a nitrophenyl (e.g., 2 or 4-nitrophenyl) ester, a dinitrophenyl (e.g., 2,4-dinitrophenyl) ester, a sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) ester or a pentafluorophenyl ester, preferably an N-hydroxysuccinimide ester; Z s is the following formula: [ka] is expressed as During the ceremony, q is an integer from 1 to 5; U is -H or SO3M; The remaining variables are as described above in any one of the first through seventh and seventeenth specific embodiments, or as in any further specific embodiments described herein.

[0348] In certain embodiments, the immunoconjugate of the first embodiment comprises: (a) reacting a cytotoxic agent with a linker compound having an amine-reactive group and a thiol-reactive group to form a cytotoxic agent-linker compound having an amine-reactive group attached thereto; and (b) by a second method, which comprises reacting a CBA with a cytotoxic drug-linker compound. It can be prepared as follows.

[0349] In one embodiment, for the second method described above, the reaction of step (a) is carried out in the presence of an imine-reactive reagent.

[0350] In one embodiment, for the second method described above, the cytotoxic agent-linker compound is reacted with the CBA without purification, or alternatively, the cytotoxic agent-linker compound is first purified before reacting with the CBA.

[0351] In certain embodiments, the immunoconjugate of the first embodiment comprises: (a) reacting a CBA with a linker compound having an amine-reactive group and a thiol-reactive group to form a modified CBA having a thiol-reactive group attached thereto; and (b) A third method includes reacting a modified CBA with a cytotoxic agent.

[0352] In one embodiment, for the third method described above, the reaction of step (b) is carried out in the presence of an imine-reactive reagent.

[0353] In certain embodiments, the immunoconjugate of the first embodiment can be prepared by a fourth method, which includes reacting a CBA, a cytotoxic compound, and a linker compound having an amine-reactive group and a thiol-reactive group.

[0354] In one embodiment, for the fourth method, the reaction is carried out in the presence of an imine-reactive agent.

[0355] In certain embodiments, for the second, third, or fourth embodiment above, the linker compound having an amine-reactive group and a thiol-reactive group has the following formula: [ka] is expressed as wherein X is a halogen and J D -SH, -SSR d or -SC(=O)R g and R d is phenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl or nitropyridyl, and R g is alkyl; the remaining variables are as described above for formulas (a1)-(a10), and the cytotoxic agent is represented by the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the variables are as described above in any one of specific embodiments 8 through 12, and 17, and as any further specific embodiments described herein.

[0356] In certain embodiments, for the second, third, or fourth method described above, the linker compound having an amine-reactive group and a thiol-reactive group is represented by any one of formulas (a1L)-(a10L), and the cytotoxic agent is represented by the following formula: [ka] is expressed as wherein the variables are as defined above in any one of specific embodiments 13 to 17. and as any further specific embodiment described herein.

[0357] In a second embodiment, the immunoconjugate of the invention comprises an oxidized CD123 / IL-3Rα binding agent (including an antibody, antigen-binding fragment thereof, or polypeptide comprising the antibody or antigen-binding fragment thereof) (e.g., an oxidized antibody or antigen-binding fragment thereof, or polypeptide thereof) covalently linked to a cytotoxic agent described herein, as described in the first aspect of the invention described herein, via one or more aldehyde groups present on the oxidized CD123 binding agent. The aldehyde groups present on the oxidized CD123 / IL-3Rα binding agent can be generated by oxidizing one or more 2-hydroxyethylamine moieties of the CD123 / IL-3Rα binding agent, where the 2-hydroxyethylamine moiety is part of a serine, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residue. In one embodiment, aldehyde groups can be generated by oxidizing the 2-hydroxyethylamine moieties of one or more N-terminal serine residue(s) present in the CD123 / IL-3Rα binding agent (e.g., one, two, three, or up to four Ser residues at the N-terminus of the light chain and / or heavy chain).

[0358] In a first particular embodiment of the second embodiment, the immunoconjugate of the invention has the following formula: [ka] is expressed as During the ceremony, CBA is an oxidized CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject oxidized antibody or antigen-binding fragment thereof as described above, or a subject oxidized polypeptide thereof as described above), Ws is 1, 2, 3, or 4, J CB ' is the aldehyde group of CBA, s1with an aldehyde-reactive group of the formula: [ka] is expressed as s1 is the site covalently attached to CBA, and s2 is the site of Cy s1 is a moiety covalently bonded to Cy s1 is the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; Z d1 is absent, -C(=O)-NR9-, or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; R a and R b is, in each occurrence, independently —H, (C1-C3) alkyl, or a charged substituent or ionic group, Q; r and r' are independently integers of 1 to 6; W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x3 is (C1-C6) alkyl, L is -NR9-(CR a R b ) r” or absent, r″ is an integer of 0 to 6.

[0359] In a second particular embodiment, for the immunoconjugate of formula (S1), Cy s1 is represented by formula (S1a) or (S1a1), and the remaining variables are as described above in the first specific embodiment.

[0360] In a third particular embodiment, for the immunoconjugate of formula (S1), Cy s1 is represented by formula (S1b) or (S1b1), and the remaining variables are as described above in the first particular embodiment. More specifically, R x3 is (C2-C4) alkyl.

[0361] In a fourth particular embodiment, for the immunoconjugate of formula (S1), R a and R b are both H, R5 and R9 are both H or Me, and the remaining variables are as described above in the first or second specific embodiment.

[0362] In a fifth particular embodiment, for the immunoconjugate of formula (S1), P is a peptide containing 2 to 5 amino acid residues, with the remaining variables being as described above in the first, second, or fourth particular embodiment. In a further particular embodiment, P is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Le u-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Ar and Met-Ala. Even more specifically, P is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-Ala, Ala-Ala, Ala-D-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0363] In a sixth particular embodiment, for the immunoconjugate of formula (S1), Q is -SO3M and the remaining variables are as described above in the first, second, fourth, or fifth particular embodiment, or as any further particular embodiment described herein.

[0364] In a seventh particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] or [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the double line between N and C [ka] represents a double bond, X is absent and Y is -H. In another further particular embodiment, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M.

[0365] In an eighth particular embodiment, the immunoconjugate of the invention has the following formula: [ka] is expressed as During the ceremony, CBA is an oxidized CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject oxidized antibody or antigen-binding fragment thereof as described above, or a subject oxidized polypeptide thereof as described above), J CB ' is the aldehyde group of CBA and Cy s2 and which has the formula: [ka] is expressed as where s1 is the site covalently attached to CBA and s2 is the site of Cys2 is a moiety covalently bonded to Cy s2 is the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is —H or (C1-C4)alkyl, and when it is a single bond, X is —H or an amine protecting moiety and Y is —OH or —SO3M; M is H + or a cation, R x1 is (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x2 is (C1-C6) alkyl, L1 is the following formula: [ka] is expressed as During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s2 is the moiety covalently bonded to the -S- group of Z a2is absent, -C(=O)-NR9- or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; Q is H, a charged substituent, or an ionic group; R a1 , R a2 , R a3 , R a4 is, in each occurrence, independently H or (C1-C3) alkyl; q1 and r1 are each independently an integer from 0 to 10, provided that q1 and r1 are not both 0.

[0366] In further particular embodiments, Z a2 is absent, q1 and r1 are each independently an integer from 0 to 3, provided that q1 and r1 are not both 0, and the remaining variables are as described above in the eighth specific embodiment. a1 , R a2 , R a3 , R a4 are all -H.

[0367] In another further particular embodiment, Z a2 is -C(=O)-NH- or -NH9-C(=O)-, q1 and r1 are each independently an integer from 1 to 6, and the remaining variables are as described above in the eighth specific embodiment. Even more specifically, R a1 , R a2 , R a3 , R a4 are all -H.

[0368] In a ninth particular embodiment, for the immunoconjugate of formula (S2), Cy s2 is represented by formula (S2a) or (S2a1), and the remaining variables are as described above in the eighth specific embodiment, or as in any further specific embodiment described herein.

[0369] In a tenth particular embodiment, for the immunoconjugate of formula (S2), Cys2 is represented by formula (S2b) or (S2b1), and the remaining variables are as described above in the eighth specific embodiment, or as in any further specific embodiment described herein.

[0370] In an eleventh particular embodiment, for the immunoconjugate of formula (S2), -L1- is of the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein R is H or —SO3M, and the remaining variables are as described above in the eighth, ninth, or tenth specific embodiment, or as any further specific embodiment described herein.

[0371] In a twelfth particular embodiment, for the immunoconjugate of formula (S2), R e is H or Me, and R x1 (CH2) p -(CR f R g )- and R x2 (CH2) p -(CR f R g )- and R f and R g are each independently —H or (C1-C4) alkyl, and p is 0, 1, 2, or 3. More specifically, R f and R g are the same or different and are selected from -H and -Me.

[0372] In a thirteenth particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the double line between N and C [ka] represents a double bond, X is absent and Y is -H. In another further particular embodiment, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M.

[0373] In a fourteenth particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is an oxidized CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject oxidized antibody or antigen-binding fragment thereof as described above, or a subject oxidized polypeptide thereof as described above), J CB ' is the aldehyde group of CBA and Cy s3 and a moiety formed by reacting an aldehyde-reactive group of the formula: [ka] is expressed as s1 is the site covalently attached to CBA, and s2 is the site of Cy s3 is a moiety covalently bonded to Cy s3 is the following formula: [ka] is expressed as During the ceremony, m' is 1 or 2, R1 and R2 are each independently H or (C1-C3) alkyl; L1 is the following formula: [ka] is expressed as During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s3 is the moiety covalently bonded to the -S- group of Z a2 is absent, -C(=O)-NR9- or -NR9-C(=O)-; R9 is —H or (C1-C3) alkyl; Q is H, a charged substituent, or an ionic group; R a1 , R a2 , R a3 , Ra4 is, in each occurrence, independently H or (C1-C3) alkyl; q1 and r1 are each independently an integer from 0 to 10, provided that q1 and r1 are not both 0.

[0374] In further particular embodiments, Z a2 is absent, q1 and r1 are each independently an integer from 0 to 3, provided that q1 and r1 are not both 0, and the remaining variables are as described above in the fourteenth specific embodiment. a 1. R a2 , R a3 , R a4 are all -H.

[0375] In another further particular embodiment, Z a2 is -C(=O)-NH- or -NH9-C(=O)-, q1 and r1 are each independently an integer from 1 to 6, and the remaining variables are as described above in the fourteenth specific embodiment. Even more specifically, R a1 , R a2 , R a3 , R a4 are all -H.

[0376] In a fifteenth particular embodiment, for the immunoconjugate of formula (S3), m' is 1, R1 and R2 are both H, and the remaining variables are as described above in the fourteenth particular embodiment or as any further particular embodiment described herein.

[0377] In a sixteenth particular embodiment, for the immunoconjugate of formula (S3), m' is 2, R1 and R2 are both Me, and the remaining variables are as described above in the fourteenth particular embodiment or as in any further particular embodiment described herein.

[0378] In a seventeenth particular embodiment, for the immunoconjugate of formula (S3), -L1- is of the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein R is H or —SO3M and M is H + or a cation.

[0379] In an eighteenth particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM is of the following formula: [ka] It is expressed as:

[0380] In a nineteenth particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] wherein: CBA is an oxidized CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject oxidized antibody or antigen-binding fragment thereof as described above, or a subject oxidized polypeptide thereof as described above), J CB ' is the aldehyde group of CBA and Cy s4 and a moiety formed by reacting an aldehyde-reactive group of the formula: [ka] is expressed as s1 is the site covalently attached to CBA, and s2 is the site of Cy s4 is a moiety covalently bonded to Cy s4 is the following formula: [ka] is expressed as L1' is of the following formula: [ka] is expressed as During the ceremony, s3 is J CB is a moiety covalently bonded to the ' group, s4 is Cy s4 is the moiety covalently bonded to the -NMe- group of Z b1 and Z b2 are both absent or Z b1 and Z b2 one of which is absent and the other is -CH2-O- or -O-CH2-; Z b1 ' and Z b2 each ' is independently absent, -CH2-O-, -O-CH2-, -NR9-C(=O)-CH2-, or -CH2-C(=O)-NR9-, where R9 is H or (C1-C3) alkyl; n1 and m1 each independently represent an integer of 1 to 6, one of E1 and E2 is -C(=O)- and the other is -NR9-, or one of E1 and E2 is -C(=O)- or -NR9- and the other is absent; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R b1 , R b2 , R b3 , R b4 , R b5 and R b6 is each independently H or (C1-C3) alkyl in each occurrence.

[0381] In a twentieth particular embodiment, for the immunoconjugate of formula (S4), R b1 , R b2 , R b3 , R b4 , Rb5 , and R b6 are all H, and the remaining variables are as described above in the nineteenth specific embodiment.

[0382] In a twenty-first particular embodiment, for the immunoconjugate of formula (S4), R9 is H, and the remaining variables are as described above in the nineteenth or twentieth particular embodiment.

[0383] In a twenty-second particular embodiment, for the immunoconjugate of formula (S4), Z b1 ' and Z b2 ' are both absent or Z b1 ' is -CH2-O-, and Z b2 ' is absent or Z b1 ' is -CH2-C(=O)-NR9-, and Z b2 is O—CH 2 — or absent, and the remaining variables are as described above in the nineteenth, twentieth, or twenty-first specific embodiment.

[0384] In a 23rd particular embodiment, for the immunoconjugate of formula (S4), P is a peptide containing 2 to 5 amino acid residues, and the remaining variables are as described above in the 19th, 20th, 21st, or 22nd particular embodiment. In further particular embodiments, P is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: No. 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala, with the remaining variables being as described above in the twenty-third specific embodiment. Even more specifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0385] In a twenty-fourth particular embodiment, the immunoconjugate of the second embodiment has the following formula: [ka] [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM has the following structural formula: [ka] It is expressed as:

[0386] In a twenty-fifth particular embodiment, for the immunoconjugate of the second embodiment, M is H + , Na + or K + and the remaining variables are as described above in any one of the first through twenty-fourth specific embodiments, or as any further specific embodiments described herein.

[0387] In any of the specific embodiments 1 through 25 described above, a subject oxidized antibody or antigen-binding fragment thereof may have one, two, three, or up to four N-terminal 2-hydroxyethylamine moieties oxidized to aldehyde group(s) for covalent conjugation to a cytotoxic agent described herein. The N-terminal 2-hydroxyethylamine moiety may be part of a serine, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residue, preferably Ser or Thr. For simplicity, the following description, including the oxidation reaction and any subsequent covalent conjugation with a linker or cytotoxic agent, may refer to Ser as a specific example of such an N-terminal 2-hydroxyethylamine moiety, but should generally be interpreted as relating to all N-terminal 2-hydroxyethylamine moieties. A subject antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region (HCVR) having the amino acid sequence set forth in SEQ ID NO: 38, and an immunoglobulin light chain variable region (LCVR) having the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). A subject antibody or antigen-binding fragment thereof can also comprise an Ig HCVR having the amino acid sequence set forth in SEQ ID NO: 32, 34, 38, 39, or 40 (preferably SEQ ID NO: 34), and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 37. A subject antibody or antigen-binding fragment thereof can comprise an Ig heavy chain (HC) region having the amino acid sequence set forth in SEQ ID NO: 53 or 56, and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 33 , 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). A subject antibody or antigen-binding fragment thereof can also comprise an Ig HC region having the amino acid sequence set forth in SEQ ID NO: 48, 50, 53, 54, 56, 59, or 60 (preferably SEQ ID NO: 53), and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, the penultimate residue to the N-terminus of SEQ ID NO: 34, 38, 50, 53, 54, or 56 is Phe, and in certain other embodiments, the penultimate residue to the N-terminus of SEQ ID NO: 34, 38, 50, 53, 54, or 56 is Val.

[0388] In a specific embodiment, the immunoconjugate of the second embodiment can be prepared by a first method comprising reacting an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde as described in the first aspect of the invention with a cytotoxic agent having an aldehyde-reactive group.

[0389] In certain embodiments, the immunoconjugate of the second embodiment can be prepared by a second method comprising reacting an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde as described in the first aspect of the invention with a linker compound having an aldehyde-reactive group to form a modified CD123 / IL-3Rα binding agent having a linker attached thereto, and subsequently reacting the modified CD123 / IL-3Rα binding agent with a cytotoxic agent.

[0390] In certain embodiments, the immunoconjugate of the second embodiment can be prepared by a third method, which comprises contacting an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde as described in the first aspect of the invention with a cytotoxic agent, followed by adding a linker compound having an aldehyde-reactive group.

[0391] In certain embodiments, the immunoconjugate of the second embodiment comprises: (a) oxidizing a CD123 / IL-3Rα binding agent having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde group; and (b) A fourth method includes reacting an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde with a cytotoxic agent having an aldehyde-reactive group.

[0392] In certain embodiments, the immunoconjugate of the second embodiment comprises: (a) oxidizing a CD123 / IL-3Rα binding agent having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde group; (b) A fifth method includes reacting an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde with a linker compound having an aldehyde-reactive group to form a modified CD123 / IL-3Rα binding agent having a linker attached thereto, and then reacting the modified CD123 / IL-3Rα binding agent with a cytotoxic agent.

[0393] In certain embodiments, the immunoconjugate of the second embodiment comprises: (a) oxidizing a CD123 / IL-3Rα binding agent having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde group; (b) An oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde can be prepared by a sixth method, which includes contacting the agent with a cytotoxic agent, followed by adding a linker compound having an aldehyde-reactive group.

[0394] In one embodiment, for the first or fourth method above, the cytotoxic agent having an aldehyde reactive group has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein J CB is the following formula: [ka] is expressed as The remaining variables are as described above in any one of the first through seventh and twenty-fifth specific embodiments, and as in any further specific embodiments described herein.

[0395] In another embodiment, for the first or fourth method above, the cytotoxic agent having an aldehyde reactive group has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein J CB are as described above, and the remaining variables are as described above in any one of specific embodiments 19 through 25, and as in any further specific embodiments described herein.

[0396] In one embodiment, for the second, third, fifth or sixth method above, the linker compound has the formula: [ka] is expressed as During the ceremony, J. D -SH, -SSR d , or -SC(=O)R g and R d is phenyl, nitrophenyl, dinitrophenyl, carboxynitrophenyl, pyridyl or nitropyridyl, and R g is alkyl, and J CB is as described above, and the cytotoxic agent has the formula: [ka] is expressed as The remaining variables are as described above in any one of specific embodiments 8 through 13 and 25, and as in any further specific embodiments described herein.

[0397] In another embodiment, for the second, third, fifth or sixth method described above, the linker compound is a compound represented by the formula (L s a) and the cytotoxic compound is represented by the following formula: [ka] is expressed as The remaining variables are as described in any one of specific embodiments 14-18 and 25, and as in any further specific embodiments described herein.

[0398] In one embodiment, for the first or fourth method described above, the cytotoxic agent is reacted with an imine-reactive reagent, such as NaHSO, to form a modified cytotoxic agent prior to reaction with the oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde. In one embodiment, the modified cytotoxic agent is not purified prior to reaction with the oxidized CBA having an N-terminal aldehyde. Alternatively, the modified cytotoxic agent is purified prior to reaction with the oxidized CBA having an N-terminal aldehyde.

[0399] In another embodiment, for the second or fifth method described above, the cytotoxic agent is reacted with an imine-reactive reagent, such as NaHSO3, to form a modified cytotoxic agent prior to reaction with a modified CD123 / IL-3Rα binding agent having a linker attached thereto. In one embodiment, the modified cytotoxic agent is purified prior to reaction with a modified CD123 / IL-3Rα binding agent having a linker attached thereto. Alternatively, the modified cytotoxic agent is not purified prior to reaction with an oxidized CD123 / IL-3Rα binding agent having an N-terminal aldehyde.

[0400] In yet another embodiment, for the third or sixth method above, the reaction of the oxidized CD123 / IL-3Rα binding agent with the cytotoxic agent and linker compound is carried out in the presence of an imine-reactive reagent, such as NaHSO3.

[0401] Any suitable oxidizing agent can be used in step (a) of the fourth, fifth, or sixth method described above. In certain embodiments, the oxidizing agent is a periodate. More specifically, the oxidizing agent is sodium periodate.

[0402] An excess of molar equivalents of oxidizing agent relative to the CD123 / IL-3Rα binding agent can be used. In certain embodiments, about 2-100, 5-80, 10-50, 1-10, or 5-10 molar equivalents of oxidizing agent can be used. In certain embodiments, about 10 or about 50 equivalents of oxidizing agent can be used. When using a large amount of oxidizing agent, a short reaction time is used to avoid over-oxidation. For example, when using 50 equivalents of oxidizing agent, the oxidation reaction is carried out for about 5 to about 60 minutes. Alternatively, when using 10 equivalents of oxidizing agent, the reaction is carried out for about 30 minutes to about 24 hours. In one embodiment, when 5 to 10 molar equivalents of oxidizing agent are used, the oxidation reaction is carried out for about 5 to about 60 minutes (e.g., about 10 to about 30 minutes, about 20 to about 30 minutes).

[0403] In certain embodiments, a catalyst is present in the reaction of the first, second, or third method described above, or in the reaction of step (b) of the fourth, fifth, or sixth method described above. Any suitable catalyst known in the art can be used. In one embodiment, the catalyst is aniline or a substituted aniline. Representative aniline catalysts include, but are not limited to, aniline, o-phenylenediamine, m-phenylenediamine, 3,5-diaminobenzoic acid, p-phenylenediamine, 2-methyl-p-phenylenediamine, N-methyl-p-phenylenediamine, o-aminophenol, m-aminophenol, p-aminophenol, p-methoxyaniline, 5-methoxyanthranilic acid, o-aminobenzoic acid, and 4-aminophenethyl alcohol. In one embodiment, the catalyst is 4-aminophenethyl alcohol. In certain embodiments, the reaction of step (b) is carried out at a pH of about 5.0 to about 6.5. In certain embodiments, the reaction of step (b) is carried out at a pH of about 5.0.

[0404] In certain embodiments, for the reaction of the first, second, or third method described above, or the reaction of step (b) of the fourth, fifth, or sixth method described above, the compound having an aldehyde-reactive group (e.g., a cytotoxic agent or a linker compound described herein) is used in molar excess relative to the oxidized cell-binding agent (e.g., an oxidized antibody or an oxidized antigen-binding moiety). In certain embodiments, the ratio of compound having an aldehyde-reactive group to oxidized cell-binding agent is about 10:1 to about 1.1:1, or about 5:1 to about 2:1. In one embodiment, the ratio is about 4:1.

[0405] In a third embodiment, the immunoconjugate of the present invention comprises a CD123 / IL-3Rα binding agent (including an antibody, an antigen-binding fragment thereof, or a polypeptide comprising the antibody or antigen-binding fragment thereof) described in the first aspect of the invention covalently attached to a cytotoxic agent as described herein via a thiol group (-SH) of one or more cysteine ​​residues present in the CD123-binding agent.

[0406] In a first particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is Cy C1 a CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, as described above), covalently linked by a cysteine ​​residue to Wc is 1 or 2, Cy C1 is the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R5 is —H or (C1-C3) alkyl; P is an amino acid residue or a peptide containing 2 to 20 amino acid residues; R a and R b is, in each occurrence, independently —H, (C1-C3) alkyl, or a charged substituent or ionic group, Q; W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x3 is (C1-C6) alkyl, Lc is [ka] is expressed as where s1 is the site covalently attached to CBA, and s2 is the site of Cy C1 is a moiety covalently bonded to the -C(=O)- group of R 19 and R 20 is, in each occurrence, independently —H or (C1-C3) alkyl; m" is an integer from 1 to 10, R h is —H or (C1-C3) alkyl.

[0407] In a second particular embodiment, for the immunoconjugate of formula (C1), Cy C1is represented by formula (C1a) or (C1a1), and the remaining variables are as described above in the first specific embodiment.

[0408] In a third particular embodiment, for the immunoconjugate of formula (C1), Cy C1 is represented by formula (C1b) or (C1b1), and the remaining variables are as described above in the first specific embodiment.

[0409] In a fourth particular embodiment, for the immunoconjugate of formula (C1), Cy C1 is represented by formula (C1a) or (C1a1), and R a and R b are both H, R5 is H or Me, and the remaining variables are as described above in the first or second specific embodiment.

[0410] In a fifth particular embodiment, for the immunoconjugate of formula (C1), P is a peptide containing 2 to 5 amino acid residues, with the remaining variables being as described above in the first, second, or fourth particular embodiment. In a further particular embodiment, P is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, and the like. ys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- In another more specific embodiment, P is selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In another more specific embodiment, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0411] In a sixth particular embodiment, for the immunoconjugate of formula (C1), Q is -SO3M and the remaining variables are as described above in the first, second, fourth or fifth particular embodiment, or as any further particular embodiment described herein.

[0412] In a seventh particular embodiment, for the immunoconjugate of formula (C1), R 19 and R 20 are both H, m″ is an integer from 1 to 6, and the remaining variables are as described above in the first, second, third, fourth, fifth, or sixth specific embodiment, or as any further specific embodiment described herein.

[0413] In an eighth particular embodiment, for the immunoconjugate of formula (C1), -LL C - is the following formula: [ka] is expressed as The remaining variables are as described above in the first, second, third, fourth, fifth, sixth or seventh specific embodiment, or as in any further specific embodiment described herein.

[0414] In a ninth particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single bond or a double bond, provided that when it is a double bond, X is absent and Y is -H, provided that when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the doublet between N and C [ka] represents a double bond, X is absent and Y is -H. In another further particular embodiment, the double bond between N and C [ka] represents a single bond, X is -H, and Y is -SO3M.

[0415] In a tenth particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is Cy C2 a CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, as described above), covalently linked by a cysteine ​​residue to Wc is 1 or 2, Cy C2 is the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein: Double line between N and C [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H or (C1-C4) alkyl; when it is a single bond, X is -H or an amine protecting moiety, Y is -OH or -SO3M, and M is H + or cation, R x1 is (C1-C6) alkyl, R e is —H or (C1-C6) alkyl, W' is -NR e’ and R e’ is -(CH2-CH2-O) n -R k and n is an integer from 2 to 6, R k is -H or -Me, R x2 is (C1-C6) alkyl, Lc' is represented by the following formula: [ka] is expressed as During the ceremony, s1 is the site covalently attached to CBA, and s2 is the site of Cy C2 is the moiety covalently bonded to the -S- group of Z is -C(=O)-NR9- or -NR9-C(=O)-; Q is -H, a charged substituent, or an ionic group; R9, R 10 , R 11 , R12 , R 13 , R 19 , R 20 , R 21 and R 22 is, in each occurrence, independently —H or (C1-C3) alkyl; q and r, in each occurrence, are independently an integer from 0 to 10; m and n each independently represent an integer of 0 to 10; R h is —H or (C1-C3) alkyl, P' is an amino acid residue or a peptide containing 2 to 20 amino acid residues.

[0416] In further specific embodiments, q and r are each independently an integer from 1 to 6, more specifically an integer from 1 to 3. Even more specifically, R 10 , R 11 , R 12 and R 13 are all H.

[0417] In another more specific embodiment, m and n are each independently an integer from 1 to 6, more specifically an integer from 1 to 3. Even more specifically, R 19 , R 20 , R 21 and R 22 are all H.

[0418] In an eleventh particular embodiment, for the immunoconjugate of formula (C2), Cy C2 is represented by formula (C2a) or (C2a1), and the remaining variables are as described above in the tenth specific embodiment, or as in any further specific embodiment described herein.

[0419] In a twelfth particular embodiment, for the immunoconjugate of formula (C2), Cy C2 is represented by formula (C2b) or (C2b1), and the remaining variables are as described above in the tenth specific embodiment.

[0420] In a thirteenth particular embodiment, for the immunoconjugate of formula (C2), P' is a peptide containing 2 to 5 amino acid residues, and the remaining variables are as described in the tenth, eleventh, or twelfth particular embodiment, or as any further particular embodiment described herein. In further particular embodiments, P' is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9 -Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Va l, Ala-Leu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg-Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val- selected from Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In another more specific embodiment, P' is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0421] In a fourteenth particular embodiment, for the immunoconjugate of formula (C2), -L C '- is the formula: [ka] It is expressed as:

[0422] In a fifteenth particular embodiment, for the immunoconjugate of formula (C2), Re is H or Me, and R x1 (CH2) p -(CR f R g )- and R x2 (CH2) p -(CR f R g )- and R f and R g are each independently —H or (C1-C4) alkyl, p is 0, 1, 2, or 3, and the remaining variables are as described above in the tenth, eleventh, twelfth, thirteenth, or fourteenth specific embodiment. More specifically, R f and R g are the same or different and are selected from -H and -Me.

[0423] In a sixteenth particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein the doublet between N and C is [ka] represents a single or double bond, with the proviso that when it is a double bond, X is absent and Y is -H, and when it is a single bond, X is -H and Y is -OH or -SO3M. In a further particular embodiment, the double line between N and C [ka] represents a double bond, X is absent, and Y is -H. In another particular embodiment, N and C The double line between [ka] represents a single bond, X is -H, and Y is -SO3M.

[0424] In a seventeenth particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] is expressed as During the ceremony, CBA is Cy C3 a CD123 / IL-3Rα binding agent according to the first aspect of the invention (e.g., a subject antibody or antigen-binding fragment thereof, or a subject polypeptide thereof, as described above), covalently linked by a cysteine ​​residue to Wc is 1 or 2, Cy C3 is the following formula: [ka] is expressed as During the ceremony, m' is 1 or 2, R1 and R2 are each independently —H or (C1-C3) alkyl; Lc' is represented by the following formula: [ka] is expressed as During the ceremony, s1 is the site covalently attached to CBA, and s2 is the site of Cy C3 is the moiety covalently bonded to the -S- group of Z is -C(=O)-NR9- or -NR9-C(=O)-; Q is H, a charged substituent, or an ionic group; R9, R 10 , R 11 , R 12 , R 13 , R 19 , R 20 , R 21 and R 22 is, in each occurrence, independently —H or (C1-C3) alkyl; q and r, in each occurrence, are each an integer from 0 to 10; m and n each independently represent an integer of 0 to 10; R h is —H or (C1-C3) alkyl, P' is an amino acid residue or a peptide containing 2 to 20 amino acid residues.

[0425] In further specific embodiments, q and r are each independently an integer from 1 to 6, more specifically an integer from 1 to 3. Even more specifically, R 10 , R 11 , R 12 and R 13 are all H.

[0426] In another more specific embodiment, m and n are each independently integers 1 and 6, more specifically integers 1 to 3. Even more specifically, R 19 , R 20 , R 21 and R 22 are all H.

[0427] In an eighteenth particular embodiment, for the immunoconjugate of formula (C3), P' is a peptide containing 2 to 5 amino acid residues, and the remaining variables are as described above in the seventeenth particular embodiment, or as in any further particular embodiment described herein. In further particular embodiments, P' is selected from the group consisting of Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-N 9 -Tosyl-Arg, Phe-N 9-Nitro-Arg, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-L eu-Ala-Leu (SEQ ID NO: 55), β-Ala-Leu-Ala-Leu (SEQ ID NO: 57), Gly-Phe-Leu-Gly (SEQ ID NO: 73), Val-Arg, Arg-Val, Arg- In another more specific embodiment, P' is selected from Arg, Val-D-Cit, Val-D-Lys, Val-D-Arg, D-Val-Cit, D-Val-Lys, D-Val-Arg, D-Val-D-Cit, D-Val-D-Lys, D-Val-D-Arg, D-Arg-D-Arg, Ala-Ala, Ala-D-Ala, D-Ala-Ala, D-Ala-D-Ala, Ala-Met, and Met-Ala. In another more specific embodiment, P' is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0428] In a nineteenth particular embodiment, for the immunoconjugate of formula (C3), -L C '- is the formula: [ka] is expressed as In the formula, M is H + or cation, and the remaining variables are as described above in the seventeenth or eighteenth specific embodiment, or as any further specific embodiment described herein.

[0429] In a twentieth particular embodiment, for the immunoconjugate of formula (C3), m' is 1, R1 and R2 are both H, and the remaining variables are as described above in the seventeenth, eighteenth, or nineteenth particular embodiments, or as any further particular embodiments described herein.

[0430] In a 21st particular embodiment, for the immunoconjugate of formula (C3), m' is 2, R1 and R2 are both Me, and the remaining variables are as described above in the 17th, 18th, or 19th particular embodiment, or as any further particular embodiment described herein.

[0431] In a twenty-second particular embodiment, the immunoconjugate of the third embodiment has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein DM is of the following formula: [ka] It is expressed as:

[0432] In a twenty-third particular embodiment, for the immunoconjugate of the third embodiment, M is H + , Na + or K + and the remaining variables are as described in any one of the first through twenty-second specific embodiments, or as any further specific embodiments described herein.

[0433] In any of the above specific embodiments 1 through 23, a subject antibody or antigen-binding fragment thereof, or a polypeptide comprising a subject antibody or antigen-binding fragment thereof, has a Cys residue at a position corresponding to an engineered Cys in the heavy chain CH3 domain (i.e., the penultimate residue) of SEQ ID NO: 54 or 56. A subject antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain region (HC) having the amino acid sequence set forth in SEQ ID NO: 54, and an immunoglobulin light chain variable region (LCVR) having the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). A subject antibody or antigen-binding fragment thereof can also comprise an Ig heavy chain region having the amino acid sequence set forth in SEQ ID NO: 56, and an Ig LCVR having the amino acid sequence set forth in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). In certain embodiments, the penultimate residue of SEQ ID NOs: 54 and 56 is Phe, and in certain other embodiments, the penultimate residue of SEQ ID NOs: 54 and 56 is Val.

[0434] The immunoconjugate of the third embodiment described above (e.g., the immunoconjugate of any one of the first through twenty-third specific embodiments, or any further specific embodiment described herein) can be prepared by reacting a CBA having one or more free cysteines with a cytotoxic agent having a thiol-reactive group described herein.

[0435] In one embodiment, the cytotoxic agent having a thiol-reactive group has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein -L C C is the following formula: [ka] is expressed as wherein the variables are as described above in any one of the first through ninth and twenty-third specific embodiments, or as any further specific embodiments described herein.

[0436] In another embodiment, the cytotoxic agent having a thiol reactive group has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, wherein L C C ' is the following formula: [ka] is expressed as wherein the variables are as described above in any one of the tenth to sixteenth and twenty-third specific embodiments, or as any further specific embodiments described herein.

[0437] In yet another embodiment, the cytotoxic agent having a thiol reactive group has the following formula: [ka] Is it expressed as or a pharmaceutically acceptable salt thereof, C C ' is as described above, and the remaining variables are as described above in any one of the seventeenth to twenty-third specific embodiments, or as any further specific embodiments described herein.

[0438] In certain embodiments, an organic solvent is used in the reaction of the CBA with the cytotoxic agent to solubilize the cytotoxic agent. Exemplary organic solvents include, but are not limited to, dimethylacetamide (DMA), propylene glycol, and the like. In one embodiment, the reaction of the CBA with the cytotoxic agent is carried out in the presence of DMA and propylene glycol.

[0439] 4. Compositions and Methods of Use The present invention includes compositions (e.g., pharmaceutical compositions) comprising a subject antibody or antigen-binding fragment thereof, or immunoconjugate thereof, as described herein (e.g., conjugates of formula (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), and a carrier (a pharmaceutically acceptable carrier). The present invention also includes compositions (e.g., pharmaceutical compositions) comprising a subject antibody or antigen-binding fragment thereof, or conjugates of formula (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3), and a carrier (a pharmaceutically acceptable carrier), further comprising a second therapeutic agent. The compositions are useful for inhibiting abnormal cell growth in a mammal (e.g., a human) or treating a proliferative disorder thereof, including a blood cancer, leukemia, or lymphoma.

[0440] In particular, the present invention provides pharmaceutical compositions comprising one or more of the CD123-binding agents or immunoconjugates thereof described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable solvent. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients, including hematological cancers, leukemia, or lymphoma.

[0441] In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or immunoconjugate thereof of the present invention with a pharmaceutically acceptable vehicle (e.g., carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition, Mack Publishing, 2000). Suitable pharmaceutically acceptable solvents include non-toxic buffers (e.g., phosphate, citrate, and other organic acids); salts (e.g., sodium chloride); antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens (e.g., methyl or propyl paraben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins (e.g., serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamic acid, hydroxybenzoates, etc.). Examples of suitable surfactants include, but are not limited to: amino acids, asparagine, histidine, arginine, or lysine; carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrins); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and non-ionic surfactants (e.g., TWEEN or polyethylene glycol (PEG)).

[0442] The pharmaceutical compositions described herein can be administered in any number of ways for local or systemic treatment. Administration can be topical (such as to mucous membranes, including vaginal and rectal delivery) (e.g., transdermal patches, ointments, lotions, creams, gels, eye drops, suppositories, sprays, liquids, and powders); pulmonary (e.g., inhalation or insufflation of powders or aerosols, including nebulizers; intratracheal, intranasal, epidermal, and transdermal); oral; or parenteral, including intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular). In some specific embodiments, administration is intravenous. The pharmaceutical compositions described herein can also be used in vitro or ex vivo.

[0443] The antibodies or immunoconjugates of the invention can be combined with a second compound (e.g., one known to be effective in treating the disease or disorder of interest) in a dosing regimen as a pharmaceutical combination formulation or combination therapy. In some embodiments, the second compound is an anti-cancer agent. In some embodiments, the method involves administering a second compound of the invention and an immunoconjugate that results in better efficacy compared to administration of the immunoconjugate alone. The second compound can be administered via any number of methods, including, for example, topical, pulmonary, oral, parenteral, or intracranial administration. In some embodiments, administration is oral administration. In some embodiments, administration is intravenous administration. In some embodiments, administration is both oral and intravenous administration.

[0444] The antibody or immunoconjugate may also be combined with analgesics or other drugs in a pharmaceutical combination formulation or administration regimen as a combination therapy.

[0445] The antibody or immunoconjugate can be combined with a second compound having anti-cancer properties in a pharmaceutical combination formulation or administration regimen as a combination therapy. The second compound in the pharmaceutical combination formulation or administration regimen can have complementary activity to the ADC of the combination so that they do not adversely affect each other. Pharmaceutical compositions containing a CD123-binding agent and a second anti-cancer agent are also provided.

[0446] The present invention includes methods for inhibiting abnormal cell growth or treating a proliferative disease in a mammal (e.g., a human), comprising administering to the mammal a therapeutically effective amount of a subject antibody or antigen-binding fragment thereof, or immunoconjugate described herein (e.g., a conjugate of formula (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), or composition thereof, alone or in combination with a second therapeutic agent.

[0447] In certain embodiments, the mammalian abnormal cell growth or proliferative disorder is a disease or condition associated with or characterized by expression of CD123 (e.g., cancer), including hematological cancer, leukemia, or lymphoma. In certain embodiments, the proliferative disorder is cancer of the lymphoid organs or a hematological tumor.

[0448] For example, cancers include acute myeloid leukemia (AML, including CD33-low AML, P-glycoprotein-positive AML, relapsed AML, or refractory AML), chronic myeloid leukemia (CML), including CML and CML-associated Abelson oncogene blast crisis (Bcr-ABL translocation), myeloma, and leukemia. and blastoid leukemia (BDSC), non-Hodgkin's lymphoma (NHL) including myeloblastic leukemia (MDS), acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) including Richter's syndrome or Richter's transformation of CLL, hairy cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disease (B-CLPD), atypical chronic lymphocytic leukemia (preferably with marked CD11c expression), blastic plasmacytoid dendritic cell neoplasm (BPDCN), mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis, and Burkitt's lymphoma.

[0449] In certain embodiments, the B-ALL is CD19-positive B-ALL. In certain other embodiments, the B-ALL is CD19-negative B-ALL.

[0450] In certain embodiments, the cancer has at least one negative prognostic factor (eg, P-glycoprotein overexpression, EVI1 overexpression, p53 alteration, DNMT3A mutation, FLT3 internal tandem duplication).

[0451] In certain embodiments, a therapeutically effective amount of a subject antibody or antigen-binding fragment thereof, or immunoconjugate described herein (e.g., a conjugate of formula (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), or composition thereof, alone or in combination with a second therapeutic agent, selectively inhibits the proliferation of leukemic stem cells (LSC), leukemic progenitor cells (LP), and / or leukemic blasts over normal hematopoietic stem cells (HSC). In certain embodiments, the IC of the subject agents described above that inhibit the proliferation of leukemic stem cells (LSC), leukemic progenitor cells (LP), and / or leukemic blasts 50 The value or half-maximal concentration is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 300, 500-fold lower or less than that of normal hematopoietic stem cells (HSCs).

[0452] In certain embodiments, the anti-leukemia treatments of the present invention not only target and kill leukemic blasts, but also preferably target and kill leukemic progenitor cells (LPs) and leukemic stem cells (LSCs). In certain embodiments, the treatments are also less selective for normal HSCs. In certain embodiments, CD123 expression on LSCs, LPs, and leukemic blasts is much higher (e.g., at least 20-25 times higher on LSCs, AML progenitors, and AML blasts) than on normal lymphocytes (which may be nearly negative). In certain embodiments, CD123 expression levels on LPs and LSCs are at least as high as on leukemic blasts.

[0453] Similarly, the present invention provides a method for inducing cell death in a selected cell population, comprising contacting target cells or tissues containing target cells with an effective amount of a subject antibody or antigen-binding fragment thereof, or immunoconjugate of the present invention, the target cells being cells to which the cell-binding agent of the conjugate can bind.

[0454] Optionally, other active agents (eg, other anti-tumor agents) can be administered together with the conjugate.

[0455] Cancer treatments and their dosages, routes of administration, and recommended uses are well known in the art and are described in literature such as the US Physicians' Desk Reference (PDR). The PDR discloses dosages of drugs that have been used in various cancer treatments. The therapeutically effective dosing regimens and dosages of the above-mentioned chemotherapy drugs depend on the particular cancer being treated, the extent of the disease, and other factors well known to physicians in the art, and can be determined by a physician. The contents of the PDR are expressly incorporated herein by reference in their entirety. Those skilled in the art will readily appreciate the use of the PDR in determining the dosing regimens and dosages of chemotherapy drugs and conjugates that can be used in accordance with the teachings of the present invention. In addition, the PDR can be reviewed using one or more of the following parameters: General Index, Manufacturer, Product (by corporate or trademarked drug name), Classification Index, Generic / Chemical Index (by non-branded common drug name), color image of the drug, Product Information consistent with FDA labeling, Chemical Information, Function / Action, Indications and Contraindications, Clinical Trials, Side Effects, and Warnings.

[0456] Examples of in vitro applications include the treatment of autologous bone marrow prior to transplant into the same patient to kill diseased or malignant cells; the treatment of bone marrow prior to its transplant to kill competent T cells and prevent graft-versus-host disease (GVHD); and the treatment of cell cultures to kill all cells except desired variants that do not express the target antigen, or to kill variants that express undesirable antigens.

[0457] Conditions for non-clinical in vitro use are readily determined by one of skill in the art.

[0458] Examples of clinical ex vivo uses include the removal of tumor cells or lymphoid cells from bone marrow prior to autologous transplantation for the treatment of cancer or autoimmune disease, or the removal of T cells and other lymphoid-like cells from autologous or allogeneic bone marrow or tissue prior to transplantation to prevent GVHD. Treatment can be performed as follows: Bone marrow is collected from a patient or other individual and incubated in serum-containing medium containing a cytotoxic agent of the present invention at a concentration ranging from about 10 μM to 1 pM at about 37°C for about 30 minutes to about 48 hours. The exact conditions of concentration and time of incubation (i.e., dosage) are readily determined by one of ordinary skill in the art. After incubation, the bone marrow cells are washed with serum-containing medium and returned intravenously to the patient according to well-known methods. In situations where the patient undergoes other treatments, such as a course of ablative chemotherapy or total body irradiation, between the time of bone marrow collection and the time of reinfusion of the treated cells, the treated bone marrow cells are stored frozen in liquid nitrogen using standard medical equipment.

[0459] For clinical in vivo use, the cytotoxic compounds or conjugates of the invention are supplied as solutions or lyophilized powders that are tested for sterility and endotoxin levels.

[0460] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those skilled in the art as appropriate for the clinical situation. Examples of suitable carriers, diluents, and / or excipients include (1) Dulbecco's phosphate buffered saline, pH about 7.4, with or without about 1 mg / mL to 25 mg / mL human serum albumin, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose, which may also contain an antioxidant such as tryptamine and a stabilizer such as Tween 20.

[0461] The methods of the present invention for inducing cell death of selected cell populations, inhibiting cell proliferation and / or treating cancer can be practiced in vitro, in vivo or ex vivo. [Example]

[0462] Example 1 Generation of mouse monoclonal antibodies against human and cynomolgus monkey CD123 antigens To generate mouse anti-CD123 antibodies (wild-type BALB / c female mice (Charles River Laboratory, Wilmington, MA)), human CD123-expressing stable 300-19 cell line (a BALB / c-derived B cell precursor line (M.G.Reth et al., 1985, Nature, 317:353-355)) in PBS was injected every 2 weeks for 5 doses at a dose of 5 × 10 6 Three days before sacrifice for hybridoma generation, the immunized mice received another dose of antigen intraperitoneally. Spleens from the mice were harvested according to standard animal protocols, ground between two sterile frosted microscope slides, and then resuspended in RPMI-1640 medium. A single-cell suspension was obtained. After erythrocyte lysis with ACK lysis buffer, spleen cells were mixed with mouse myeloma P3X63Ag8.653 cells (P3 cells) (J.F. Kearney et al., 1979, J. Immunol., 123:1548-1550) at a P3 cell:spleen cell ratio of 1:3. The spleen and P3 cell mixture was washed and treated with pronase in fusion medium (0.3 M mannitol / D-sorbitol, 0.1 mM CaCl2, 0.5 mM MgCl2, and 1 mg / mL BSA) for 3 minutes at room temperature. The reaction was stopped by adding fetal bovine serum (FBS, Invitrogen). The cells were then washed, resuspended in 2 mL of cold fusion medium, and fused in a BTX ECM 2001 electrofusion machine (Harverd Apparatus). The fused cells were slowly added to hypoxanthine-aminopterin-thymidine (HAT)-containing RPMI-1640 selection medium (Sigma-Aldrich), incubated at 37°C for 20 minutes, and then seeded at 200 μL / well into flat-bottom 96-well plates. The plates were then incubated at 37°C in a 5% CO2 incubator until hybridoma clones were available for antibody screening. Other techniques for immunization and hybridoma generation can also be used, including those described by J. Langone and H. Vunakis (Eds., Methods in Enzymology, Vol. 121, Immunochemical Techniques, Part I, Academic Press, Florida) and E. Harlow and D. Lane (Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, New York, NY).

[0463] Hybridoma Screening and Selection Hybridoma screening was performed using a flow cytometry binding assay with the human CD123-expressing stable 300-19 cell line and wild-type 300-19 cells. Briefly, wild-type 300-19 cells were first labeled with CELLTRACE™ far-red DDAO-SE (Invitrogen), mixed with untreated cells at a 1:1 ratio, and incubated with hybridoma supernatant for 2 hours on ice. The cells were then washed, incubated with PE-labeled anti-mouse IgG (Jackson Immunoresearch), washed, fixed with formalin, and analyzed using a FACS array (BD Bioscience). Hybridomas specifically reactive to the human CD123 antigen were expanded, and the supernatants were rescreened in a flow cytometry binding assay using three independent cell lines: the human CD123-expressing stable 300-19 cell line, the cynomolgus monkey CD123-expressing stable 300-19 cell line, and the wild-type 300-19 cell line. Hybridomas that showed positive binding to human and cynomolgus CD123 antigens but negative binding to wild-type 300-19 cells were further subcloned by limiting dilution. One subclone from each hybridoma that showed specific binding to human and cynomolgus CD123 antigens was selected for further analysis.

[0464] A total of six fusions were performed during this study. Approximately 6,000 hybridomas were screened to generate 33 hybridomas specific for human and cynomolgus monkey CD123 antigens, and 18 hybridomas were subcloned. Stable subclones were cultured, and the isotypes of the monoclonal antibodies were identified using commercially available mouse IgG isotyping reagents (e.g., IsoStrip Mouse Monoclonal Antibody Isotyping Kit, product number 11493027001, Roche Diagnostics GmbH, Germany).

[0465] Antibody purification Antibodies were purified from hybridoma subclone supernatants using standard methods, such as protein A or G chromatography (HiTrap protein A or G HP, 1 mL, Amersham Biosciences). Briefly, supernatants were purified by centrifugation for 1 The supernatant was prepared for chromatography by adding 1 / 10 volume of 1M Tris / HCl buffer (pH 8.0). The pH-adjusted supernatant was filtered through a 0.22 μm filter membrane and loaded onto a column equilibrated with binding buffer (PBS, pH 7.3). The column was washed with binding buffer until a stable baseline was obtained with no absorbance at 280 nm. The antibody was eluted with 0.1 M acetate buffer containing 0.15 M NaCl (pH 2.8) using a flow rate of 0.5 mL / min. Fractions of approximately 0.25 mL were collected and neutralized by adding 1 / 10 volume of 1 M Tris / HCl (pH 8.0). Peak fraction(s) were dialyzed twice overnight against 1× PBS and sterilized by filtration through a 0.2 μm filter membrane. The purified antibody was quantified by absorbance at A280.

[0466] Purified protein A fractions were further polished using ion exchange chromatography (IEX) with quaternary ammonium chloride (Q) chromatography for mouse antibodies. Briefly, samples from protein A purification were buffer exchanged into binding buffer (10 mM Tris, 10 mM sodium chloride, pH 8.0) and filtered through a 0.22 μm filter. The prepared sample was then loaded onto a Q fast flow resin (GE Lifesciences) equilibrated with binding buffer at a flow rate of 120 cm / h. The column size was selected to have sufficient capacity to bind all MAbs in the sample. The column was then washed with binding buffer until a stable baseline with no absorbance at 280 nm was obtained. The antibody was eluted by starting a 20-column-volume (CV) gradient from 10 mM to 500 mM sodium chloride. Peak fractions were collected based on absorbance at 280 nm (A280). The monomer content was assessed by size-exclusion chromatography (SEC) using an Agilent HPLC1100 system (Agilent, Santa Clara, CA) with a TSK Gel G3000SWXL, 7.8 x 300 mm, equipped with a SWXL guard column, 6.0 x 40 mm (Tosoh Bioscience, Montgomeryville, PA). Fractions with greater than 95% monomer content were pooled, buffer-exchanged into PBS (pH 7.4) using a TFF system, and sterilized by filtration through a 0.2 μm filter membrane. The IgG concentration of the purified antibody was measured at A280 using an extinction coefficient of 1.47. Alternative methods, such as ceramic hydroxyapatite (CHT), were also used to polish the antibody with good selectivity. Type II CHT resin with a 40 μm particle size (Bio-Rad Laboratories) was used with a similar protocol as described for IEX chromatography. The binding buffer for CHT corresponded to 20 mM sodium phosphate (pH 7.0) and the antibody was eluted with a gradient of 20 to 160 mM sodium phosphate over 20 CV.

[0467] Example 2 V of anti-CD123 antibody L and VH Cloning and sequencing of the region V L and V H Region cloning Total cellular RNA was extracted from 5 × 10 CD123 hybridomas using the RNeasy kit (QIAgen) according to the manufacturer's protocol. 6 cDNA was then synthesized from total RNA using the Superscript II cDNA synthesis kit (Invitrogen).

[0468] The PCR procedure for amplifying antibody variable region cDNA from hybridoma cells was based on the method described by Wang et al. ((2000) J Immunol Methods. 233:167-77) and Co et al. ((1992) J Immunol. 148:1149-54). L and V H Sequences were amplified with a degenerate primer at the 5' end and either a mouse kappa or IgG1 constant region specific primer at the 3' end, respectively. Purified amplicons were sent to Beckman Coulter Genomics for sequencing.

[0469] V L and V H Because the degenerate primers used to clone the cDNA sequence altered the 5' end, additional sequencing attempts were necessary to verify the complete cDNA sequence. The preliminary sequence was entered into a search query on the NCBI IgBlast site to identify the mouse germline sequence from which the antibody sequence was derived. PCR primers were then designed to anneal to the germline-linked leader sequence of the mouse antibody, such that this new PCR reaction would yield the complete variable region cDNA sequence unaltered by the PCR primers.

[0470] Mass measurement for sequence confirmation The variable region cDNA sequences obtained for each of the anti-CD123 antibodies were combined with the germline constant region sequences to obtain the full-length antibody cDNA sequences. The molecular weights of the heavy and light chains were then calculated from translation of the cDNA sequences and compared with the molecular weights obtained by LC / MS analysis of purified murine anti-CD123 antibodies. The molecular weights observed for each of the light and heavy chains were consistent with the expected values.

[0471] Example 3 Humanization of antibodies Expression of recombinant antibodies The confirmed variable region amino acid sequence of the murine CD123 antibody was codon-optimized, synthesized, and cloned in frame with a human antibody constant region by Blue Heron Biotechnology to construct a chimeric version of the CD123 antibody. The vector, constant region, and cloning method used for the chimeric CD123 antibody were identical to those used for the human CD123 antibody described below. The chimeric antibody chCD123-6 consists of the mouse variable region sequences of SEQ ID NOs: 28 and 29, respectively, along with human IgG1 and κ constant sequences in the heavy and light chains, respectively. The light chain variable region was cloned into the EcoRI and BsiWI sites of the pAbKZeo plasmid, and the heavy chain variable region was cloned into the HindIII and Apa1 sites of the pAbG1Neo plasmid. These expression constructs were transiently produced in either adherent HEK-293T cells or suspension-adapted HEK-293T cells using a modified PEI procedure in shake flasks. PEI transient transfections were performed as previously described (Durocher et al., Nucleic Acids Res. 30(2):E9 (2002)), except that HEK-293T cells were grown in Freestyle 293 (Invitrogen) and the culture medium volume was left undiluted after addition of the PEI-DNA complexes. Transfections were incubated for 1 week, and then the cleared supernatant was purified by standard protein A chromatography followed by a polishing chromatography procedure.

[0472] Antibody humanization The murine CD123-6 antibody was humanized using a complementarity-determining region (CDR) grafting procedure substantially as described in Jones et al., Nature 321:604-608, 1986; Verhoeyen et al., Science 239:1534-1536, 1988; U.S. Patent No. 5,225,539; and U.S. Patent No. 5,585,089. CDR grafting generally involves replacing the Fv framework regions (FR) of a murine antibody with t...

Claims

[Claim 1] An antibody or antigen-binding fragment thereof, (a) binds an epitope within amino acids 101-346 of the human CD123 / IL3-Rα antigen; and (b) the antibody or antigen-binding fragment thereof, which inhibits IL3-dependent proliferation of antigen-positive TF-1 cells.

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