Anti-CD123 antibody, as well as its complex and derivatives.

Abstract

This invention provides an antitumor agent that demonstrates efficacy against cancer cells expressing CD123, which has been reported to be overexpressed in malignant cells of a wide range of hematological malignancies. [Solution] Provided are an antibody or its antigen-binding fragment, an immune complex containing the antibody, and a method for using such CD123-binding molecules to diagnose and treat diseases such as B-cell malignancies, (a) an epitope within amino acids 101-346 of the human CD123 / IL3-Rα antigen, and (b) an antibody or its antigen-binding fragment that inhibits IL3-dependent proliferation of antigen-positive TF-1 cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims, under Section 119 of the United States Patent Act, the benefit of the filing dates of U.S. Provisional Patent Application No. 62 / 186,161 filed June 29, 2015, U.S. Provisional Patent Application No. 62 / 338,203 filed May 18, 2016, and U.S. Provisional Patent 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 generally relates to antibodies, their antigen-binding fragments, polypeptides, and immune complexes that bind to the CD123 antigen (the α chain of the interleukin-3 receptor or IL-3Rα). The present invention also relates to methods of using such CD123-binding molecules for diagnosing and treating diseases such as B-cell malignancies. [Background technology]

[0003] CD123 (interleukin-3 receptor α, 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 the early differentiation of pluripotent stem cells in erythrocytes, myeloids, and lymphoid progenitor cells. CD123 is related to CD34 + It is expressed in determined progenitor cells, but is not the same as CD34. + / CD38 - CD123 is not expressed by hematopoietic stem cells (HSCs). CD123 is expressed by basophils, mast cells, and plasmacytoid dendritic cells, to some extent by mononuclear cells, macrophages, and eosinophils, and is low-expressed or not expressed at all by neutrophils and meganuclear cells. Some non-hematopoietic tissues (e.g., placenta, Leydig cells of the testis, certain brain cell components, and some endothelial cells) also express CD123. However, its expression there is almost always 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 the normal human precursor cell population, CD123 is expressed by a subset of hematopoietic progenitor cells (HPCs), rather than by normal HSCs. CD123 has also been reported to be 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 in malignant cells of a wide range of hematological malignancies, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) (Munoz et al., Haematologica 86(12):1261-1269, 2001). CD123 overexpression is associated with a poor prognosis in AML (Tettamanti et al., Br.J.Haematol. 161:389-401, 2013). AML and MDS are thought to be caused by a small population of leukemic stem cells (LSCs) that are persistent, and LSCs are usually 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, while CD123 is absent in 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 associated with several other malignancies / pre-malignancies, chronic myeloid leukemia (CML) progenitor cells (including acutely transformed CML), Hodgkin's Reedsternberg (RS) cells, transformed non-Hodgkin lymphoma (NHL), Some chronic lymphocytic leukemias (CLL) (CD11c + ), a subset of acute T lymphoblastic leukemia (T-ALL) (16%, mostly immature, mostly adult), plasmacytoid dendritic cell (pDC) (DC2) malignancies, and CD34+ / CD38 - It is also associated with myelodysplastic syndrome (MDS), a type of 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 show worse treatment outcomes than younger patients (Robak). et al., Clin.Ther.2:2349-2370, 2009). Unfortunately, the vast majority of adults currently diagnosed with AML die from the disease.

[0007] Treatment for AML initially focuses on inducing remission (induction therapy). Once remission is achieved, treatment is shifted to focus on ensuring remission of this species (post-remission therapy or consolidation therapy), and, if necessary, maintenance therapy. The standard remission induction strategy for AML is either anthracycline / cytarabine combination chemotherapy followed by consolidation chemotherapy with high doses of the same drugs usually used during the induction phase or during human stem cell transplantation, depending on the patient's ability to tolerate intensive care and the possibility of a cure with chemotherapy alone (see Roboz, Curr. Opin. Oncol. 24:711-719, 2012).

[0008] Drugs frequently 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 reduced leukocytosis, bleeding due to reduced 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 the higher-order structure of DNA, and generation of cytotoxic oxygen free radicals. The most serious side effect of anthracyclines is cardiotoxicity, which limits the lifetime dose administered and, to some extent, significantly limits their usefulness.

[0009] Therefore, unfortunately, despite substantial progress 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 their first complete remission are expected to relapse within three years. The optimal approach for patients at relapse or with refractory disease remains unclear. Stem cell transplantation has been established as the most effective form of antileukemia treatment in AML patients in their first or subsequent remission (Roboz, 2012).

[0010] Antibody-drug conjugates (ADCs) and other cell-binding agent-drug conjugates have emerged as a potent class of antitumor agents with efficacy against various cancers. Cell-binding agent-drug conjugates (e.g., ADCs) generally consist of three distinct elements: a cell-binding agent (e.g., an antibody), a linker, and a cytotoxic moiety. Traditionally, the cytotoxic drug moiety has been covalently bonded to lysine or cysteine ​​residues on the antibody, resulting in a heterogeneous mixture of ADCs that carry a varying number of drugs attached to different positions on the antibody molecule, obtained by reducing the interchain disulfide bonds. [Overview of the project]

[0011] The present invention is based on the remarkable discovery that the complex of the present invention is highly potent against various CD123-expressing cancer cells, particularly leukemias having at least one negative prognostic factor.

[0012] One aspect of the present invention is (a) amino acids 101-34 of the human CD123 / IL3-Rα antigen. The present invention provides an antibody or its antigen-binding fragment that binds to an epitope within 6 and inhibits (b) IL3-dependent proliferation of antigen-positive TF-1 cells.

[0013] In a particular embodiment, the antibody or its antigen-binding fragment binds to an epitope within amino acids 101-204 of the human CD123 antigen. In another embodiment, the antibody or its antigen-binding fragment binds to an epitope within amino acids 205-346 of the human CD123 antigen.

[0014] Related aspects of the present invention include (a) attaching an epitope within amino acids 1 to 100 of human CD123, and (b) 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 its antigen-binding fragment that inhibits IL3-dependent proliferation of antigen-positive TF-1 cells.

[0015] In certain embodiments, the antibody or its antigen-binding fragment inhibits the proliferation of leukemic stem cells or leukemic blast cells, rather than hematopoietic stem cells.

[0016] In certain embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of 0.3 nM or less, for example, 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 ) binds to human CD123 antigen-positive cells.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus 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 and can bind to cynomolgus CD123. In certain embodiments, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus CD123 with substantially similar binding affinities. For example, 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, or 0.05 nM to 0.1 nM and can bind to human and cynomolgus CD123. K can be measured by flow cytometry, surface plasmon resonance, or radioimmunoassay. d In certain embodiments, the antibody or antigen-binding fragment thereof inhibits at least 50% of the IL3-dependent growth of antigen-positive TF-1 cells at a concentration of 0.5 nM or less. d K d is measured by flow cytometry, surface plasmon resonance, or radioimmunoassay.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof binds to cynomolgus 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 and can bind to cynomolgus CD123. In certain embodiments, the antibody or antigen-binding fragment thereof binds to both human and cynomolgus CD123 with substantially similar binding affinities. For example, 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, or 0.05 nM to 0.1 nM and can bind to human and cynomolgus CD123. K

[0019] In certain embodiments, the antibody or its antigen-binding fragment is a) at least one heavy chain variable region or fragment comprising three consecutive 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: 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 The fragment may also include: b) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein, with the exception of 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 its antigen-binding fragment a) comprises at least one heavy element comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3 b) At least one heavy chain variable region or fragment thereof, wherein, excluding one, two, or three conserved 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, each comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein, excluding one, two, or three conserved 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, the conservative amino acid substitution includes the substitution of at least one Lys in the CDR by Arg.

[0022] In a particular embodiment, the antibody is a CDR-implanted humanized antibody containing a mouse CDR region, wherein one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) heavy chain and / or light chain framework region vernier zone residues of the antibody are derived from mouse.

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

[0024] In certain embodiments, the antibody or its antigen-binding fragment may include a) an immunoglobulin heavy chain variable region having the amino acid sequence described in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence described 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 its antigen-binding fragment may include a) an immunoglobulin heavy chain variable region having the amino acid sequence described 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 described in SEQ ID NO: 41.

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

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

[0028] In certain embodiments, the antibody or its antigen-binding fragment may include a) an immunoglobulin heavy chain region having the amino acid sequence described 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 described in SEQ ID NO: 41, except that the N-terminal residue is Ser.

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

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

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

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

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

[0034] In certain embodiments, the antibody or its antigen-binding fragment may include a) an immunoglobulin heavy chain region having the amino acid sequence described 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 described in SEQ ID NO: 41.

[0035] In certain embodiments, the antibody or its antigen-binding fragment may include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence described 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 its antigen-binding fragment may include an immunoglobulin heavy chain variable region comprising a) a CDR1 having the amino acid sequence described in SEQ ID NO: 1, a CDR2 having the amino acid sequence described in SEQ ID NO: 2 or 3, and a CDR3 having the amino acid sequence described in SEQ ID NO: 4, and b) an immunoglobulin light chain variable region comprising a CDR1 having the amino acid sequence described in SEQ ID NO: 16, a CDR2 having the amino acid sequence described in SEQ ID NO: 17, and a CDR3 having the amino acid sequence described in SEQ ID NO: 18.

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

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

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

[0041] In certain embodiments, the antibody is mouse, non-human mammalian, chimeric, humanized, or human antibody. For example, a humanized antibody may be a CDR-transplanted antibody or a resurfacing antibody. In certain embodiments, the antibody is a full-length antibody. In certain embodiments, its antigen-binding fragment is Fab, Fab', F(ab')2, F dThese include single-chain Fv or scFv, disulfide-bonded Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, small antibody, F(ab')3, quadruple-specific antibody, triple-specific antibody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0042] Another aspect of the present invention is the V of either the target antibody or its antigen-binding fragment. H and V L A polypeptide containing a sequence is provided. The polypeptide may be fused with a protein other than a Pseudomonas toxin.

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

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

[0045] Another aspect of the present invention is the following formula: [ka] An immune complex having, During the ceremony, CBA is Cy L1 The antibody of the present invention or its anti- The original binding fragment, or the polypeptide of the present invention, W L is an integer between 1 and 20, Cy L1 The formula is as follows: [ka] Represented by, Provides an immune complex or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected 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 between 2 and 6. R k is -H or -Me, R x3 It is an (C1-C6) alkyl group, L' is given by 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 In each occurrence, Q is independently -H, (C1-C3) alkyl, or a charged substituent or ionic group. m is an integer between 1 and 6. Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is H + Or it is a cation.

[0046] In a particular embodiment, R a and R b Both are H, and R5 is either H or Me.

[0047] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues. For example, P may 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 may be selected from Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Al It is a.

[0048] In a particular embodiment, Q is -SO3M.

[0049] In a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where W L The integers are from 1 to 10, and the double line between N and C [ka] The expression represents a single or double bond, provided 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] The relevant aspect is given by the following formula: [ka] An immune complex having, During the ceremony, CBA is formed by the heliidine residue Cy L2The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the antibody of the present invention. W L is an integer between 1 and 20, Cy L2 The formula is as follows: [ka] Represented by, Provides an immune complex or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected moiety and Y is -OH or -SO3M. R x1 and R x2 These are (C1-C6) alkyl groups, 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 between 2 and 6. R k is -H or -Me, Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is -H + Or it is a cation.

[0051] In a particular embodiment, R eis H or Me, and R x1 and R x2 It is independently -(CH2) p -(CR f R g )- and in the formula, R f and R g Each of these is independently -H or (C1-C4) alkyl, and p is 0, 1, 2, or 3.

[0052] In a particular embodiment, R f and R g They are either identical or different, and are selected from -H and -Me.

[0053] In a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where W L The integers are from 1 to 10, and the double line between N and C [ka] The expression represents a single or double bond, provided 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 a particular embodiment, a double line between N and C [ka] The symbol represents a double bond, where X is absent and Y is -H. In certain embodiments, the double line between N and C [ka] represents a single bond, where X is -H and Y is -SO3M. In certain embodiments, M is H + na + or K + That is the case.

[0055] Another related aspect of the present invention is the following formula: [ka] Provides an immune complex having, During the ceremony, CBA is derived from Lys residues by Cy L3 The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the antibody of the present invention. In the formula, W L is an integer between 1 and 20, Cy L3 The formula is as follows: [ka] Represented by, m' is either 1 or 2. R1 and R2 are each independently H or (C1-C3) alkyl. Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is H + Or it is a cation.

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

[0057] In a particular embodiment, the immune complex is expressed by the following formula: [ka] is represented by, or a pharmaceutically acceptable salt thereof, wherein W L is an integer from 1 to 10.

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

[0059] Another aspect of the present invention is the following formula:

Chemical formula

Chemical formula

Chemical formula

[0060] For simplicity, when listing Ser as the N-terminal residue in each of the following examples, it should be understood that other 2-hydroxyethylamine moieties, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residues as part of serine are intended, where applicable, particularly with respect to Thr.

[0061] In a particular embodiment, R a and R b Both are H, and both R5 and R9 are either H or Me.

[0062] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues. For example, P may 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 , can be selected from the group consisting of 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 a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] [ka] [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The expression represents a single or double bond, provided 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 the following formula: [ka] An immune complex having, During the ceremony, CBA is J CB The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the base. J CB ' is an aldehyde group obtained from the 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 This is the part formed by reacting the aldehyde reactive group, J CB ' is expressed by the following formula: [ka] It is represented as, In the formula, s1 is the site that is covalently bonded to CBA, and s2 is Cy s2 It is a site that is covalently bonded to, Cy s2 The formula is as follows: [ka] Represented by, Provides an immune complex or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected moiety and Y is -OH or -SO3M. M is H + or it is a cation, R x1 It is an (C1-C6) alkyl group, 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 between 2 and 6. R k is -H or -Me, R x2 It is an (C1-C6) alkyl group, L1 is given by the following formula: [ka] Represented by, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s2 It is a site that is covalently bonded to the -S- group, Z a2 is either 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 In each occurrence, it is independently H or (C1-C3) alkyl. q1 and r1 are independent integers between 0 and 10, provided that neither q1 nor r1 is 0.

[0065] In a particular embodiment, -L1- is the following expression: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where R is H or -SO3M.

[0066] In a particular embodiment, 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 Each is independently -H or (C1~C4)alkyl, and p is 0, 1, 2 or It is 3. In a particular embodiment, R f and R g They are either identical or different, and are selected from -H and -Me.

[0067] In a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The expression represents a single or double bond, provided 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 a particular embodiment, a double line between N and C [ka] The symbol represents a double bond, where X is absent and Y is -H.

[0069] In a particular embodiment, a double line between N and C [ka] represents a single bond, where X is -H and Y is -SO3M. In certain embodiments, M is H + na + or K + That is the case.

[0070] Another aspect of the present invention is the following formula: [ka] Provides an immune complex having, During the ceremony, CBA is J CB The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to a base. J CB ' is an aldehyde group obtained from the oxidation of the 2-hydroxyethylamine moiety of 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 This is a portion formed by reacting the aldehyde reactive group, and J CB ' is expressed by the following formula: [ka] It is represented as, In the formula, s1 is the site that is covalently bonded to CBA, and s2 is Cy s3 It is a site that is covalently bonded to, Cy s3 The formula is as follows: [ka] It is represented as, During the ceremony, m' is either 1 or 2. R1 and R2 are each independently H or (C1-C3) alkyl. L1 is given by the following formula: [ka] It is represented as, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s3 It is a site that is covalently bonded to the -S- group, Z a2 is either 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 In each occurrence, it is independently H or (C1-C3) alkyl. q1 and r1 are independent integers between 0 and 10, provided that neither q1 nor r1 is 0.

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

[0072] In a particular embodiment, -L1- is the following expression: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where R is H or -SO3M, and M is H + Or it is a cation.

[0073] In a particular embodiment, the immune complex is expressed by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where DM is given by the following formula: [ka] It is represented as follows.

[0074] Another aspect of the present invention is the following formula: [ka] Provides an immune complex having, During the ceremony, CBA is J CB The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to a base. J CB ' is an aldehyde group obtained from the oxidation of the 2-hydroxyethylamine moiety of 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 This is a portion formed by reacting the aldehyde reactive group, and J CB ' is expressed by the following formula: [ka] It is represented as, In the formula, s1 is the site that is covalently bonded to CBA, and s2 is Cy s4 It is a site that is covalently bonded to, Cy s4 The formula is as follows: [ka] It is represented as, L1' is given by the following formula: [ka] It is represented as, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s4 It is a site that is covalently bonded to the -NMe- group, Z b1 and Z b2 Both are absent, or Z b1 and Z b2 One of them is absent, and the other is -CH2-O- or -O-CH2-. Z b1 'and Z b2' are either independently absent, or -CH2-O-, -O-CH2-, -NR9-C(=O)-CH2-, or -CH2-C(=O)-NR9-, R9 is H or (C1-C3) alkyl. n1 and m1 are each independent integers between 1 and 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 In each occurrence, each element is independently either H or (C1-C3) alkyl.

[0075] In a particular embodiment, R b1 , R b2 , R b3 , R b4 , R b5 , and R b6 All of these are H. In certain embodiments, R9 is H.

[0076] In a particular embodiment, Z b1 'and Z b2 ' is either absent in both cases, or Z b1 ' is -CH2-O-, Z b2 ' is absent, or Z b1 ' is -CH2-C(=O)-NR9-, Z b2 ' is either O-CH2- or absent.

[0077] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues. For example, P may 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 These are la-Ala, Ala-D-Ala, D-Ala-Ala, and D-Ala-D-Ala.

[0078] In a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where DM has the following structural formula: [ka] It is represented as follows.

[0079] Another aspect of the present invention is the following formula: [ka] An immune complex represented by, During the ceremony, CBA is formed by cysteine ​​residues. C1 The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the antibody of the present invention. Wc is 1 or 2, Cy C1 The formula is as follows: [ka] Represented by, Provides an immune complex or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, however, 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 amine protecting moiety, Y is -OH or -SO3M, and M is H + or provided that it is a 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 In each occurrence, independently, -H, (C1~C3) alkyl, or charged It is a substituent or an ionic group Q, W' is -NR e’ And, R e’ is -(CH2-CH2-O) n -R k And, n is an integer between 2 and 6. R k is -H or -Me, Rx3 It is an (C1-C6) alkyl group, Lc is [ka] Represented as, s1 is the site that is covalently bound to CBA, and s2 is Cy C1 This is the site that is covalently bonded to the -C(=O)- group, and in the formula, R 19 and R 20 In each occurrence, it is independently -H or (C1~C3)alkyl, m'' is an integer between 1 and 10. R h is -H or (C1~C3) alkyl.

[0080] In a particular embodiment, R a and R b Both are H, and R5 is either H or Me.

[0081] In certain embodiments, P is a peptide containing 2 to 5 amino acid residues. For example, P may 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- The following can 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 a particular embodiment, R 19 and R 20 Both are H, and m'' is an integer between 1 and 6.

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

[0084] In a particular embodiment, the immune complex is expressed by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The expression represents a single or double bond, provided 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 the following formula: [ka] An immune complex represented by, During the ceremony, CBA is formed by cysteine ​​residues. C2 The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the antibody of the present invention. Wc is 1 or 2, Cy C2 The formula is as follows: [ka] Represented by, Provides an immune complex or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, however, 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 amine protecting moiety, Y is -OH or -SO3M, and M is H + or provided that it is a cation, R x1 It is an (C1-C6) alkyl group, 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 between 2 and 6. R kis -H or -Me, R x2 It is an (C1-C6) alkyl group, L C ' is expressed by the following formula: [ka] It is represented as, During the ceremony, s1 is the site that is covalently bound to CBA, and s2 is Cy C2 It is a site that is covalently bonded to the -S- group, 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 In each occurrence, it is independently -H or (C1~C3)alkyl, q and r are, in each occurrence, independent integers between 1 and 10. m and n are independent integers between 0 and 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' may 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 a particular embodiment, P' is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0087] In certain embodiments, immune complex-L C '- is represented by the following expression: [ka] It is represented as follows.

[0088] In a particular embodiment, 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 in the formula, R f and R g Each is independently -H or (C1-C4)alkyl, and p is 0, 1, 2, or 3. In certain embodiments, R f and R g They are either identical or different, and are selected from -H and -Me.

[0089] In a particular embodiment, the immune complex is expressed by the following formula: [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The expression represents a single or double bond, provided 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 a particular embodiment, a double line between N and C [ka] The symbol represents a double bond, where X is absent and Y is -H.

[0091] In a particular embodiment, a double line between N and C [ka] represents a single bond, where X is -H and Y is -SO3M. In certain embodiments, M is H + na + or K + That is the case.

[0092] Another aspect of the present invention is the following formula: [ka] Provides an immune complex having, During the ceremony, CBA is formed by cysteine ​​residues. C3 The antibody of the present invention or its antigen-binding fragment, or the polypeptide of the present invention, is covalently bound to the antibody of the present invention. Wc is 1 or 2, Cy C3 The formula is as follows: [ka] It is represented as, During the ceremony, m' is either 1 or 2. R1 and R2 are each independently -H or (C1~C3) alkyl. L C ' is expressed by the following formula: [ka] It is represented as, During the ceremony, s1 is the site that is covalently bound to CBA, and s2 is Cy C3 It is a site that is covalently bonded to the -S- group, 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 In each occurrence, it is independently -H or (C1~C3)alkyl, q and r are, in each occurrence, independent integers between 1 and 10. m and n are independent integers between 0 and 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' may be Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Lle-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- The following are 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 represented by the following expression: [ka] It is represented as, In the formula, M is H + Or it is a cation.

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

[0096] In a particular embodiment, the immune complex is expressed by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where DM is given by the following formula: [ka] This is the drug portion, represented by [the symbol].

[0097] Another aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide or immune complex 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, the method comprising contacting cells with the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide of the present invention, or the immune complex of the present invention, or the pharmaceutical composition of the present invention.

[0099] In certain embodiments, the cells are tumor cells. In certain embodiments, the cells are leukemia cells or lymphoma cells.

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

[0101] In certain embodiments, cancer or cytoproliferative disorders are leukemia or lymphoma. In certain embodiments, cancer or cytoproliferative disorders include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), including B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), and pilocytic cell leukemia (H). The cancer is selected from the group consisting of CL), myelodysplastic syndrome, blastic plasmacytoid dendritic cell neoplasm (BPDCN) leukemia, non-Hodgkin lymphoma (NHL) including mantle cell lymphoma, and Hodgkin 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 for treating a target cell proliferative disorder, wherein the cells of the cell proliferative disorder express CD123, and the method comprises administering to the target an effective amount of the antibody or antigen-binding fragment thereof of the present invention, or the polypeptide of the present invention, or the immune complex of the present invention, or the pharmaceutical composition of the present invention, in an amount sufficient to treat the cell proliferative disorder.

[0103] Any embodiment described herein, including those described in only one aspect of the present invention (but not otherwise described or repeated elsewhere) and those described only in examples, is intended to be combined with any one or more other embodiments of the present invention, unless expressly denied or inappropriate. [Brief explanation of the drawing]

[0104] [Figure 1] This study demonstrates the inhibition of IL-3-dependent proliferation of TF-1 cells by a chimeric CD123-6 antibody (chCD123-6) and its CDR-transplanted huCD123-6 antibodies (huCD123-6Gv4.6 and huCD123-6Gv4.7). [Figure 2A] We demonstrate that three mouse anti-CD123 antibodies (muCD123-3, -6, and -14) inhibit IL-3-dependent proliferation of TF-1 cells, at least similarly to 7G3. We show inhibition of TF-1 cells cultured in the presence of IL-3 (1 ng / mL) by various anti-CD123 antibodies, including CD123-conjugated control antibodies 7G3, 6H6, and 9F5. [Figure 2B]Three mouse anti-CD123 antibodies (muCD123-3, -6, and -14) inhibit IL-3-dependent proliferation of TF-1 cells, at least similarly to 7G3. The same anti-CD123 antibodies also inhibit TF-1 cells cultured in the presence of GM-CSF (2 ng / mL). [Figure 3] The mouse anti-CD123 antibodies muCD123-3, -6, and -14 inhibit dose-dependent IL-3 (1 ng / mL)-dependent proliferation of TF-1 cells to a higher degree than the 7G3 antibody. muCD123-16 is a negative control anti-CD123 antibody that binds to CD123 but does not inhibit IL-3-dependent proliferation of TF-1 cells. [Figure 4A] This study demonstrates that antibodies against mouse muCD123-3, -6, and -14 have higher binding affinity to CD123-positive AML cells than the 7G3 antibody used on CD123-expressing TF-1 cells. [Figure 4B] This study demonstrates that antibodies against mouse muCD123-3, -6, and -14 have higher binding affinity to CD123-positive AML cells than antibodies against 7G3 cells expressed by CD123-expressing HNT-34 cells. [Figure 5A] This study demonstrates that the chimeric anti-CD123 antibodies chCD123-3, -6, and -14 maintain high binding affinity to their mouse control using HNT-34 cells. A chimeric antibody chKTI, which does not bind to CD123, was included as a negative control. [Figure 5B] This study demonstrates that the chimeric anti-CD123 antibodies chCD123-3, -6, and -14 maintain high binding affinity to their mouse control using the CD123-positive acute myeloid leukemia (AML) cell line MOLM-13. A CD123-inactive chimeric antibody chKTI was included as a negative control. [Figure 6] The chimeric chCD123-3, -6, and -14 anti-CD123 antibodies retain the functional activity of the mouse control, 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 mouse (muCD123-6), chimeric (chCD123-6), and CDR-transplanted huCD123-6 antibodies (huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3) all exhibit higher affinity for CD123-expressing HNT-34 cells than 7G3. A chimeric antibody chKTI, which does not bind to CD123, was included as a negative control. [Figure 7B] Figures 7B and 7C show that the covalent binding of huCD123-6Gv4.7S3 or -Gv4.7 antibodies to D1 or D2 compounds via Lys-, Ser-, or Cys binding only moderately affects the binding affinity of these ADC complexes, namely Ser-bound huCD123-6Gv4.7S3-SeriMab-sD1 (see structure in Figure 17) and huCD123-6Gv4.7S3-SeriMab-D8 in Figure 7B, and Lys-bound huCD123-6Gv4.7S3-sSPDB-D1 and huCD123-6Gv4.7S3-D2, as well as Cys-bound huCD123-6Gv4.7-CysMab-D4 and huCD123-6Gv4.7-CysMab-D5 in Figure 7C. In Figure 7C, the non-complexed huCD123-6Gv4.7 antibody has the heavy chain sequence of SEQ ID NO: 54, where Xaa is Val. The complex containing "S3-SeriMab" has cytotoxic (in this case, indolinobenzodiazepine or "IGN" compound) binding via the oxidized N-terminal Ser of the light chain. The complex containing "CysMab" has cytotoxic (in this case, IGN compound) binding via the manipulated Cys of the heavy chain (i.e., the Cys corresponding to the fifth-to-last Cys of SEQ ID NO: 54). [Figure 7C]Figures 7B and 7C show that the covalent binding of huCD123-6Gv4.7S3 or -Gv4.7 antibodies to D1 or D2 compounds via Lys-, Ser-, or Cys binding only moderately affects the binding affinity of these ADC complexes, namely Ser-bound huCD123-6Gv4.7S3-SeriMab-sD1 (see structure in Figure 17) and huCD123-6Gv4.7S3-SeriMab-D8 in Figure 7B, and Lys-bound huCD123-6Gv4.7S3-sSPDB-D1 and huCD123-6Gv4.7S3-D2, as well as Cys-bound huCD123-6Gv4.7-CysMab-D4 and huCD123-6Gv4.7-CysMab-D5 in Figure 7C. In Figure 7C, the non-complexed huCD123-6Gv4.7 antibody has the heavy chain sequence of SEQ ID NO: 54, where Xaa is Val. The complex containing "S3-SeriMab" has cytotoxic (in this case, indolinobenzodiazepine or "IGN" compound) binding via the oxidized N-terminal Ser of the light chain. The complex containing "CysMab" has cytotoxic (in this case, IGN compound) binding via the manipulated Cys of the heavy chain (i.e., the Cys corresponding to the fifth-to-last Cys of SEQ ID NO: 54). [Figure 8A] The study shows that the huCD123-6 antibody inhibits IL-3-dependent proliferation of TF-1 cells more effectively than the 7G3 antibody when used in chimeric (chCD123-6) and CDR transplantation (huCD123-6Gv4.7S2 and huCD123-6Gv4.7S3) cells. [Figure 8B] Since these antibodies did not have an inhibitory effect when cells proliferated in the presence of GM-CSF, it is indicated that the inhibition is IL-3 dependent. [Figure 9A] The expression construct of the IL-3Rα (CD123) extracellular domain, as well as the chimeric receptor protein containing the IL-3Rα (gray) and GMRα domains (white), are shown. [Figure 9B] This shows that the CD123-6 antibody primarily binds to the CRM domain of IL-3Rα (residues 101-306). [Figure 9C]This shows that the CD123-3 antibody primarily binds to the CRM domain of IL-3Rα (residues 101-306). [Figure 9D] This shows that the CD123-14 antibody binds only to the N-terminal domain of IL-3Rα (residues 1-100). [Figure 9E] This shows that the 7G3 antibody binds only to the N-terminal domain of IL-3Rα (residues 1-100). [Figure 9F] This demonstrates 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 1-100). [Figure 10] This study demonstrates that the resurface-forming huCD123-6Rv1.1 antibody, specifically the mytansinoid DM1 complex of huCD123-6Rv1.1-CX1-1-DM1, exhibits dose-dependent cytotoxicity on the growth factor-independent CD123-expressing AML cell line OCI-AML4. The cytotoxicity is CD123-dependent, as evidenced by the ability of excessive non-complexed huCD123-6 antibody (500 nM) to inhibit cytotoxicity. [Figure 11A] This study demonstrates the in vitro cytotoxicity of various resurface-forming lysine-binding huCD123-6Rv1.1-IGN complexes on multiple CD123-positive malignant cell lines of different origins. [Figure 11B] This study demonstrates the in vitro cytotoxicity of various lysine or cysteine-bound huCD123-6-IGN complexes on multiple CD123-positive B-ALL cell lines. Unbound KTI antibody complexes are included as a negative control. [Figure 11C] This study demonstrates that various Lys or Cys-binding IGN compounds are highly active in P-gp (P-glycoprotein) positive AML cell lines Kasumi-3 and MOLM-1. The control curve, represented by white data points, is created in the presence of excess non-complex-matched huCD123 antibody. [Figure 11D]Almost all of the various Lys or Cys-bound CD123-IGN complexes of the present invention demonstrate that they kill 90% of AML progenitor cells from nine AML patient samples at nM or sub-nM concentrations. [Figure 11E] The Cys-binding huCD123-6Gv4.7-CysMab-D5 complex kills normal blood cells at concentrations more than 100 times higher than those required to kill AML progenitor cells. In contrast, Mylotarg does not exhibit this preferential killing effect. [Figure 12A] This study demonstrates the in vitro cytotoxicity of various lysine-binding huCD123-6Rv1.1-IGN complexes in primary cells from AML patients. Results from a standard CFU assay in one primary patient sample are shown. [Figure 12B] This study shows the in vitro cytotoxicity of various lysine-binding huCD123-6Rv1.1-IGN complexes in primary cells from AML patients. The IC90 values ​​for all AML patient samples treated with the complex are also shown. [Figure 13A] The IGN complex of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, black circle) is shown to be at least as active against AML cell line EOL-1 (Figure 13A), B-ALL cell line KOPN-8 (Figure 13B), and CML cell line MOLM-1 (Figure 13C) as the lysine-binding complex of the same antibody (huCD123-6Gv4.6-D2, black circle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Gv4.6-CysMab-D5 and the white square for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed chCD123-6 antibody. [Figure 13B]The IGN complex of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, black circle) is shown to be at least as active against AML cell line EOL-1 (Figure 13A), B-ALL cell line KOPN-8 (Figure 13B), and CML cell line MOLM-1 (Figure 13C) as the lysine-binding complex of the same antibody (huCD123-6Gv4.6-D2, black circle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Gv4.6-CysMab-D5 and the white square for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed chCD123-6 antibody. [Figure 13C] The IGN complex of huCD123-6 CysMab (huCD123-6Gv4.6-CysMab-D5, black circle) is shown to be at least as active against AML cell line EOL-1 (Figure 13A), B-ALL cell line KOPN-8 (Figure 13B), and CML cell line MOLM-1 (Figure 13C) as the lysine-binding complex of the same antibody (huCD123-6Gv4.6-D2, black circle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Gv4.6-CysMab-D5 and the white square for huCD123-6Gv4.6-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed chCD123-6 antibody. [Figure 14A] The seriMab of huCD123-6 (huCD123-6Rv1.1S2-SeriMab-D8, black circle) is at least as active as the lysine-binding complex of the same antibody for AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B), and CML cell line MOLM-1 (Figure 14C) (huCD123-6Rv1.1-D2, black downward triangle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Rv1.1S2-SeriMab-D8 and the white downward triangle for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed huCD123-6 antibody. [Figure 14B]The seriMab of huCD123-6 (huCD123-6Rv1.1S2-SeriMab-D8, black circle) is at least as active as the lysine-binding complex of the same antibody for AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B), and CML cell line MOLM-1 (Figure 14C) (huCD123-6Rv1.1-D2, black downward triangle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Rv1.1S2-SeriMab-D8 and the white downward triangle for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed huCD123-6 antibody. [Figure 14C] The seriMab of huCD123-6 (huCD123-6Rv1.1S2-SeriMab-D8, black circle) is at least as active as the lysine-binding complex of the same antibody for AML cell lines SHI-1 (Figure 14A) and HNT-34 (Figure 14B), and CML cell line MOLM-1 (Figure 14C) (huCD123-6Rv1.1-D2, black downward triangle). The dotted curves connecting to the white data points in each figure represent the activity of each complex (i.e., the white circle for huCD123-6Rv1.1S2-SeriMab-D8 and the white downward triangle for huCD123-6Rv1.1-D2) in the presence of an inhibitory concentration (500 nM) of the non-complexed huCD123-6 antibody. [Figure 15] A schematic diagram shows a general process that can be used to synthesize the Ser-binding complex of the present invention. [Figure 16] A schematic diagram shows a general process that can be used to synthesize the Ser-binding complex of the present invention. [Figure 17] A schematic diagram shows a general process that can be used to synthesize the Ser-binding complex of the present invention. [Figure 18] This study demonstrates that the Cys-binding huCD123-6Gv4.7-CysMab-D5 complex exhibits higher activity than gemtuzumab ozogamicin (GO) (also known as Mylotarg) from arbitrarily extracted AML patient samples. [Figure 19]The Cys-binding huCD123-6Gv4.7-CysMab-D5 complex kills normal progenitor cells at concentrations more than 100 times higher than those required to kill AML progenitor cells. In contrast, Mylotarg and huCD123-6G4.7-CysMab-D5' (an ADC of the DNA crosslinking agent D5') do not exhibit this preferential killing effect. [Figure 20] This study demonstrates the in vivo efficacy of the CD123-IGN complex in a subcutaneous model of MV4-11AML. [Figure 21] This study demonstrates that the Cys-binding huCD123-6Gv4.7-CysMab-D5 complex is highly active against various CD123-positive AML cells that possess poor prognostic factors. [Figure 22] This image shows in vivo bioluminescence imaging of mice treated with the huCD123-6Gv4.7-CysMab-D5 complex compared to mice treated with solvent and control on day 26. Treatment with the complex significantly reduces tumor burden in the mice. [Figure 23] Treatment with the huCD123-6Gv4.7-CysMab-D5 complex extends the survival rate of 6 / 6 mice compared to mice treated with the solvent and control. [Figure 24] Incubation of MV4-11 cells with the huCD123-6Gv4.7-CysMab-D5 complex induces DNA damage, S-phase arrest of the cell cycle, and apoptosis-mediated cell death. [Figure 25] This study demonstrates the in vivo efficacy of the CD123-IGN complex in a Molm-13AML seeding model. [Figure 26] This study demonstrates the in vivo efficacy of the CD123-IGN complex in a subcutaneous EOL-1 model. [Figure 27] This study demonstrates the in vivo efficacy of the huCD123-CysMab-D5 complex at various dosages in a subcutaneous EOL-1 model. [Figure 28]This paper demonstrates the in vivo efficacy of the huCD123-CysMab-D5 conjugate in an EOL-1 subcutaneous model, compared to effective load (FGN849 or D5), naked antibody, control, cytarabine, and the corresponding free drug form of azacitidine. [Figure 29] This study demonstrates the in vivo efficacy of the CD123-IGN complex in an MV4-11AML seeding model. [Figure 30] This study demonstrates the in vivo efficacy of the CD123-IGN complex in a subcutaneous model of MV4-11AML. [Figure 31] Treatment with the huCD123-CysMab-D5 complex extends the survival of mice compared to mice treated with the solvent and controls. [Figure 32] This study demonstrates the in vivo tolerability of huCD123-CysMab-D5 and huCD123-SeriMab-sD1 in mice. [Figure 33] This demonstrates the in vivo tolerability of the huCD123-lysine-binding-D2 complex in mice. [Modes for carrying out the invention]

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

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

[0107] All such terms above relating to CD123 mean either the protein or the nucleic acid sequence as described herein. The term “CD123 / IL-3Rα” includes “full length,” untreated CD123 / IL-3Rα, and any form of CD123 / IL-3Rα resulting from intracellular processing. The same terms also encompass naturally occurring variants of the 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 other sources) or prepared by recombinant or synthetic methods. Examples of 3 / 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 mutant 1), and NP_001254642 and NM_001267713 (protein and nucleic acid sequences of human CD123 mutant 2).

[0108] The term “antibody” means an immunoglobulin molecule that recognizes a target in particular (e.g., protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combination thereof) and specifically binds to it at at least one antigen-recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” includes 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 the antigen-detection 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 any of the five major classes of immunoglobulins, namely IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), based on the identity of their heavy chain constant domains, which are called α, δ, ε, γ, and μ. Different classes of immunoglobulins have different known subunit structures and three-dimensional shapes. Antibodies can be naked or conjugate to other molecules (e.g., toxins, radioisotopes, etc.).

[0109] In some embodiments, the antibody is a non-natural antibody. In some embodiments, the antibody is purified from a natural component. In some embodiments, the antibody is synthesized by recombinant DNA. In some embodiments, the antibody is synthesized by hybridoma.

[0110] A “blocking” antibody or “antagonist” antibody inhibits or reduces the biological activity of the antigen to which it binds (e.g., CD123 / IL-3Rα). In certain embodiments, the blocking antibody or antagonist antibody substantially or completely inhibits the biological activity of the antigen. Preferably, the 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 antibodies capable of binding to CD123 / IL-3Rα with sufficient affinity so that they are useful as diagnostic and / or therapeutic agents when targeting CD123 / IL-3Rα. Unless otherwise specified, the degree to which an anti-CD123 / IL-3Rα antibody binds to unrelated non-CD123 / IL-3Rα proteins is less than approximately 10% of the binding of the antibody to CD123 / IL-3Rα as measured, for example, by radioimmunoassay (RIA). In certain embodiments, antibodies that bind to CD123 / IL-3Rα have a dissociation constant (K) of ≤0.5 nM, ≤0.3 nM, ≤0.1 nM, ≤0.05 nM, or ≤0.01 nM. d ) has. 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 well-known commercially available CD123 antibody (e.g., 7G3(mouse IgG). 2a It does not bind to the same epitope of CD123 that is 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 antibody and its antigen-binding fragment of the present invention are shown in Table 1-6 below. Nomenclature for the various antibodies and immune complexes of the present invention is provided separately below.

[0113] The term "antibody fragment" refers to a portion of an intact antibody, and also to the antigen-detection variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and F v Examples include, but are not limited to, fragments, linear antibodies, single-chain antibodies, and multispecific antibodies formed from antibody fragments. The term “antigen-binding fragment of an antibody” means 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 a specific fragment of a full-length antibody. Examples of binding fragments included in the term “antigen-binding fragment of an antibody” are (i) Fab fragments, V L, V H , C L , and C H1 (ii) A monovalent fragment consisting of domains (for example, an antibody digested by papain yields three fragments: two antigen-binding Fab fragments and one non-antigen-binding Fc fragment), (ii) a bivalent fragment containing an F(ab')2 fragment and two Fab fragments linked by disulfide crosslinks at the hinge region (for example, an antibody digested by pepsin yields two fragments: a bivalent antigen-binding F(ab')2 fragment and a non-antigen-binding pFc' fragment), and its associated F(ab') monovalent unit, (iii) V H and C H1 F consisting of domains d (iv) V of a single arm of the antibody, (iv) the fragment (i.e., the heavy chain portion contained in Fab) L and V H F consisting of domains v Fragments, and associated disulfide bonds F v (v)V H (vi) comprising (but not limited to) domain-based dAb (domain antibody) or sdAb (single-domain antibody) fragments (Ward et al., Nature 341:544-546, 1989), and (vi) isolation complementarity-determining regions (CDRs).

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

[0115] The term "humanized antibody" refers to a form of non-human (e.g., mouse) antibody that is a specific immunoglobulin chain, chimeric immunoglobulin, or fragment thereof, containing the smallest non-human (e.g., mouse) sequence. Generally, a humanized antibody is a human immunoglobulin in which residues from the complementarity-determining region (CDR) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) that has the desired specificity, affinity, and capability (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 with corresponding residues in non-human species antibodies that have the desired specificity, affinity, and capability. Humanized antibodies are F v Further modification by substituting additional residues within the framework region and / or substituted non-human residues can improve and optimize the specificity, affinity, and / or capabilities of the antibody. Generally, humanized antibodies contain at least one, usually two or three, variable domains, all or nearly all of the CDR region corresponding to non-human immunoglobulins, while all or nearly all of the FR region is a human immunoglobulin sequence. Humanized antibodies also contain the immunoglobulin constant region or domain (F C ) may include at least a portion of, usually human immunoglobulin. Examples of methods used to produce humanized antibodies include 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 Rogu This is described in ska et al., Protein Eng. 9(10):895-904, 1996 (all incorporated herein by reference). In some embodiments, “humanized antibody” includes resurface-forming antibody. In some embodiments, “humanized antibody” is CDR-implantable 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 variable regions of the heavy and light chains each consist of four framework regions (FRs) linked by three complementarity-determining regions (CDRs) (also known as hypervariable regions). The CDRs of each chain are held in close proximity to CDRs from other chains by the FRs, contributing to the formation of the antibody's antigen-binding site. At least two techniques exist for determining CDRs: (1) an approach based on interspecies sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological There are two approaches: (1) Interest, 5th ed., 1991, National Institutes of Health, Bethesda Md., and (2) an approach based on crystallographic analysis 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 CDR.

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

[0119] Kabat-like amino acid position numbering refers to the numbering system used for the heavy-chain or light-chain variable domains of antibody compilations in 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 include a few or additional amino acids corresponding to the shortening or insertion into the FR or CDR of the variable domain. For example, the heavy-chain variable domain may include a single amino acid insertion after H2 residue 52 (residue 52a by Kabat), as well as insertion residues after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c by Kabat). The Kabat numbering of residues can be determined for a given antibody by sequence comparison in the homology region of the sequence of an antibody having a “standard” Kabat numbering sequence. Chothia, on the other hand, relates to the position of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917,1987). The end of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is 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 region represents a compromise between the Kabat CDR and the Chothia structural loop and is 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 prepared using any technique well known in the art that has an amino acid sequence corresponding to a human-produced antibody. In certain embodiments, a human antibody does not have a non-human sequence. This definition of a human antibody includes intact antibodies, full-length antibodies, or antigen-binding fragments thereof.

[0121] The term "chimeric antibody" refers to an antibody whose immunoglobulin molecule's amino acid sequence originates from two or more species. Generally, both the light and heavy chain variable regions correspond to the variable regions of an antibody derived from one mammalian species (e.g., mouse, rat, rabbit) having the desired specificity, affinity, and ability, while the constant region is homologous to the sequence in an antibody derived from another species (usually human), thus avoiding or reducing the possibility of inducing an immune response in that species (e.g., human). In certain embodiments, a chimeric antibody may include an antibody or its antigen-binding fragment, comprising at least one human heavy chain and / or light chain polypeptide (e.g., an antibody comprising a mouse light chain and a human heavy chain polypeptide).

[0122] The terms “epitope” and “antigenic determinant” are used herein interchangeably and refer to a portion of an antigen that is recognized and specifically bound to a particular antibody. When the antigen is a polypeptide, the epitope can be formed from both continuous amino acids and discontinuous amino acids juxtaposed by the tertiary folding of the protein. Epitopes formed from continuous amino acids are usually retained even when the protein denatures, while epitopes formed by tertiary folding are usually lost when the protein denatures. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids in a specific spatial arrangement.

[0123] "Binding affinity" generally refers to the total strength 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 specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that indicates a 1:1 interaction between the elements of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for partner Y is generally expressed by the dissociation constant (K). d ) or half-effective concentration (EC 50 Affinity can be expressed by the following: Affinity can be measured by common methods well known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate rapidly, while high-affinity antibodies generally tend to bind quickly to antigens and maintain binding for a longer period. Various methods for measuring binding affinity are well known in the art, and any of them can be used in the present invention. Specific exemplary embodiments are described herein.

[0124] As used herein, "or higher" refers to binding affinity, meaning a stronger bond between a molecule and its binding partner. As used herein, "or higher" refers to a smaller number K d This refers to a stronger binding, expressed as a value. For example, an antibody having an affinity for an antigen of "0.3 nM or higher" means that the antibody's affinity for the antigen is 0.3 nM or less, for example, 0.29 nM, 0.28 nM, 0.27 nM, or any value of 0.3 nM or less. In one embodiment, K d The affinity of the antibody, determined by this, is approximately 10 -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 It is M.

[0125] "Specifically binding" generally means that an antibody binds to an epitope via its antigen-binding domain, and that such 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 easily than it would to bind to a random, unrelated epitope. The term "specificity" is used herein to limit the relative affinity with which a particular antibody binds to a particular epitope. For example, antibody "A" may be considered to have higher specificity for a given epitope than antibody "B", or antibody "A" may bind to epitope "C" with higher specificity than it has for related epitope "D".

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

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

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

[0129] As used herein, the terms “substantially similar” or “substantially identical” mean that a person skilled in the art will know that the difference between two values ​​is such that the difference between the two values ​​(e.g., K d Within the context of the biological properties measured by the values, a sufficiently high degree of similarity between the two numerical values ​​(generally one related to the antibody of the present invention and the other related to the reference / comparative antibody) is considered to be little or no biological and / or statistically significant. The difference between the two values ​​is less than approximately 50%, less than approximately 40%, less than approximately 30%, less than approximately 20%, or less than approximately 10%, depending on the value of the reference / comparative antibody.

[0130] "Isolated" polypeptides, antibodies, polynucleotides, vectors, cells, or compositions are polypeptides, antibodies, polynucleotides, vectors, cells, or compositions in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to a degree that they are not found in nature. In some embodiments, isolated antibodies, polynucleotides, vectors, cells, or compositions are substantially pure.

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

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

[0133] A "linker" is any chemical site capable of stably covalently binding a compound, usually a drug, such as a cytotoxic agent as described herein (e.g., a mytansinoid or IGN (indolinobenzodiazepine) compound), to a cell-binding agent (e.g., an anti-CD123 / IL-3Rα antibody or a fragment thereof). The linker may be sensitive to or substantially resistant to acid-induced cleavage, photo-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage under conditions in which the compound or antibody maintains its activity. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid-unstable groups, photosensitive groups, peptidase-unstable groups, and esterase-unstable groups. Linkers also include charged linkers and their hydrophilic forms as described herein and as well 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 samples containing high levels of CD123 expression. CD123 may be elevated, increased, or overexpressed compared to control levels (e.g., expression levels in biological samples, tissues, or cells from non-cancer subjects, samples or cancers known to not express CD123 / IL-3Rα or to have low levels of its expression, or normal samples or cancers that do not have elevated CD123 / IL-3Rα levels). For example, samples with increased expression (e.g., from hematological malignancies such as leukemia and lymphoma) may include increases of at least 2, 3, 4, 5, 10, 15, 20, 25, 30, or at least 50 times compared to control / normal levels.

[0135] A “reference sample” can be used to correlate and compare results obtained from a test sample using the method of the present invention. The reference sample may be cells (e.g., cell line, cell pellet) or tissue. The CD123 / IL-3Rα level of the “reference sample” may be the absolute or relative amount, range of amounts, minimum and / or maximum amount, average amount, and / or median amount of CD123 / IL-3Rα. The “reference sample” can also serve as a baseline for CD123 / IL-3Rα expression on which the test sample is compared. The “reference sample” may include a prior 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. The CD123 / IL-3Rα level may also be expressed as a standard curve value. A standard curve is a quantitative test method that plots assay data to measure the CD123 / IL-3Rα concentration of a sample. In one embodiment, the reference sample is purified CD123 / IL This is an antigen standard containing -3Rα. The diagnostic method of the present invention may 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 may be a predetermined value and may be measured from a reference sample (e.g., a control biological sample or reference sample) tested together with the test sample. The reference value may be a single cutoff value such as the median or mean, or a range of values ​​such as a confidence interval. The reference value may be set for various individual subgroups.

[0136] In this specification, 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 the IHC procedure.

[0137] In this specification, the term “secondary antibody” refers to an antibody that specifically binds to a primary antibody, thereby forming a crosslink or bond, if any, between the primary antibody and a subsequent reagent. A secondary antibody is generally the second antibody used in an immunohistochemical procedure.

[0138] The “sample” or “biological sample” of the present invention is of biological origin, and in certain embodiments, for example, of eukaryotic origin. In some embodiments, the sample is a human sample, but animal samples may also be used. Non-limited 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 urogenital 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 described above can be used to test the expression patterns or state of a sample, including, but not limited to, comparing different types of cells or tissues, comparing different developmental stages, 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 capable of binding to and capturing a target molecule in a sample so that, under appropriate conditions, the capture reagent-target molecule complex can be separated from the rest of the sample. In one embodiment, the capture reagent is immobilized. In one embodiment, the capture reagent in a sandwich immunoassay is an antibody against a target antigen or a mixture of different antibodies.

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

[0141] As used herein, the term “detection means” refers to a portion or technique used to detect the presence of a detectable antibody, and includes a detection agent for amplifying an immobilized label (e.g., a label captured on a microtiter plate). In one embodiment, the detection means is a fluorescence quantitative detection agent (e.g., avidin or streptavidin).

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

[0143] As used herein, the term “labeled” refers to a detectable compound or composition that is directly or indirectly conjugated to an antibody in order to create a “labeled” antibody. The substance itself may be detectable (e.g., by radioactive isotope labeling or fluorescent labeling), or, in the case of enzyme labeling, it may catalyze a chemical change in a detectable substrate compound or composition.

[0144] "To correlate" or "to be correlated" means, in any case, to compare the performance and / or results of a first analysis with the performance and / or results of a second analysis. For example, the results of a first analysis can be used in conducting a second analysis, and / or to determine whether a second analysis should be performed, and / or the results of a first analysis can be compared with the results of a second analysis. In one embodiment, increased expression of CD123 / IL-3Rα correlates with an increased likelihood that CD123 / IL-3Rα targeted therapy will be effective.

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

[0146] Examples of cancers include lymphoma and leukemia. 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 antibody, its antigen-binding fragment, or its immune complex) include AML, CML, ALL (e.g., B-ALL), CLL, myelodysplastic syndrome, blastic plasmacytoid DC tumor (BPDCN) leukemia, non-Hodgkin lymphoma (NHL), progenitor B-cell lymphoblastic leukemia / lymphoma and mature B-cell tumors (e.g., B-cell chronic lymphocytic leukemia (B-CLL) / small lymphocytic lymphoma (SLL), B-cell prelymphocytic leukemia). Examples include B-cell lymphomas, lymphoplasmacytic lymphomas, mantle cell lymphoma (MCL), follicular lymphomas (FL) including low-grade, intermediate-grade and high-grade FL, cutaneous follicular central lymphoma, marginal zone B-cell lymphomas (MALT type, nodal and splenic marginal zone), pilocytic cell leukemia (HCL), diffuse large B-cell lymphoma, Burkitt lymphoma, plasmacytoma, plasmacytomyeloma, post-transplant lymphoproliferative disorders, Waldenström macroglobulinemia, anaplastic large cell lymphoma (ALCL), and Hodgkin's leukemia (HL).

[0147] Cancer includes cancers that contain cells with increased CD123 / IL-3Rα expression levels. 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 cells,” “tumor cells,” and grammatically equivalent terms refer to the entire cell population derived from a tumor or precancerous lesion, including non-tumor-forming cells (which include many tumor cell populations) and oncogeneic stem cells (cancer stem cells). As used herein, the term “tumor cells” is modified by the term “non-tumor-forming” when it simply refers to tumor cells that lack the ability to regenerate and differentiate to be distinguished from cancer stem cells.

[0149] The term "subject" refers to any animal (e.g., mammal) that is the recipient of a particular treatment, including but not limited to humans, non-human primates, and rodents. In this specification, the terms "subject" and "patient" are generally used interchangeably when referring to human subjects.

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

[0151] The term "pharmaceutical preparation" refers to a preparation that is in a form that produces the biological activity of an active ingredient and does not contain additional ingredients that are unacceptably toxic to the person to whom the preparation is administered. Yes. Such preparations can be sterile.

[0152] The “effective amount” of the antibody or immune complex disclosed herein is an amount sufficient to perform the specific purpose described. The “effective amount” can be determined empirically and conventionally with respect to the purpose described.

[0153] The term “therapeutic dose” refers to the amount of an antibody or other drug that is effective in “treating” a disease or disorder in a subject or mammal. In the case of cancer, a therapeutic dose of a drug may reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow to some extent and halt in certain embodiments) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent and halt in certain embodiments) tumor metastasis; inhibit tumor growth to some extent; alleviate to some extent one or more symptoms associated with cancer, and / or result in 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 possibly 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 that a drug can prevent growth and / or kill existing cancer cells, it may be cell proliferation inhibitory and / or cytotoxic.

[0154] "Prophylactically effective dose" refers to the effective dose required for the desired prophylactic outcome and for the required duration. Generally, but not necessarily, because prophylactic doses are used for subjects before or in the early stages of disease, the prophylactically effective dose is less than the therapeutically effective dose.

[0155] The term "responds favorably" generally refers to the occurrence of a favorable state in the subject. In the context of cancer treatment, the term refers to providing a therapeutic effect to the subject. Positive therapeutic effects of cancer can be measured in various ways (see WAWeber, J.Nucl.Med. 50:1S-10S (2009)). For example, tumor growth inhibition, molecular marker expression, serum marker expression, and molecular imaging techniques can all be used to evaluate the therapeutic effectiveness of anti-cancer treatments. Regarding tumor growth inhibition, according to NCI standards, T / C < 42% is the lowest level of antitumor activity. In T / C (%) = median treated tumor volume / median control tumor volume × 100, T / C < 10% is considered a high level of antitumor activity. A favorable response can be evaluated, 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 according to 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 generally measured using the Kaplan-Meier method and the Response Evaluation Criteria in Solid Tumors (RECIST) 1.1. Generally, progression-free survival refers to the period during which a patient survives without further progression of their cancer.

[0158] "Complete response," "complete remission," or "CR" refers to the disappearance of all signs of a tumor or cancer that has responded to treatment. This does not necessarily mean that the cancer is 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, that responds to treatment.

[0160] "Stable disease" refers to a disease that does not progress or recur. In a stable disease, there is neither sufficient tumor reduction to be considered a partial response nor sufficient tumor growth to be considered a progressive disease.

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

[0162] "Disease-free survival" (DFS) refers to the period during and after treatment when a patient is disease-free.

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

[0164] "Chemotherapy drugs" are chemical compounds useful in cancer treatment, regardless of their mechanism of action. The terms "to treat," "to cure," "to treat," or "to alleviate" refer to therapeutic means that treat, reduce, or halt the progression of a diagnosed condition or disorder. Therefore, those requiring treatment include those who have already been diagnosed with the disease and may also include those with minimal residual disease, resistant lesions, or replacement lesions. In a particular embodiment, if a patient exhibits one or more of the following: a reduction in the number of cancer cells or their complete absence; a reduction in tumor size; inhibition or absence of cancer cell infiltration into peripheral organs, including, for example, the spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduction of one or more symptoms associated with a particular cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity, tumorigenicity, or tumorigenic capacity of the tumor; a reduction in the number or expression frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumoric 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), progressive disease (PD), time to progression (TTP), or any combination thereof, the subject is considered to have been successfully "treated" for cancer by the method of the present invention.

[0165] Preventive measures or precautionary measures mean means that prevent and / or delay the onset of a target condition or disease. Therefore, those who require preventive measures or precautionary measures include those who are susceptible to the disease and those who must prevent the disease.

[0166] Preventive measures or preventive measures mean therapeutic measures that prevent and / or delay the onset of a target condition or disease. Therefore, those who require preventive measures or preventive measures include those who are susceptible to the disease and those who must prevent the disease.

[0167] As used herein, the term “healthcare worker” refers to an individual or institution that has direct contact with and provides assistance to a living person (e.g., a human patient). Examples of healthcare workers, in no particular order, include physicians, nurses, technicians, therapists, pharmacists, counselors, alternative medicine professionals, medical facilities, clinics, hospitals, emergency rooms, clinics, emergency treatment centers, alternative medicine clinics / facilities, and any other entity that provides general and / or specialized care, assessment, maintenance, therapy, pharmacotherapy, and / or advice to all or any part of a patient’s condition, including but not limited to general, specialized, surgical, and / or any other type of treatment, assessment, maintenance, therapy, pharmacotherapy, and / or advice.

[0168] In some embodiments, a healthcare professional may manage or direct another healthcare professional administering treatment to treat cancer. As used herein, “application” of treatment includes prescribing treatment to a subject, and adding, applying, or giving treatment to a subject. Healthcare professionals may perform the following actions: obtain a sample, process a sample, submit a sample, receive a sample, transport a sample, analyze or measure a sample, quantify a sample, provide results obtained after analyzing / measuring / quantifying a sample, receive results obtained after analyzing / measuring / quantifying a sample, compare / score results obtained after analyzing / measuring / quantifying one or more samples, provide comparisons / scores from one or more samples, obtain comparisons / scores from one or more samples, apply a treatment or therapeutic agent (e.g., CD123 / IL-3Rα conjugate), initiate the application of a treatment, stop the application of a treatment, continue the application of a treatment, temporarily suspend the application of a treatment, increase the dose of a therapeutic agent, decrease the dose of a therapeutic agent, continue administering a dose of a therapeutic agent, increase the frequency of administration of a therapeutic agent, decrease the frequency of administration of a therapeutic agent, maintain the same dose frequency of a therapeutic agent, replace a treatment or therapeutic agent with at least one other treatment or therapeutic agent, or combine a treatment or therapeutic agent with at least one other treatment or additional therapeutic agent, or have another healthcare professional or patient perform or instruct another healthcare professional or patient to do so. These procedures can be performed automatically by healthcare professionals using computer-based methods (e.g., via web services or standalone computer systems).

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

[0170] The term "vector" refers to a structure capable of delivering and expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensers, liposome-encapsulated DNA or RNA expression vectors, and certain eukaryotic cells (e.g., producing cells).

[0171] The terms “polypeptide,” “peptide,” and “protein” are used herein to mean the same thing to refer to polymers of amino acids of any length. The polymers may be linear or branched, and they may contain modified amino acids and may be interposed by non-amino acids. The terms also include naturally or indirectly modified amino acid polymers (e.g., disulfide bond formation, glycosylation, lipidization, acetylation, phosphorylation, or any other operation or modification (e.g., covalent bonding with a labeling component)). Furthermore, polypeptides containing one or more analogues of amino acids (including, for example, non-natural amino acids), and other modifications well known in the art, are also included within the definitions. Since the polypeptides of the present invention are antibody-based, it is understood that in certain embodiments, polypeptides may arise as single chains or accompanying chains. In some embodiments, polypeptides, peptides, or proteins are of non-natural origin. In some embodiments, polypeptides, peptides, or proteins are purified from other naturally occurring components. In some embodiments, polypeptides, peptides, or proteins are produced by recombinant means.

[0172] With respect to two or more nucleic acids or polypeptides, "identical" or "identity" % means that when compared and aligned for maximum agreement (with gaps introduced as necessary), without considering any conserved amino acid substitutions as part of sequence identity, two or more sequences or subsequences are identical or have a certain percentage of identical nucleotides or amino acid residues. Identity % can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software available for obtaining amino acid or nucleotide sequence alignments are well known in the art. One such unrestricted 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) is an additional publicly available software program that can be used to align sequences. In certain embodiments, identity % between two nucleotide sequences can be determined using the GAP program of GCG software (e.g., NWSgapdna.CM). The P matrix, along with gap weights of 40, 50, 60, 70, or 90 and length weights of 1, 2, 3, 4, 5, or 6, is used to measure the identity % between two amino acid sequences (e.g., using either the Blossum62 matrix or the PAM250 matrix, along with 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, the identity % between nucleotides or amino acid sequences is measured using the Myers and Miller algorithm (CABIOS, 4:11-17, 1989). For example, identity % can be measured using the ALIGN program (version 2.0) and a residue table with a gap length penalty of 12 and a gap penalty of 4, and PAM120. Appropriate parameters for maximum sequencing by specific sequencing software can be determined by those skilled in the art. In specific embodiments, the default parameters of the sequencing software are used. In specific embodiments, the identity % "X" of the first amino acid sequence to the second amino acid sequence is calculated as 100 × (Y / Z), where Y is the number of amino acid residues recorded as a perfect match in the alignment of the first and second sequences (aligned by visual inspection or a specific 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 identity % of the first sequence to the second sequence is greater than the identity % of the second sequence to the first sequence.

[0173] As a non-limiting example, whether a particular polynucleotide has a certain percentage of sequence identity with respect to a reference sequence (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) can be determined in certain embodiments using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI53711). Bestfit uses the Smith and Waterman local homology algorithm (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 measure whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and that a homology gap of up to 5% of the total number of nucleotides in the reference sequence is permitted.

[0174] In some embodiments, two nucleic acids or polypeptides of the present invention are substantially identical, meaning that when compared and aligned for maximum match, as measured using a sequence comparison algorithm or by visual inspection, 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%, and 99% nucleotide or amino acid residue identity. In certain embodiments, the identity exists over a range of sequences whose length is at least about 10, about 20, about 40–60 residues or any integer value between them, or over a region longer than 60–80 residues, at least about 90–100 residues, or the sequences are substantially identical over the full length of the sequences being compared (e.g., the coding region of a nucleotide sequence).

[0175] A "conservative amino acid substitution" is a substitution in which one amino acid residue is replaced by another amino acid residue with a similar side chain. A group of amino acid residues with similar side chains is the group in question. These are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), unloaded electrode side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, the substitution of phenylalanine for tyrosine is a conservative substitution. In certain embodiments, conservative substitutions in the polypeptide and antibody sequences of the present invention do not inhibit the binding of the amino acid sequence-containing polypeptide or antibody to the antigen(s), i.e., to CD123 / IL-3Rα to which the polypeptide or antibody binds. Methods for confirming conserved nucleotide and amino acid substitutions that do not exclude antigen binding are well known in the art (see, for example, 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] P-glycoprotein 1, also known as permeable glycoprotein, P-gp or Pgp, multidrug resistance protein 1 (MDR1), ATP-binding cassette subfamily B member 1 (ABCB1), or differentiation antigen group 243 (CD243) as used herein, is an ABC transporter of the MDR / TAP subfamily that transports a wide variety of substrates across the extracellular and intracellular membranes. It is an ATP-dependent efflux pump with broad substrate specificity. P-gp is widely distributed and expressed in the intestinal epithelium, which delivers xenobiotics (e.g., toxins or drugs) back into the intestinal lumen, in hepatocytes, which deliver them into the bile ducts, in cells of the proximal tubules of the kidney, which deliver them into the urethral ducts, and in capillary endothelial cells, including those at the blood-brain barrier and blood-testis barrier, which deliver 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" means a saturated linear or branched monovalent hydrocarbon group having 1 to 20 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl, 2-methyl-1-propyl, -CH2CH(CH3)2, 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, and 1-octyl. Preferably, alkyl groups have 1 to 10 carbon atoms. More preferably, the alkyl group has 1 to 4 carbon atoms.

[0178] The number of carbon atoms in the group is determined by the prefix "C". x-xx This specification may specify by ", in which case x and xx are integers. For example, "C 1-4 "Alkyl" refers to an alkyl group that has 1 to 4 carbon atoms.

[0179] The terms “compound” and “cytotoxic compound” are used interchangeably. These terms aim to include compounds in which a structure or formula, or any derivative thereof, is disclosed in the present invention, or into which a structure or formula, or any derivative thereof, is incorporated by reference. The terms also include stereoisomers, geometric isomers, tautomers, solvates, metabolites, and salts (e.g., pharmaceutically acceptable salts) of all compounds of the formulas disclosed in the present invention. The terms also include any solvates, hydrates, and polymorphs of any of the foregoing. The specific detailed descriptions of “stereoisomers,” “geometric isomers,” “tautomers,” “solvates,” “metabolites,” “salts,” “complexes,” “hydrates,” or “polymorphs” of the particular aspects of the Invention described herein should not be construed as an intentional omission of these forms of other aspects of the Invention, where the term “compound” is used without detailed descriptions of these other forms.

[0180] The term "chiral" refers to a molecule that cannot be superimposed on its mirror image partner, while the term "achiral" refers to a molecule that can be superimposed on its mirror image partner.

[0181] The term "stereoisomer" refers to compounds that have the same chemical structure and bonding properties, but differ in the spatial arrangement of atoms, and cannot be interconverted by rotation around a single bond.

[0182] A "diastereomer" refers to a stereoisomer that has two or more chirality centers, and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting point, boiling point, spectral characteristics, and reactivity. Mixtures of diastereomers can be separated using high-resolution analytical techniques such as crystallization, electrophoresis, and chromatography.

[0183] An "enantiomer" refers to two stereoisomers of a compound that are mirror images of each other and cannot be superimposed.

[0184] The definitions and conventions of stereochemistry used herein generally follow those of SP. Parkered., 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 as various stereoisomers. All stereoisomers of the compounds of the present invention, including diastereomers, enantiomers, and atropisomers, as well as mixtures thereof such as 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 optically active compounds, the prefixes D and L, or R and S, are used to indicate the absolute stereochemistry of the molecule with respect to its chiral center(s). The prefixes d and I, or (+) and (-), are used to indicate the rotation of plane polarization by a compound, with (-) or l meaning the compound is levorotatory. Compounds prefixed with (+) or d are dextrorotatory. In a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. Certain stereoisomers are sometimes called enantiomers, and mixtures of such isomers are often called enantiomer mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemic compound, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemic compound” refer to equimolar mixtures of two enantiomer species that lack optical activity.

[0185] The term "tautomer" or "tautomer" refers to structural isomers of different energies that are interconvertible across low-energy barriers. For example, proton tautomers (also known as prototropic tautomers) involve interconversion via the transfer of protons (e.g., keto-enol and imine-enamine isomerization). Valence tautomers involve interconversion via the rearrangement of several bonding electrons.

[0186] The term "imine-reactive reagent" refers to a reagent that can react with an imine group. Examples of imine-reactive reagents include sulfites (H2SO3, H2SO2, or HSO3 formed by a cation). - , SO3 2- Or HSO2 - (salt of), metabisulfite (H2S2O5, or S2O5 formed by a cation) 2- Salts of PO3S, mono, di, tri and tetra-thiophosphates (PO3SH3, PO2S2H3, POS3H3, PS4H3, and PO3S formed with cations) 3- , PO2S2 3- POS3 3- Or PS4 3- (Salt of), thiophosphate ester (R i O)2PS(OR i ), R i SH, R i SOH, 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 by cations) 2- (salt of), dithionite (H2S2O4, or S2O4 formed by a cation) 2- (Salt of), phosphorodithioate (P(=S)(OR k (SH)(OH), or salts formed with cations), hydroxamic acid (R kC(=O)NHOH, or a salt formed with a cation), hydrazide (R k CONHNH2), formaldehyde sulfoxylate (HOCH2SO2H, or HOCH2SO2 - Na + HOCH2SO2 is formed by cations such as these. - Examples include, but are not limited to, salts of, glycated nucleotides (e.g., GDP-mannose), fludarabine, or mixtures thereof, where R i and R i ' is a linear or branched alkyl group each independently having 1 to 10 carbon atoms, and -N(R J ) substituted with at least one substituent selected from -CO2H, -SO3H, and -PO3H; R i and R i’ Furthermore, it may be optionally substituted with alkyl substituents as described herein; R j R is a linear or branched alkyl group having 1 to 6 carbon atoms; 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 linear or branched alkyl having 1 to 4 carbon atoms, more preferably R k (The cation is methyl, ethyl, or propyl). Preferably, the cation is Na + or K + These are monovalent cations such as . Preferably, the imine-reactive reagent is selected from sulfites, hydroxylamines, urea, and hydrazine. More preferably, the imine-reactive reagent is NaHSO3 or KHSO3.

[0187] As used herein, the term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the present invention. Examples of salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbic acid, succinates, maleates, gentians, fumarates, glucons, glucurons, sucroses, formates, benzoates, glutamates, methanesulfons, "mesylates," ethanesulfons, benzenesulfons, p-toluenesulfons, pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmacopoeia-acceptable salts may include the inclusion of other molecules, such as acetate ions, succinate ions, or other counterions. The counterion may be an organic or inorganic part that stabilizes the charge in the parent compound. Furthermore, pharmaceutically acceptable salts may have two or more charged atoms in their structure. Examples of pharmaceutically acceptable salts where multiple charged atoms are part of the salt may have multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

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

[0189] If 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), alkali metal hydroxide, or alkaline earth metal hydroxide. 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” means a compound further comprising 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.) bonded by non-covalent intermolecular forces. The solvate or hydrate of a compound is immediately prepared by adding at least 1 molar equivalent of a hydroxyl solvent (e.g., methanol, ethanol, 1-propanol, 2-propanol, or water) to the compound, resulting in the solvation or hydration of the imine moiety.

[0191] A “metabolite” or “catabolic product” is a product created by the metabolism or catabolism of a particular compound, its derivative, or its complex, or its salt, within the body. Metabolites of a compound, its derivative, or its complex 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 may be produced, for example, from oxidation, hydroxylation, reduction, hydrolysis, amidation, amidation, esterification, esterification, enzymatic cleavage, etc. of the administered compound. Accordingly, the present invention includes metabolites of a compound, its derivative, or its complex, which are prepared by a method comprising contacting the compound, its derivative, or its complex of the present invention with a mammal for a period of time sufficient to obtain the metabolite.

[0192] The term "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammals it treats.

[0193] The term “protecting group” or “protecting moiety” refers to a substituent commonly used to react with other functional groups on a compound, its derivatives, or complexes, and to block or protect specific functional groups. For example, an “amine protecting group” or “amino protecting moiety” is a substituent bonded to an amino group that blocks or protects an amino functional group of a 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, FMOCs, substituted ethyl carbamates, 1,6-β elimination (also called “self-destructing”), and carbamates cleaved by urea, amides, peptides, alkyl and aryl derivatives). Preferred amino protection The protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and 9-fluorenylmethyleneoxycarbonyl (Fmoc). For a general description of protecting groups and their use, see PGMWuts & T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 2007.

[0194] The term "amino acid" refers to amino acids of natural or non-natural origin. In one embodiment, the amino acid is NH2-C(R aa’ R aa It is expressed as )-C(=O)OH, where R aa and R aa’ Each of these is independently a linear, branched, or cyclic alkyl, alkenyl, or alkynyl, aryl, heteroaryl, or heterocyclyl, each having H, 1 to 10 carbon atoms, or R aa The N-terminal nitrogen atom can form a heterocycle together (for example, in proline). The term "amino acid residue" refers to a single hydrogen atom in an amino acid (e.g., -NHC(R). aa 'R aa When removed from the amine and / or carboxyl terminus of )-C(=O)O-), it refers to the corresponding residue.

[0195] The term "cation" refers to an ion with a positive charge. A cation is monovalent (for example, Na). + , K + NH4 + (e.g.), divalent (for example, Ca 2+ Mg 2+ (e.g., Al) or polyvalent (e.g., Al 3+ (and so on). Preferably, the cation is monovalent.

[0196] The term "reactive ester group" refers to an ester group that can react immediately with an amine group to form an amide bond. Typical reactive ester groups include, but are not limited to, N-hydroxysuccinimide esters, N-hydroxyphthalimide esters, N-hydroxysulfosuccinimide esters, p-nitrophenyl esters, dinitrophenyl esters, pentafluorophenyl esters, and their derivatives, the derivatives of which promote amide bond formation. In certain embodiments, the reactive ester group is N-hydroxysuccinimide ester or 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. Typical amine-reactive groups include, but are not limited to, reactive ester groups, acyl halides, sulfonyl halides, imide esters, or reactive thioester groups. 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-hydroxysulfosuccinimide ester.

[0198] The term "thiol-reactive group" refers to a group that can react with a thiol (-SH) group to form a covalent bond. Typical 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] Where used in this disclosure and in these claims, the singular forms "a," "an," and "the" include the plural forms, unless otherwise stated.

[0200] Whenever an embodiment is described herein in the words “including,” it is understood that other similar embodiments described in the words “consisting of” and / or “essentially consisting of” are also provided.

[0201] In this specification, the term "and / or" as used 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 In this specification, the names used for anti-CD123 antibodies, cytotoxic compounds, and their immune complexes 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 mouse 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, along with the corresponding SEQ ID NOs. 1-25. The heavy chain variable region (HCVR) sequences are provided in Table 3A, along with the corresponding SEQ ID NOs. 26, 28, and 30. Their light chain variable region (LCVR) sequences are provided in Table 4A, along with the corresponding SEQ ID NOs. 27, 29, and 31. The full-length heavy chain (HC) sequences of the mouse antibodies are provided in Table 5 (SEQ ID NOs. 42, 44, and 46), and the full-length light chain (LC) sequences of the mouse 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 possessing mouse heavy and light chain variable regions and human constant region sequences. For example, the chimeric antibody chCD123-6 consists of mouse HCVR and LCVR of SEQ ID NOs. 28 and 29, respectively, along with human IgG1 and κ constant sequences in the heavy and light chains. See Example 3.

[0205] huCD123-3, -6, and -14 are the corresponding humanized antibodies. When humanization is performed by CDR transplantation of six corresponding mouse CDR regions (HC and LC CDR1-3), the letter "G" follows immediately after the clonal designation, followed by a version number indicating the origin of the human light chain and heavy chain variable region sequences. Therefore, huCD123-6Gv4.6 means a humanized CD123 antibody based on the transplantation ("G") of six CDR regions onto the human light chain variable region Gv4 and heavy chain variable region Gv6 from the corresponding muCDR123-6 antibody. 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 (sequences 32 and 34, where sequence 34 differs only in the second residue Xaa, thus representing -Gv6 and -Gv7), and their DNA coding sequences are provided in Table 3B (sequences 62, 64, and 66). Three full-length HC sequences, huCD123-6Gv1, -Gv6, and -Gv7, are provided in Table 5 (sequences 48 and 50, where sequence 50 differs only in the second residue Xaa, thus representing 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 performed by resurfacing, the resurfacing heavy chain sequence is denoted by "rh" immediately following the mouse CD123 antibody clone number, and is further denoted by one of two versions of the resurfacing sequence, v1.0 or v1.1. Therefore huCD123-6rhv1.0 and -rhv1.1, along with their version designations 1.0 and 1.1 respectively, are resurfaced heavy chain sequences containing the CDR region corresponding to the muCD123-6 antibody. See HCVR SEQ ID NOs. 39 and 40 in Table 3A, and SEQ ID NOs. 68 and 69 in Table 3B. Also see 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 sequence number 41 in Table 4A and full-length LC sequence number 61 in Table 6.

[0210] The resurface-forming antibody possessing huCD123-6rhv1.0 and huCD123-6rlv1.0 is huCD123-6Rv1.0, and the resurface-forming antibody possessing huCD123-6rhv1.1 and huCD123-6rlv1.0 is huCD123-6Rv1.1.

[0211] NTS2, or simply "S2," refers to an antibody that has a modified ser at the N-terminus of its heavy chain. The S2 variant of huCD123-6Gv6 / 7 has the HCVR sequence shown in Table 3A, SEQ ID NO: 38, and the full-length HC protein sequence shown in Table 5, SEQ ID NO: 53.

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

[0213] Antibodies containing an engineered N-terminal ser (S2 or S3) can be covalently bound by cytotoxic drugs / agents via oxidized N-terminal ser or "conventional" Lys binding. When drug binding is via oxidized N-terminal ser, the name of the complex includes the notation "SeriMab". When drug binding is via Lys, the name of the complex does not include SeriMab (despite the fact that there is an S2 or S3 notation indicating the presence of an engineered N-terminal ser). The specific type of binding is also evident from the cytotoxic reactive group. For example, huCD123-6Gv4.7S3-SeriMab-D8 refers to a complex between D8 and the humanized CD123 antibody huCD123-6Gv4.7S3, via an oxidized N-terminal ser on the light chain. The humanized CD123 antibody has a transplanted mouse CD123-6CDR region, human LC Gv4 and heavy chain Gv7, and the N-terminus of the light chain has an engineered ser (S3). In contrast, huCD123-6Gv4.7S3-sSPDB-D1 refers to a complex between huCD123-6Gv4.7S3, the same humanized CD123 antibody as D1, and huCD123-6Gv4.7S3, which is linked by Lys binding via a sulfonated SPDB linker.

[0214] In certain embodiments, if both the light chain and the heavy chain N-terminus contain manipulated Ser, the antibody name may appear as "S2S3" or "S2S3-SeriMab".

[0215] The specific antibodies of the present invention have an engineered Cys gene in the heavy chain CH3 domain at a position corresponding to the same Kabat position as the fifth-to-last Cys gene in SEQ ID NO: 54. Such HCs, or antibodies containing such HCs, are denoted as CysMab. Thus, huCD123-6Gv4.6-CysMab is a humanized CD123 antibody transplanted with the muCD123-6CDR region, and based on the human light chain Gv4 and heavy chain Gv6 sequences, the engineered Cys gene is located in the HC CH3 domain at a position corresponding to the fifth-to-last Cys gene in SEQ ID NO: 54. Similarly, its heavy chain sequence is huCD123-6Gv6-CysMab. Furthermore, 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 resurfacing antibodies described above may be further modified to contain either a light chain (S3 variant of the resurfacing antibody), a heavy chain (S2 variant of the resurfacing antibody), or both (see below) as the N-terminal ser. Alternatively, the resurfacing antibody may have a modified Cys at the same Kabat position corresponding to the fifth-to-last Cys in SEQ ID NO: 54 (CysMab version of the resurfacing antibody). The resurfacing antibody may have both the modified Cys and the N-terminal ser.

[0217] However, in the complex formed between such a CysMab and cytotoxicity, the cytotoxicity can, at least theoretically, bind to the CysMab by Cys or conventional Lys. However, as used herein, unless otherwise specified, the complex with the CysMab designation refers to the complex between CysMab and cytotoxicity, even if it is a Cys bond (and not a Lys bond). The specific type of binding is also evident based on the cytotoxic reactive group.

[0218] Other variations or combinations of the above-mentioned general nomenclature have also been considered and will be readily apparent to those skilled in the art. For example, huCD123-6Rv1.1-CysMab is a resurface-formed version of huCD123-6(v1.1) having an operated Cys located in the HC CH3 region at the position corresponding to the fifth-to-last Cys of sequence number 54.

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

[0220] It should be noted that several cytotoxins differ only slightly due to the different binding chemistry required to bind them to different antibody side chains (i.e., Lys bonds, Cys bonds, oxidized N-terminal Ser bonds). Nevertheless, these related cytotoxins are assigned different "D" designations. See D1 and D4, as well as D2, D5, and D8.

[0221] The conjugate of the target antibody and cytotoxic agent generally follows the names of the antibody and cytotoxic agent as described above.

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

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

[0224] 2. CD123 binder 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 conjugates.” In certain embodiments, the CD123 / IL-3Rα conjugate is an antibody or its antigen-binding fragment (or simply “antibody”), its immune complex, or its polypeptide.

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

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

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

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

[0229] Such sequences from non-human species can be aligned with human sequences using one of the many sequence alignment tools approved in the art (e.g., those described herein and above), thereby easily obtaining any amino acid residue or nucleotide that "corresponds" to any given human sequence or region of a sequence.

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

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

[0232] In certain embodiments, CD123 / IL-3Rα conjugates (e.g., antibodies) inhibit IL-3-dependent signaling (e.g., IL-3-dependent proliferation of CD123-positive TF-1 cells). While not bound by any particular theory, the CD123 / IL-3Rα conjugates (e.g., antibodies) of the present invention bind to CD123, such as within the CD123 region corresponding to residues 101-346 (e.g., residues 101-204, or 205-346) of human CD123 / IL-3Rα, and productive binding between CD123 and IL-3 ligand and / or This prevents, reduces, weakens, or inhibits productive binding between CD123 and the common β-chain CD131, resulting in a decrease or elimination of IL-3-dependent signaling.

[0233] In a related embodiment, the present invention (a) binds an epitope within amino acids 1 to 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 its antigen-binding fragment that inhibits IL3-dependent proliferation of antigen-positive TF-1 cells having a specific value.

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

[0235] Inhibition of cell proliferation can be carried out using any standard assay well 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 differences in the expression of cell surface markers, and the relative number of viable or residual cells can be quantitatively measured and compared after incubation with the test agent.

[0236] Inhibition of cell proliferation in LSCs, LPs, or leukemic blasts compared to normal HSCs can also be measured using in vitro efficacy assays of primary cancer cells (e.g., primary AML cells). For example, AML cells (or a normal human bone marrow sample containing normal HSCs) can be exposed for 24 hours to various concentrations of a target anti-CD123 antibody, its antigen-binding fragment, its immune complex, or a polypeptide containing the antibody or antigen-binding fragment. Untargeted (isotype-matched) antibodies or immune complex (ADC) controls can also be used in the assay. Samples can be divided into short-term liquid culture (STLC) assays to measure cytotoxicity to LSCs, LPs, or leukemic blasts, and then divided into long-term liquid culture (LTLC) assays to measure the effects on LSCs and normal HSCs. Using STLC, colony-forming units can be measured for 10–14 days in cells after seeding, for example, in semi-solid MethoCult H4230 medium (Stemcell Technologies). The LTLC assay can be similarly performed by adding growth factors during a long-term culture of 5–7 weeks. In both assays, the number of colonies can be counted to measure the colony-forming units per initially seeded cell. LTLC colonies were further analyzed for the presence of molecular markers for cancer (e.g., AML) using PCR, FISH, or both.

[0237] In certain embodiments, the antibody or its antigen-binding fragment has a dissociation constant (K) of 0.3 nM or less. d) binds to human CD123 antigen-positive cells. In certain embodiments, the antibody or its antigen-binding fragment has a K content of 0.05nM to 0.3nM, or 0.05nM to 0.2nM, or 0.05nM to 0.1nM, or 0.01nM to 0.3nM, or 0.01nM to 0.2nM, or 0.01nM to 0.1nM. d Then, it binds to human CD123.

[0238] In certain embodiments, the antibody or its antigen-binding fragment binds to cynomolgus monkey CD123. In certain embodiments, the antibody or its antigen-binding fragment has a K content 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 Then, it binds to the cynomolgus macaque CD123.

[0239] In certain embodiments, the antibody or its antigen-binding fragment binds to both human and cynomolgus monkey CD123 with substantially similar binding affinities. In certain embodiments, the antibody or its antigen-binding fragment has a K content 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 It binds to both human and cynomolgus monkey CD123.

[0240] In a particular embodiment, K d The values ​​are based on cell-based binding assays. In specific embodiments, K d The value is measured by flow cytometry. In certain embodiments, K d The value is measured by surface plasmon resonance (e.g., using a BIOCORE® surface plasmon resonance system). In certain embodiments, K d The value is measured by radioimmunoassay (RIA). In certain embodiments, K d This is measured by any other method approved in the relevant technical field.

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

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

[0243] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are a) at least one heavy chain variable region or fragment thereof comprising three consecutive 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: 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 at least one heavy chain variable region. A light chain variable region or a fragment thereof, and b) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein, excluding 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 a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are a) at least one heavy chain variable region or fragment comprising three consecutive 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: 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. a) a heavy chain variable region or fragment thereof, and b) at least one light chain variable region or fragment thereof, each comprising three consecutive complementarity-determining regions (CDRs) CDR1, CDR2, and CDR3, wherein, excluding 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 substitutions include the substitution of at least one Lys in the CDR by Arg (e.g., the Lys-for-Arg substitutions in SEQ ID NOs. 6 and 7, 8 and 9, and 19 and 20). In certain embodiments, the antibody comprises a CDR-implanted humanized antibody containing a mouse CDR region, with one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) heavy chains and / or Alternatively, the light chain framework region vernier zone residues are derived from mouse.

[0246] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include: a) an immunoglobulin heavy chain variable region comprising CDR1 having the amino acid sequence described in SEQ ID NO: 1, CDR2 having the amino acid sequence described in SEQ ID NO: 2 or 3, and optionally CDR3 having the amino acid sequence described in SEQ ID NO: 4; and 2) an immunoglobulin light chain variable region comprising CDR1 having the amino acid sequence described in SEQ ID NO: 16, CDR2 having the amino acid sequence described in SEQ ID NO: 17, and optionally CDR3 having the amino acid sequence described 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, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include: a) an immunoglobulin heavy chain variable region comprising CDR1 having the amino acid sequence described in SEQ ID NO: 5, CDR2 having the amino acid sequence described in SEQ ID NO: 6, 7, 8, 9, or 10, and optionally CDR3 having the amino acid sequence described in SEQ ID NO: 11; and 2) an immunoglobulin light chain variable region comprising CDR1 having the amino acid sequence described in SEQ ID NO: 19 or 20, CDR2 having the amino acid sequence described in SEQ ID NO: 21, and optionally CDR3 having the amino acid sequence described 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, the heavy chain variable region CDR2 is sequence number 9, and the light chain variable region CDR1 is sequence number 19. In certain embodiments, the heavy chain variable region CDR2 is sequence number 10, and the light chain variable region CDR1 is sequence number 19. In certain embodiments, the heavy chain variable region CDR2 is sequence number 6, and the light chain variable region CDR1 is sequence number 20. In certain embodiments, the heavy chain variable region CDR2 is sequence number 7, and the light chain variable region CDR1 is sequence number 20. In certain embodiments, the heavy chain variable region CDR2 is sequence number 8, and the light chain variable region CDR1 is sequence number 20. In certain embodiments, the heavy chain variable region CDR2 is sequence number 9, and the light chain variable region CDR1 is sequence number 20. In certain embodiments, the heavy chain variable region CDR2 is sequence number 10, and the light chain variable region CDR1 is sequence number 20. In the paired combinations of heavy chain variable region CDR2 and light chain variable region CDR1 described above, heavy chain variable regions CDR1 and CDR3 are sequence numbers 5 and 11, respectively, and light chain variable regions CDR2 and CDR3 are sequence numbers 21 and 22, respectively.

[0248] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include an immunoglobulin heavy chain variable region comprising: a) a CDR1 having the amino acid sequence described in SEQ ID NO: 12, a CDR2 having the amino acid sequence described in SEQ ID NO: 13 or 14, and optionally a CDR3 having the amino acid sequence described in SEQ ID NO: 15; and 2) an immunoglobulin light chain variable region comprising a CDR1 having the amino acid sequence described in SEQ ID NO: 23, a CDR2 having the amino acid sequence described in SEQ ID NO: 24, and optionally a CDR3 having the amino acid sequence described in SEQ ID NO: 25. In certain embodiments, the CDR2 of the heavy chain variable region is SEQ ID NO: 13. In certain embodiments, the 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 yet another antibody (e.g., SEQ ID NOs. 15 and 25). All possible combinations (e.g., all six light and heavy chain CDRs) are shown based on SEQ ID NOs. 1-25 in Table 1, particularly those related to the same antibody number. (wherein CD123-3, or CD123-6, or CD123-14) are intended herein without exhaustively listing all specific combinations.

[0250] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment preserve amino acid substitutions on one, two, or three consecutive residues of any one or more CDR sequences described above. That is, in some embodiments, the target antibody and its antigen-binding fragment may preserve amino acid substitutions on one, two, or three consecutive residues in any one or more SEQ ID NOs. 1 to 25.

[0251] In certain embodiments, the CD123 / IL-3Rα conjugate is a CD123 / IL-3Ra antibody or its antigen-binding fragment, comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), in which case the HCVR and LCVR are one of the sequences shown in Tables 3A and 4A below. Selected corresponding nucleic acid sequences encoding the HCVR and LCVR are shown in Tables 3B and 4B. [Table 5] [Table 6-1] [Table 6-2] [Table 7] [Table 8]

[0252] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are (a) reference V selected from the group having 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 The array and V which is at least 95% identical. H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The array and V which is at least 95% identical. L Includes arrays.

[0253] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are selected from the group having the amino acid sequence represented by (a) 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 HThe array and V which is at least 96% identical. H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The array and V which is at least 96% identical. L Includes arrays.

[0254] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are (a) reference V selected from the group having 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 The array and V which is at least 97% identical. H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The array and V which is at least 97% identical. L Includes arrays.

[0255] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are (a) reference V selected from the group having 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 The array and V which is at least 98% identical. H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The array and V which is at least 98% identical. L Includes arrays.

[0256] In a particular embodiment, the anti-CD123 antibody and its antigen-binding fragment are (a) reference V selected from the group having 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 The array and V which is at least 99% identical. H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The array and V which is at least 99% identical. L Includes arrays.

[0257] In certain embodiments, the CD123 / IL-3Rα antibody / antigen-binding fragment, having a specific percentage of sequence identity with 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), differs 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 conserved amino acid substitutions (e.g., 1, 2, or 3 conserved amino acid substitutions). In certain embodiments, a conservative amino acid substitution is the substitution of one, two, or three conservative amino acids in one or more CDR regions of the heavy chain and / or light chain.

[0258] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are (a) reference V selected from the group having 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 The same V as the array H A reference V selected from the group having the amino acid sequence and / or (b) the amino acid sequence represented by SEQ ID NOs: 27, 29, 31, 33, 35, 37, and 41 (or SEQ ID NOs: 27, 29, 31, 35, and 37) L The same V as the array L Includes arrays.

[0259] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are V as described in SEQ ID NO: 26. H Sequence and / or V as described in Sequence ID No. 27 L Includes arrays.

[0260] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are V as described in SEQ ID NO: 28. H Sequence and / or V as described in Sequence ID No. 29 L Includes arrays.

[0261] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are described in SEQ ID NO: 30. H V as described in sequence and / or sequence number 31 L Includes arrays.

[0262] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are described in SEQ ID NO: 34. H Sequence and / or V as described in Sequence ID No. 35 L Includes arrays.

[0263] In a particular embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are combined with an SEQ ID NO selected from the group consisting of 32 / 33, 34 / 33, 38 / 33, 39 / 33, 40 / 33, 32 / 35, 34 / 35, 38 / 35, 39 / 35, 40 / 35, 32 / 37, 34 / 37, 38 / 37, 39 / 37, 40 / 37, 39 / 33, 39 / 35, 39 / 37, 39 / 41, 40 / 33, 40 / 35, 40 / 37, and 40 / 41, V H Array and V L Includes arrays.

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

[0265] In related forms, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain variable region having the amino acid sequence described in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence described 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 antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain variable region having the amino acid sequence described 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 described in SEQ ID NO: 41.

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

[0268] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 59 or 60, except that the N-terminal residue is Ser and 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 described in SEQ ID NO: 41.

[0269] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are: a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 59 or 60, except that the fifth-to-last residue of SEQ ID NO: 54 is Cys; and b) an immunoglobulin having the amino acid sequence described in SEQ ID NO: 41, except that the N-terminal residue is Ser. Includes the variable region of the robulin light chain.

[0270] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain variable region having the amino acid sequence described in SEQ ID NO: 38, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 35. In a particular embodiment, Xaa in SEQ ID NO: 38 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 38 is Val(V).

[0271] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain variable region having the amino acid sequence described in SEQ ID NO: 34, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 37. In a particular embodiment, Xaa in SEQ ID NO: 34 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 34 is Val(V).

[0272] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 56, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 35. In a particular embodiment, Xaa in SEQ ID NO: 56 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 56 is Val(V).

[0273] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 37. In a particular embodiment, Xaa in SEQ ID NO: 54 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 54 is Val(V).

[0274] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described 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 described in SEQ ID NO: 41.

[0275] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 35. In a particular embodiment, Xaa in SEQ ID NO: 54 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 54 is Val(V).

[0276] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 56, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 35. In a particular embodiment, Xaa in SEQ ID NO: 56 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 56 is Val(V).

[0277] In another embodiment, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment include a) an immunoglobulin heavy chain region having the amino acid sequence described in SEQ ID NO: 54, and b) an immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 37. In a particular embodiment, Xaa in SEQ ID NO: 54 is Phe(F). In a particular embodiment, Xaa in SEQ ID NO: 54 is Val(V).

[0278] In certain embodiments, the anti-CD123 / IL-3Rα antibody and its antigen-binding fragment are CD1 It specifically binds to CD123 / IL-3Rα. In certain embodiments, the CD123 / IL-3Rα antibody or its antigen-binding fragment is a mouse, chimeric, humanized, or non-human antibody or its antigen-binding fragment that specifically binds to CD123 / IL-3Rα. In certain embodiments, the humanized antibody or its antigen-binding fragment is a CDR implantation or resurfacing antibody or its antigen-binding fragment.

[0279] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a full-length antibody. A full-length antibody may include any of the antibodies described above, defined by 1 to 4 CDRs (e.g., CDR1 and CDR2 of the heavy chain, CDR1 and CDR2 of the heavy and light chains), 1 to 6 CDR sequences (e.g., CDR1 to CDR3 of the heavy chain, CDR1 to CDR3 of the heavy and light chains), or any of the antibodies described above, defined by LCVR and / or HCVR, or any of the full-length antibodies having the heavy chain sequences of Table 5, or any of the full-length antibodies having the light chain sequences of Table 6, or any of the full-length antibodies having the heavy chain sequences of Table 5 and the light chain sequences of 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 sequence numbers 42 / 43, 44 / 45, 46 / 47, 48 / 49, 50 / 49, 53 / 4 A full-length antibody containing a combination of a full-length heavy chain sequence and a 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, or an antibody having at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with either the full-length heavy chain sequence and / or light chain sequence.

[0281] In certain embodiments, the anti-CD123 / IL-3Rα antibody is a full-length antibody comprising a combination of a full-length heavy chain sequence and a full-length light chain sequence 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 either the full-length heavy chain sequence and / or light chain sequence. Such an antibody may further contain an N-terminal Ser / Thr manipulated in the light chain, heavy chain, or both. Such an antibody may further contain a manipulated Cys at the position corresponding to the fifth-to-last Cys in SEQ ID NOs. 54.

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

[0283] In certain embodiments, the antigen-binding fragment is any of the antibodies described above: Fab, Fd, Fab', F(ab')2, Fd, single-chain Fv or scFv, disulfide-linked Fv, V-NAR domain, IgNar, intracellular antibody, IgGΔCH2, small antibody, F(ab')3, quadruspecific antibody, triplicate antibody, bispecific antibody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc, or comprises these.

[0284] In a related embodiment, the present invention relates to either an antibody or an antigen-binding fragment thereof, as described above V H and / or V L We also provide polypeptides comprising any of the sequences, either of the HCVR and / or LCVR sequences described above, or any of the CDR sequences of the HCVR and / or LCVR sequences described above. The polypeptide may be a fusion with, for example, a non-antibody protein or domain. In certain embodiments, the fusion protein is not a fusion with Pseudomonas toxin.

[0285] The affinity or binding activity of an antibody to an antigen can be experimentally measured using any suitable method known in the art (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or kinetics (e.g., BIACORE® analysis)). Direct binding assays and competitive binding assays are readily available. See, for example, Berzofsky et al., “Antibody-Antigen Interactions,” in Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984), Kuby, Janis Immunology, WH 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). Therefore, affinity and other antigen-binding parameters may vary. (For example, K D , or K d , K on , K off The measurement of ) is performed using standardized solutions of antibodies and antigens, as well as standardized buffers known in the art, such as the buffers described herein.

[0287] In one embodiment, the binding assay can be performed using flow cytometry of cells expressing the CD123 / IL-3Rα antigen on their surface. For example, CD123 / IL-3Rα-positive cells can be expressed in 100 μL of FACS buffer (e.g., RPMI-1640 medium supplemented with 2% normal goat serum) per sample, yielding 1 × 10⁶ cells. 5Cells can be incubated with various concentrations of anti-CD123 / IL-3Rα antibody. The cells can then be pelleted, washed, and incubated for 1 hour 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). The cells are then pelleted again, washed with FACS buffer, and resuspended in 200 μL of PBS-containing 1% formaldehyde. Samples can be obtained using a FACSCalibur flow cytometer, e.g., with an HTS multiwell sampler, and analyzed using CellQuest Pro (all from BD Biosciences (San Diego, US)). For each sample, the mean fluorescence intensity of FL1 (MFI) can be exported and plotted against antibody concentration in a semilog plot to create a binding curve. The sigmoid dose-response curve is fitted to the binding curve, and the EC 50 The value is calculated using a program (for example, GraphPad Prism v4 with default parameters (GraphPad software, San Diego, CA)). 50 The value is the apparent dissociation constant "K" for each antibody. d " or "K D It can be used as a standard for ".

[0288] Monoclonal antibodies can be prepared using the hybridoma method (e.g., Kohler and Milstein (1975) Nature 256:495). Using the hybridoma method, mice, hamsters, or other suitable host animals are immunized to induce lymphocyte production of antibodies that specifically bind to immune antigens. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated to form hybridoma cells, which can then be selected apart from non-fusion lymphocytes and myeloma cells, and fused with suitable myeloma cell lines, for example, using polyethylene glycol. Hybridomas that produce monoclonal antibodies specifically targeted to a selected antigen, as measured by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA)), can then be transmitted in vitro 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. Patent No. 4,816,567. The polynucleotide encoding the monoclonal antibody is isolated from mature B cells or hybridoma cells by RT-PCR using oligonucleotide primers that specifically amplify, for example, the genes encoding the heavy and light chains of the antibody, and its sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned in a suitable expression vector 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), at which point the monoclonal antibody is produced by the host cells. Furthermore, recombinant monoclonal antibodies or fragments of the desired species can be isolated from phage display libraries expressing the CDR of the desired species, as described (McCafferty e 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, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody may be replaced with 1) the region of a human antibody that generates a chimeric antibody, or 2) a non-immunoglobulin polypeptide that generates a fusion antibody. In some embodiments, the constant region is truncated or removed to generate a desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc., of the monoclonal antibody.

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

[0292] Methods for manipulating, humanizing, or resurfacing non-human or human antibodies are also available and are well known in the art. Humanized, resurfaced, or similarly manipulated antibodies may 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 substituted with residues referred to as "implant" residues, which are typically taken from "implant" variable, constant, or other domains of well-known human sequences.

[0293] Using such transfer sequences, it is possible to reduce immunogenicity, or to reduce, enhance, or alter binding, affinity, binding rate, dissociation rate, binding activity, specificity, half-life, or any other desirable properties, as is well known in the art. Generally, CDR residues are directly and almost substantially involved in influencing CD123 / IL-3Rα binding. Therefore, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences in the variable and constant regions may be substituted with human or other amino acids.

[0294] Antibodies can optionally be humanized, resurfaced, manipulated, or humanized, while retaining high affinity for the antigen CD123 / IL-3Rα and other desirable biological properties. To achieve this objective, humanized (or human) or manipulated anti-CD123 / IL-3Rα antibodies and resurfaced antibodies can optionally be prepared by analytical processes of the parent sequence and various conceptual humanized and manipulated products using three-dimensional models of the parent sequence, manipulated sequence, and humanized sequence. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display likely three-dimensional structures for selected candidate immunoglobulin sequences. By examining these representations, it is possible to analyze the roles of residues that may affect the function of the candidate immunoglobulin sequence, i.e., the residues that affect the ability of the candidate immunoglobulin to bind to its antigen (e.g., CD123 / IL-3Rα). In this way, framework (FR) residues can be selected and combined from common sequences and imported sequences to achieve desirable antibody characteristics, such as increased affinity for target antigens (multiple antigens are possible).

[0295] Humanization, resurfacing, or manipulation of the antibodies of the present invention are 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 implemented using well-known methods, such as those described in 8 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755, WO90 / 14443, WO90 / 14424, WO90 / 14430, European Patent No. 229246, No. 7,557,189, No. 7,538,195 and No. 7,342,110, each of which includes its cited references, which are incorporated herein by reference in their 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 isolating individuals that produce antibodies against the target antigen (e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985), Boemer et al., 1991, J. Immunol, 147(l):86-95 and U.S. Patent No. 5,750,3 (See Issue 73). Furthermore, human antibodies can be selected from phage libraries, such as Vaughan et al., Nat. Biotech. 14:309-314, 1996, Sheets et al., Proc. Nat'l. Acad. Sci. 95:6157-6162, 1998, Hoogenboom. It expresses human antibodies as described in and Winter, J.Mol.Biol.227:381, 1991 and Marks et al., J.Mol.Biol.222:581, 1991. Techniques for generating and using antibody phage libraries are described in U.S. Patents 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 in Rothe et al., J.Mol.Bio.doi:10.1016 / j.jmb.2007.12.018,2007 (each of these is incorporated herein by reference in its entirety). Affinity maturation and chain shuffling methods (Marks et al., Bio / Technology 10:779-783, 1992, incorporated in their entirety by reference) are well known in the art and can be used to produce high-affinity human antibodies.

[0297] Humanized antibodies can also be produced in transgenic mice containing human immunoglobulin loci, which are capable of generating a complete repertoire of human antibodies during immunization in the absence of endogenous immunoglobulin production. This method is described in U.S. Patents 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016.

[0298] In certain embodiments, antibody fragments are provided that increase, for example, tumor invasion. Various techniques for generating antibody fragments are well known. Conventionally, these fragments have been obtained via the proteolysis of complete 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 arise by recombination. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus enabling the generation of large quantities of these fragments. Such antibody fragments can also be isolated from antibody phage libraries. The antibody fragments may further be linear antibodies, as described, for example, in U.S. Patent No. 5,641,870, and may be monospecific or bispecific. Other techniques for generating antibody fragments are apparent to those skilled in the art.

[0299] In this invention, it should be understood that a modified antibody may contain any type of variable region that provides association of an antibody having the human CD123 / IL-3Rα polypeptide. In this regard, the variable region may contain or be derived from any type of mammal that can be induced to increase the humoral response and produce an immunoglobulin against a desired tumor-associated antigen. Thus, the variable region of a modified antibody may be derived from, for example, humans, mice, non-human primates (e.g., cynomolgus monkeys, macaques, etc.) or peas. In some embodiments, the variable and constant regions of the modified immunoglobulin are human. In other embodiments, the variable region of a compatible antibody (usually derived from a non-human source) may be manipulated or specifically modified to improve the binding properties of the molecule or to reduce its immunogenicity. In this regard, variable regions useful in this invention may be humanized or otherwise modified by the inclusion of an imported amino acid sequence.

[0300] In certain embodiments, both the heavy and light chain variable domains are modified by at least partial substitution of one or more CDRs, and optionally by partial framework region substitution and sequence exchange. The CDRs may originate from antibodies of the same class or subclass as the antibody from which the framework region originates, but it is assumed that the CDRs are obtained from antibodies of different classes, and in certain embodiments from different species. It may not be necessary to replace all CDRs with complete CDRs derived from the donor variable region in order to transfer the antigen-binding ability of one variable domain to another. Rather, it may only be necessary to transfer these residues as required to maintain the activity of the antigen-binding site. Considering the descriptions in U.S. Patents 5,585,089, 5,693,761, and 5,693,762, obtaining functional antibodies with reduced immunogenicity by either performing general experiments or trial-and-error testing is well within the capabilities of those skilled in the art.

[0301] Despite the modification to the variable region, those skilled in the art will understand that the modified antibody of the present invention comprises an antibody (e.g., a full-length antibody or an immunoreactive fragment thereof) in which at least one or more fractions of the constant region domain are deleted or otherwise altered to provide desirable biochemical properties such as increased tumor localization or reduced serum half-life, compared to an antibody of substantially the same immunogenicity containing the native or unaltered constant region. In some embodiments, the constant region of the modified antibody comprises the human constant region. Modification to the constant region compatible with the present invention comprises the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include modifications or alterations to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or light chain constant domains (CL). In some embodiments, an altered constant region is intended in which one or more domains are partially or completely deleted. In some embodiments, the modified antibody is a domain deletion structure or variant in which all CH2 regions are removed. (ACH2 structure) is included. In some embodiments, the excluded 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 may be manipulated to directly fuse the CH3 domain to the hinge region of each modified antibody. In other structures, it may be desirable to provide peptide spacers between the hinge region and the modified CH2 and / or CH3 domains. For example, a compatible structure may be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is fused to a hinge region having 5-20 amino acid spacers. Such spacers may be added, for example, to ensure that the regulatory elements of the constant domain remain free and accessible, or that the hinge region remains flexible. However, it should be noted that amino acid spacers may be immunogenic in some cases and may prove to induce an unwanted immune response against the structure. Therefore, in certain embodiments, any spacers added to the structure are either relatively non-immunogenic or excluded entirely in order to maintain the desired biochemical properties of the modified antibody.

[0303] It should be understood that, in addition to the deletion of an entire constant region domain, the antibodies of the present invention may also be provided by partial deletions or substitutions of several or a single amino acid. For example, a single amino acid mutation 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 a portion of one or more constant region domains that control the modulation of effector function (e.g., complementing C1Q binding). Such partial deletions of the constant region can improve selected properties of the antibody (serum half-life) while leaving other desirable functions associated with the target constant region domain intact. Furthermore, as described above, the constant region of the disclosed antibody can be modified, for example, by mutations or substitutions of one or more amino acids, which can enhance the properties of the resulting structure. In this regard, it may be possible to inhibit the activity provided by the conserved binding site (e.g., Fc binding) while substantially maintaining the composition and immunogenicity properties of the modified antibody. Certain embodiments may include the addition of one or more amino acids to the constant region to enhance a desired feature (e.g., reducing or increasing effector function) or to provide more cytotoxicity or carbohydrate adhesion. In such embodiments, it may be desirable to insert or replicate a specific sequence derived from a selected constant region domain.

[0304] The present invention further encompasses variants and equivalents that are substantially homologous to the chimeras, humanized and human antibodies, or antibody fragments thereof, described herein. These may 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 means substituting one amino acid within the same general class with another (e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid). The intent of conservative amino acid substitutions is well known in the art (e.g., as defined herein above).

[0305] The polypeptides of the present invention may be recombinant polypeptides, native polypeptides, or synthetic polypeptides containing an antibody or fragment 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 significant impact on the structure or function of the protein. Accordingly, the present invention further includes variants of the polypeptide that exhibit considerable activity against the CD123 antigen (e.g., human CD123) or contain a region of the antibody or fragment thereof. Such variants include deletions, insertions, inversions, repeats, and type substitutions.

[0306] Polypeptides and analogues can be further modified to include additional chemical portions that are not normally part of the protein. Such derivatized portions can improve the solubility, biological half-life, or absorption of the protein. The portions can also reduce or eliminate any desirable side effects, such as those of the protein. An overview of the portions 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 transforming host. In some embodiments, the DNA sequence is constructed using recombination techniques by isolating or synthesizing a DNA sequence encoding the wild-type protein of interest. Optionally, the sequence can be mutagenicated by site-directed mutagenesis to provide functional analogs. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066, 1984 and U.S. Patents 4,588,585.

[0308] In some embodiments, the DNA sequence encoding the target polypeptide (e.g., the antibody, antigen-binding fragment, or polypeptide of the present invention) is constructed entirely or partially by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed by selecting preferred codons in a host cell producing the recombinant polypeptide of interest, based on the amino acid sequence of the desired polypeptide. An isolated polynucleotide sequence encoding the target isolated polypeptide can be synthesized by applying standard methods. For example, a back-translated gene can be constructed using the complete amino acid sequence. Furthermore, DNA oligomers containing nucleotide sequences encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated together. Individual oligonucleotides typically contain a 5' or 3' overhang for complementary aggregation.

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

[0310] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding an antibody or fragment thereof against human CD123 / IL-3Rα. The recombinant expression vector is a replicable DNA structure having a synthetic or cDNA-derived DNA fragment encoding a polypeptide chain of the anti-CD123 / IL-3Rα antibody or fragment thereof, and is operably linked to an appropriate transcriptional or translational regulatory element derived from a mammalian, microorganism, virus, or insect gene. The transcriptional unit typically includes (1) a genetic element(s) having a regulatory role in gene expression (e.g., a transcription promoter or enhancer), (2) a structure or coding sequence transcribed into mRNA and translated into a protein, and (3) an assembly of appropriate transcriptional and translational start and stop sequences. Such regulatory elements may include operator sequences to control transcription. Selective genes that facilitate replication in the host and recognition of the transformant, typically conferred by the origin of replication, may be added. DNA regions can be operably ligated when they are functionally related to one another. For example, the DNA of a signal peptide (secretion leader) is operably ligated to the DNA of a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide; a promoter is operably ligated to a coding sequence if it controls the transcription of the sequence; or a ribosome-binding site is operably ligated to a coding sequence if it is positioned to enable translation. Structural elements intended for use in yeast expression systems include a leader sequence that enables the extracellular secretion of translated proteins by the host cell. Alternatively, when a recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide the final product.

[0311] The selection of expression regulatory sequences and expression vectors depends on the host selection. A wide variety of expression host / vector combinations can be used. Useful expression vectors in eukaryotic hosts include, for example, vectors containing expression regulatory sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors in bacterial hosts include well-known bacterial plasmids containing pCR1, pBR322, pMB9, and their derivatives (e.g., plasmids from Escherichia coli), and broad-host-range plasmids (M13 and filamentous single-strand DNA phages).

[0312] Suitable host cells for the expression of CD123 / IL-3Rα-binding polypeptides or antibodies (or CD123 / IL-3Rα proteins used as antigens) include prokaryotes, yeasts, insects, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include Gram-negative or Gram-positive organisms (e.g., E. coli or bacilli). Higher eukaryotic cells include mammalian cell lines (e.g., CHO cells). Cell-free translation systems are also available. Suitable cloning and expression vectors 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 disclosures of which are incorporated herein by reference. Further information regarding methods of protein synthesis, including antibody production, can be found, for example, in U.S. Patent Publication No. 2008 / 0187954, U.S. Patents No. 6,413,746 and 6,660,501, and International Patent Publication No. WO04009823, each of which is incorporated herein by reference in its entirety.

[0313] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant proteins. Such proteins are generally correctly folded, appropriately modified, and fully functional, making them suitable for expression in mammalian cells. Examples of suitable mammalian host cell systems include HEK-293T, the COS-7 strain of monkey kidney cells described by Gluzman (Cell 23:175, 1981), and other cell systems, such as L cells, CI27, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines. Mammalian expression vectors may include non-transcription elements (e.g., replication start sites, appropriate promoters and enhancers linked to the gene to be expressed), other 5' or 3' adjacent non-transcription sequences, and 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 generating heterologous proteins in insect cells are outlined in Luckow and Summers, Bio / Technology 6:47, 1988.

[0314] Accordingly, one aspect of the present invention also provides cells that produce either a target antibody or its antigen-binding fragment, or any one of the target polypeptides. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are HEK-293 or HEK-2 These are 93T cells, COS-7 cells, L cells, CI27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, or BHK cells. In certain embodiments, the cells are CHO cells.

[0315] Proteins produced by the transformed host can be purified according to any preferred 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 labeling (e.g., hexahistidine, maltose-binding domains, influenza coating sequences, and glutathione S-transferase) can be bound to the protein to allow for simple purification by passage on a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0316] For example, the supernatant from a system secreting recombinant protein into a culture medium can be first concentrated using a commercially available protein concentration filter (e.g., an Amicon or Millipopore Pellicon ultrafiltration system). After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin (e.g., a matrix or substrate having pendant diethylaminoethyl (DEAE) groups) can be used. This matrix may 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, the CD123 / IL-3Rα binder can be further purified using one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps with hydrophobic RP-HPLC medium (e.g., silica gel having pendant methyl or other aliphatic groups). Some or all of the above purification steps can also be used in various combinations to form a homogeneous recombinant protein.

[0317] Recombinant proteins prepared in bacterial cultures can be isolated, for example, by initial extraction from a cell pellet, 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 the final purification step. Microbial cells used for recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysis agents.

[0318] Methods well known in the art for purifying antibodies and other proteins include, for example, those described in U.S. Patent Publications 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 binder of the present invention has an N-terminal serine, which can be oxidized with an oxidizing agent to form an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde group.

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

[0321] An excess molar equivalent of oxidizing agent can be used relative to the CD123 / IL-3Rα binder. In certain embodiments, about 2 to 100, 5 to 80, 10 to 50, 1 to 10, or 5 to 10 molar equivalents of oxidizing agent can be used. In certain embodiments, about 10 or Approximately 50 equivalents 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 approximately 5 to 60 minutes. Alternatively, when using 10 equivalents of oxidizing agent, the reaction is carried out for approximately 30 minutes to 24 hours. In one embodiment, 5 to 10 molar equivalents of oxidizing agent are used, and the oxidation reaction is carried out for approximately 5 to 60 minutes (e.g., approximately 10 to 30 minutes, or approximately 20 to 30 minutes).

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

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

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

[0325] In the first embodiment, the immune complex of the present invention comprises a CD123 / IL-3Rα binder (including an antibody, an antigen-binding fragment thereof, or a polypeptide comprising an antibody or an antigen-binding fragment thereof) which is covalently bound to the cytotoxic agent described herein by the ε-amino groups of one or more lysine residues present in the CD123 / IL-3Rα binder.

[0326] In a first specific embodiment of the first embodiment, the immune complex of the present invention is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy L1 A CD123 / IL-3Rα binder (for example, the target antibody or its antigen-binding fragment, or the target polypeptide mentioned above) that is covalently bound by a heridine residue, W L is an integer between 1 and 20, Cy L1 The formula is as follows: [ka] Cytotoxic compounds represented by, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected 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 between 2 and 6. R k is -H or -Me, R x3 It is an (C1-C6) alkyl group, L' is given by 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 In each occurrence, Q is independently -H, (C1-C3) alkyl, or a charged substituent or ionic group. m is an integer between 1 and 6. Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is H + Or it is a cation.

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

[0328] In a third specific embodiment, for the complex of formula (L1), Cy L1 R is expressed by equation (L1b) or (L1b1), and the remaining variable elements are as described above in the first specific embodiment. More specifically, R x3 It is a (C2-C4) alkyl group.

[0329] In a fourth specific embodiment, for the complex of formula (L1), Cy L1 is expressed by equation (L1a), R a and R b Both are H, R5 is either H or Me, and the remaining variable elements 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, and the remaining variable elements are as described above in the first, second, or fourth specific embodiments. In further specific embodiments, P is 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 the sixth embodiment, Q is -SO3M, and the remaining variable elements are as described above in the first, second, fourth, or fifth specific embodiment, or as in any further specific embodiment described herein.

[0332] In a seventh specific embodiment, the immune complex of the first embodiment is given by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] or [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where W L is an integer from 1 to 10, and N and C and Double line between them [ka] The ∫ represents a single or double bond, provided 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 further specific embodiments, the double line between N and C [ka] represents a double bond, where X is absent and Y is -H. In another further specific embodiment, the double line between N and C [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0333] In the eighth specific embodiment, the immune complex of the first embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy L2 A CD123 / IL-3Rα binder according to the first aspect of the present invention, which is covalently bound by a helixine residue (for example, the target antibody or its antigen-binding fragment, or the target polypeptide described above), W L is an integer between 1 and 20, Cy L2 The formula is as follows: [ka] Cytotoxic compounds represented by, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected moiety and Y is -OH or -SO3M. R x1 and R x2 These are (C1-C6) alkyl groups, 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 between 2 and 6. R k is -H or -Me, Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is -H + Or it is a cation.

[0334] In a ninth specific embodiment, for the immune complex of formula (L2), Cy L2 This is represented by equation (L2a) or (L2a1), and the remaining variable elements are as described above in the eighth specific embodiment.

[0335] In a tenth specific embodiment, for the immune complex of formula (L2), Cy L2 This is represented by equation (L2b) or (L2b1), and the remaining variable elements are as described above in the eighth specific embodiment.

[0336] In the 11th specific embodiment, for the immune complex of formula (L2), R e is H or Me, and R x1 and Rx2 It is independently -(CH2) p -(CR f R g )- and R f and R g Each is independently -H or (C1-C4)alkyl, p is 0, 1, 2, or 3, and the remaining variable elements are as described above in the eighth, ninth, or tenth specific embodiment. More specifically, R f and R g They are either identical or different, and are selected from -H and -Me.

[0337] In a twelfth specific embodiment, the immune complex of the first embodiment is given by the following formula: [ka] [ka] or [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where W L The integers are from 1 to 10, and the double line between N and C [ka] The ∫ represents a single or double bond, provided 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 further specific embodiments, the double line between N and C [ka] represents a double bond. In another further specific embodiment, a double line between N and C [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0338] In a 13th specific embodiment, the immune complex of the first embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy L3 A CD123 / IL-3Rα binder according to the first aspect of the present invention, which is covalently bound by a Lys residue (for example, the target antibody or its antigen-binding fragment, or the target polypeptide described above), W L is an integer between 1 and 20, Cy L3 The formula is as follows: [ka] It is represented as, m' is either 1 or 2. R1 and R2 are each independently H or (C1-C3) alkyl. Z s1 The formula is as follows: [ka] One of the following has been selected: During the ceremony, q is an integer between 1 and 5. M is H + Or it is a cation.

[0339] In the 14th specific embodiment, for the immune complex of formula (L3), m' is 1, R1 and R2 are both H, and the remaining variable elements are as described above in the 13th specific embodiment.

[0340] In the 15th specific embodiment, for the immune complex of formula (L3), m' is 2, R1 and R2 are both Me, and the remaining variable elements are as described above in the 13th specific embodiment.

[0341] In a specific sixteenth embodiment, the immune complex of the first embodiment is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where W L The integers are between 1 and 10.

[0342] In the 17th specific embodiment, with respect to the immune complex of the first embodiment, M is H + na + or K + The remaining variable elements are as described above in any one of the first to sixteenth specific embodiments, or as in any further specific embodiments described herein.

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

[0344] The immune complexes described in the first embodiment or any particular embodiment thereof can be prepared according to any method well 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 immunocomplex of the first embodiment can be prepared by a first method, which includes the step of reacting a cytotoxic agent having an amine reactive group with CBA.

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

[0347] In one embodiment, for the first method described above, the cytotoxic agent having an amine-reactive reagent is given by the following formula: [ka] It is represented by or a pharmaceutically acceptable salt thereof, in the formula, L c ' is expressed by 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) is represented as, C(=O)E is a reactive ester group, such as N-hydroxysuccinimide ester, N-hydroxysulfosuccinimide ester, nitrophenyl (e.g., 2 or 4-nitrophenyl) ester, dinitrophenyl (e.g., 2,4-dinitrophenyl) ester, sulfo-tetrafluorophenyl (e.g., 4-sulfo-2,3,5,6-tetrafluorophenyl) ester, or pentafluorophenyl ester, preferably N-hydroxysuccinimide ester. Z s The formula is as follows: [ka] It is represented as, During the ceremony, q is an integer from 1 to 5, U is -H or SO3M, The remaining variable elements are as described above in any one of the first to seventh and seventeenth specific embodiments, or as in any further specific embodiments described herein.

[0348] In a particular embodiment, the immune complex of the first embodiment is (a) A step of 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 that binds thereto, and (b) by a second method, which includes the step of reacting CBA with a cytotoxic agent-linker compound. It can be prepared by [method].

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

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

[0351] In a particular embodiment, the immune complex of the first embodiment is (a) A step of reacting 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 to which it is bound, and (b) It can be prepared by a third method, which includes the step of reacting modified CBA with a cytotoxic agent.

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

[0353] In certain embodiments, the immunocomplex of the first embodiment can be prepared by a fourth method, which includes reacting 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 a particular embodiment, for the second, third, or fourth embodiment described above, the linker compound having an amine-reactive group and a thiol-reactive group is given by the following formula: [ka] It is represented as, In the formula, 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 variable elements are as described above for formulas (a1) to (a10), and the cytotoxic agent is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, wherein the variable element is as described above in any one of the eighth to twelfth and seventeenth specific embodiments, and as in any further specific embodiments described herein.

[0356] In a particular embodiment, 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 one of the formulas (a1L) to (a10L), and the cytotoxic agent is represented by the following formula: [ka] It is represented as, In the formula, the variable element is as described above in any one of the 13th to 17th specific embodiments. and as described in any further specific embodiments herein.

[0357] In a second embodiment, the immune complex of the present invention comprises an oxidized CD123 / IL-3Rα binder (including an antibody, its antigen-binding fragment, or a polypeptide comprising an antibody or its antigen-binding fragment) (e.g., an oxidized antibody or its antigen-binding fragment, or its polypeptide) which is covalently bound to the cytotoxic agent described herein as described in the first aspect of the present invention by one or more aldehyde groups present in the oxidized CD123 binder. The aldehyde groups present in the oxidized CD123 / IL-3Rα binder can be generated by oxidizing one or more 2-hydroxyethylamine moieties of the CD123 / IL-3Rα binder, the 2-hydroxyethylamine moieties being part of serine, threonine, hydroxylysine, 4-hydroxyornithine, or a 2,4-diamino-5-hydroxyvaleric acid residue. In one embodiment, the aldehyde group can be generated by oxidizing the 2-hydroxyethylamine moiety of one or more N-terminal serine residues (for example, the 1st, 2nd, 3rd, or up to 4th Ser residues at the N-terminus of the light chain and / or heavy chain) present in the CD123 / IL-3Rα binder.

[0358] In a first specific embodiment of the second embodiment, the immune complex of the present invention is given by the following formula: [ka] It is represented as, During the ceremony, CBA is an oxidized CD123 / IL-3Rα binder according to the first aspect of the present invention (for example, the target oxidized antibody or its antigen-binding fragment, or the target oxidized polypeptide described above), Ws is 1, 2, 3, or 4. J CB ' is the aldehyde group of CBA and Cy s1This is the part formed by reacting with the aldehyde reactive group, and is given the following formula: [ka] It is represented as, s1 is the site that is covalently bound to CBA, and s2 is Cy s1 It is a site that is covalently bonded to, Cy s1 The formula is as follows: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, however, 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 amine protecting moiety, Y is -OH or -SO3M, and M is H + or provided that it is a 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 either absent, -C(=O)-NR9-, or -NR9-C(=O)-, R9 is -H or (C1~C3) alkyl, R a and R b In each occurrence, Q is independently -H, (C1-C3) alkyl, or a charged substituent or ionic group. r and r' are independent integers between 1 and 6. W' is -NR e’ And, R e’ is -(CH2-CH2-O) n -R k And, n is an integer between 2 and 6. R k is -H or -Me, R x3 It is an (C1-C6) alkyl group, L is -NR9-(CR a R b ) r” or absent, r'' is an integer between 0 and 6.

[0359] In a second specific embodiment, for the immune complex of formula (S1), Cy s1 This is expressed by equation (S1a) or (S1a1), and the remaining variable elements are as described above in the first specific embodiment.

[0360] In a third specific embodiment, for an immune complex of formula (S1), Cy s1 R is expressed by equation (S1b) or (S1b1), and the remaining variable elements are as described above in the first specific embodiment. More specifically, R x3 It is a (C2-C4) alkyl group.

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

[0362] In a fifth specific embodiment, for the immunocomplex of formula (S1), P is a peptide containing 2 to 5 amino acid residues, and the remaining variable elements are as described above in the first, second, or fourth specific embodiments. In further specific embodiments, P is 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 The group is selected from g, 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. More specifically, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

[0363] In the sixth specific embodiment, for the immune complex of formula (S1), Q is -SO3M, and the remaining variable elements are as described above in the first, second, fourth, or fifth specific embodiment, or as in any further specific embodiment described herein.

[0364] In a seventh specific embodiment, the immune complex of the second embodiment is given by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] or [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The ∫ represents a single or double bond, provided 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 further specific embodiments, the double line between N and C [ka] represents a double bond, where X is absent and Y is -H. In another further specific embodiment, the double line between N and C [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0365] In the eighth specific embodiment, the immune complex of the present invention is given by the following formula: [ka] It is represented as, During the ceremony, CBA is an oxidized CD123 / IL-3Rα binder according to the first aspect of the present invention (for example, the target oxidized antibody or its antigen-binding fragment, or the target oxidized polypeptide described above), J CB ' is the aldehyde group of CBA and Cy s2 This is the part formed by reacting the aldehyde reactive group, and it is represented by the following formula: [ka] It is represented as, In the formula, s1 is the site that is covalently bonded to CBA, and s2 is Cys2 It is a site that is covalently bonded to, Cy s2 The formula is as follows: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, provided 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-protected moiety and Y is -OH or -SO3M. M is H + or it is a cation, R x1 It is an (C1-C6) alkyl group, 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 between 2 and 6. R k is -H or -Me, R x2 It is an (C1-C6) alkyl group, L1 is given by the following formula: [ka] It is represented as, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s2 It is a site that is covalently bonded to the -S- group, Z a2is either 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 In each occurrence, it is independently H or (C1-C3) alkyl. q1 and r1 are independent integers between 0 and 10, provided that neither q1 nor r1 is 0.

[0366] In further specific embodiments, Z a2 There is no such thing as q1 and r1 being integers between 0 and 3, respectively, provided that neither q1 nor r1 is 0, and the remaining variable elements are as described above in the eighth specific embodiment. More specifically, R a1 , R a2 , R a3 , R a4 All of them are -H.

[0367] In another further specific embodiment, Z a2 is -C(=O)-NH- or -NH9-C(=O)-, where q1 and r1 are each independently integers from 1 to 6, and the remaining variable elements are as described above in the eighth specific embodiment. More specifically, R a1 , R a2 , R a3 , R a4 All of them are -H.

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

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

[0370] In the 11th specific embodiment, for an immune complex of formula (S2), -L1- is the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where R is H or -SO3M, and the remaining variable elements are as described above in the eighth, ninth or tenth specific embodiment, or as in any further specific embodiment described herein.

[0371] In a 12th specific embodiment, for an immune complex 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 Each of them is independently -H or (C1~C4) alkyl, and p is 0, 1, 2, or 3. More specifically, R f and R g They are either identical or different, and are selected from -H and -Me.

[0372] In a 13th specific embodiment, the immune complex of the second embodiment is given by the following formula: [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The ∫ represents a single or double bond, provided 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 further specific embodiments, the double line between N and C [ka] represents a double bond, where X is absent and Y is -H. In another further specific embodiment, the double line between N and C [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0373] In a specific 14th embodiment, the immune complex of the second embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is an oxidized CD123 / IL-3Rα binder according to the first aspect of the present invention (for example, the target oxidized antibody or its antigen-binding fragment, or the target oxidized polypeptide described above), J CB ' is the aldehyde group of CBA and Cy s3 This is the part formed by reacting the aldehyde reactive group, and is given the following formula: [ka] It is represented as, s1 is the site that is covalently bound to CBA, and s2 is Cy s3 It is a site that is covalently bonded to, Cy s3 The formula is as follows: [ka] It is represented as, During the ceremony, m' is either 1 or 2. R1 and R2 are each independently H or (C1-C3) alkyl. L1 is given by the following formula: [ka] It is represented as, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s3 It is a site that is covalently bonded to the -S- group, Z a2 is either 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 In each occurrence, it is independently H or (C1-C3) alkyl. q1 and r1 are independent integers between 0 and 10, provided that neither q1 nor r1 is 0.

[0374] In further specific embodiments, Z a2 There is no such thing as q1 and r1 being integers between 0 and 3, respectively, provided that neither q1 nor r1 is 0, and the remaining variable elements are as described above in the 14th specific embodiment. More specifically, R a 1. R a2 , R a3 , R a4 All of them are -H.

[0375] In another further specific embodiment, Z a2 is -C(=O)-NH- or -NH9-C(=O)-, where q1 and r1 are each independently integers from 1 to 6, and the remaining variable elements are as described above in the 14th specific embodiment. More specifically, R a1 , R a2 , R a3 , R a4 All of them are -H.

[0376] In the 15th specific embodiment, for the immunocomplex of formula (S3), m' is 1, R1 and R2 are both H, and the remaining variable elements are as described above in the 14th specific embodiment or in any further specific embodiment described herein.

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

[0378] In the 17th specific embodiment, for an immune complex of formula (S3), -L1- is the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where R is H or -SO3M and M is H + Or it is a cation.

[0379] In the 18th specific embodiment, the immune complex of the second embodiment is given by the following formula: [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where DM is given by the following formula: [ka] It is represented as follows.

[0380] In a specific embodiment of the 19th, the immune complex of the second embodiment is given by the following formula: [ka] It is expressed as, in the formula, CBA is an oxidized CD123 / IL-3Rα binder according to the first aspect of the present invention (for example, the target oxidized antibody or its antigen-binding fragment, or the target oxidized polypeptide described above), J CB ' is the aldehyde group of CBA and Cy s4 This is the part formed by reacting the aldehyde reactive group, and is given the following formula: [ka] It is represented as, s1 is the site that is covalently bound to CBA, and s2 is Cy s4 It is a site that is covalently bonded to, Cy s4 The formula is as follows: [ka] It is represented as, L1' is given by the following formula: [ka] It is represented as, During the ceremony, s3 is J CB It is a site that is covalently bonded to the base, s4 is Cy s4 It is a site that is covalently bonded to the -NMe- group, Z b1 and Z b2 Both are absent, or Z b1 and Z b2 One of them is absent, and the other is -CH2-O- or -O-CH2-. Z b1 'and Z b2 Each of the following is either 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 are each independent integers between 1 and 6. Either 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 In each occurrence, each element is independently either H or (C1-C3) alkyl.

[0381] In a 20th specific embodiment, for the immunoconjugate of formula (S4), R b1 , R b2 , R b3 , R b4 , Rb5 , and R b6 All of these are H, and the remaining variable elements are as described above in the 19th specific embodiment.

[0382] In the 21st specific embodiment, for the immunoconjugate of formula (S4), R9 is H, and the remaining variable elements are as described above in the 19th or 20th specific embodiment.

[0383] In a 22nd specific embodiment, with respect to the immunoconjugate of formula (S4), Z b1 'and Z b2 ' is either absent in both cases, or Z b1 ' is -CH2-O-, Z b2 ' is absent, or Z b1 ' is -CH2-C(=O)-NR9-, Z b2 ' is either O-CH2- or absent, and the remaining variable elements are as described above in the 19th, 20th, or 21st specific embodiment.

[0384] In the 23rd specific embodiment, for the immune complex of formula (S4), P is a peptide containing 2 to 5 amino acid residues, and the remaining variable elements are as described above in the 19th, 20th, 21st, or 22nd specific embodiment. In further specific embodiments, P is 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) P is selected from the group consisting of 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 variable elements being as described above in the 23rd specific embodiment. 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 specific embodiment 24, the immune complex of the second embodiment is given by the following formula: [ka] [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, DM has the following structural formula: [ka] It is represented as follows.

[0386] In a specific 25th embodiment, with respect to the immune complex of the second embodiment, M is H + na + or K + The remaining variable elements are as described above in any one of the first to twenty-fourth specific embodiments, or as in any further specific embodiments described herein.

[0387] In any of the first to twenty-fifth specific embodiments described above, the target oxidative antibody or its antigen-binding fragment may have one, two, three, or up to four N-terminal 2-hydroxyethylamine moieties oxidized to an aldehyde group for covalent bonding with the cytotoxic agents described herein. The N-terminal 2-hydroxyethylamine moieties may be part of serine, threonine, hydroxylysine, 4-hydroxyornithine, or 2,4-diamino-5-hydroxyvaleric acid residues, preferably Ser or Thr. For simplicity, the following description, including the oxidation reaction and any subsequent covalent bonding with the 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. The target antibody or its antigen-binding fragment may include an immunoglobulin heavy chain variable region (HCVR) having the amino acid sequence described in SEQ ID NO: 38, and an immunoglobulin light chain variable region (LCVR) having the amino acid sequence described in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). The target antibody or its antigen-binding fragment may also include an Ig HCVR having the amino acid sequence described in SEQ ID NO: 32, 34, 38, 39, or 40 (preferably SEQ ID NO: 34), and an Ig LCVR having the amino acid sequence described in SEQ ID NO: 37. The target antibody or its antigen-binding fragment may include an Ig heavy chain (HC) region having the amino acid sequence described in SEQ ID NO: 53 or 56, and SEQ ID NO: 33 The target antibody or its antigen-binding fragment may also include an Ig LCVR having the amino acid sequence described in SEQ ID NOs. 35, 37, or 41 (preferably SEQ ID NOs. 55 or 37). The target antibody or its antigen-binding fragment may also include an Ig HC region having the amino acid sequence described in SEQ ID NOs. 48, 50, 53, 54, 56, 59, or 60 (preferably SEQ ID NOs. 53), and an Ig LCVR having the amino acid sequence described in SEQ ID NOs. 37. In certain embodiments, the second residue from the N-terminus of SEQ ID NOs. 34, 38, 50, 53, 54, or 56 is Phe, and in certain other embodiments, the second residue from the N-terminus of SEQ ID NOs. 34, 38, 50, 53, 54, or 56 is Val.

[0388] In certain embodiments, the immune complex of the second embodiment can be prepared by a first method, which involves reacting an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde as described in the first aspect of the present invention with a cytotoxic agent having an aldehyde-reactive group.

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

[0390] In certain embodiments, the immune complex of the second embodiment can be prepared by a third method comprising contacting an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde as described in the first aspect of the present invention with a cytotoxic agent, followed by the addition of a linker compound vesicle having an aldehyde-reactive group.

[0391] In a particular embodiment, the immune complex of the second embodiment is (a) A step of oxidizing a CD123 / IL-3Rα binder having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde group, and (b) A fourth method can be prepared, comprising the step of reacting an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde with a cytotoxic agent having an aldehyde-reactive group.

[0392] In a particular embodiment, the immune complex of the second embodiment is (a) A step of oxidizing a CD123 / IL-3Rα binder having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde group, (b) This can be prepared by a fifth method, which includes reacting an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde with a linker compound having an aldehyde-reactive group to form a modified CD123 / IL-3Rα binder having a linker to which the linker binds, and then reacting the modified CD123 / IL-3Rα binder with a cytotoxic agent.

[0393] In a particular embodiment, the immune complex of the second embodiment is (a) A step of oxidizing a CD123 / IL-3Rα binder having an N-terminal 2-hydroxyethylamine moiety (e.g., Ser / Thr) with an oxidizing agent to form an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde group, (b) A sixth method can be prepared, comprising contacting an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde with a cytotoxic agent, followed by adding a linker compound cell having an aldehyde-reactive group.

[0394] In one embodiment, for the first or fourth method described above, the cytotoxic agent having an aldehyde-reactive group is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where J CB The formula is as follows: [ka] It is represented as, The remaining variable elements are as described above in any one of the first to 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 described above, the cytotoxic agent having an aldehyde-reactive group is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, J CB The above is true, and the remaining variable elements are as described above in any one of the 19th to 25th specific embodiments, and as in any further specific embodiments described herein.

[0396] In one embodiment, for the second, third, fifth, or sixth method described above, the linker compound is of the following formula: [ka] It is represented 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 It is an alkyl group, and J CB As stated above, the cytotoxic agent is given by the following formula: [ka] It is represented as, The remaining variable elements are as described above in any one of the eighth to thirteenth and twenty-fifth specific embodiments, 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 of the above formula (L s Represented by a), the cytotoxic compound is given by the following formula: [ka] It is represented as, The remaining variable elements are as described in any one of the 14th to 18th and 25th specific embodiments, and as in any further specific embodiments described herein.

[0398] In one embodiment, in the first or fourth method described above, the cytotoxic agent is reacted with an imine-reactive reagent such as NaHSO3 to form a modified cytotoxic agent before reacting with an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde. In one embodiment, the modified cytotoxic agent is not purified before reacting with oxidized CBA having an N-terminal aldehyde. Alternatively, the modified cytotoxic agent is purified before reacting with 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, and then reacted with a modified CD123 / IL-3Rα binder having a linker to which it binds. In one embodiment, the modified cytotoxic agent is purified and then reacted with a modified CD123 / IL-3Rα binder having a linker to which it binds. Alternatively, the modified cytotoxic agent is not purified before reacting with an oxidized CD123 / IL-3Rα binder having an N-terminal aldehyde.

[0400] In yet another embodiment, the reaction of the oxidized CD123 / IL-3Rα binder with the cytotoxic agent and linker compound in the third or sixth method described above 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 molar equivalent of oxidizing agent can be used relative to the CD123 / IL-3Rα binder. In certain embodiments, about 2 to 100, 5 to 80, 10 to 50, 1 to 10, or 5 to 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. Or, when using 10 equivalents of oxidizing agent, 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).

[0403] In certain embodiments, the 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 in the art can be used. In one embodiment, the catalyst is aniline or substituted aniline. Typical 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 in step (b) is carried out at a pH of approximately 5.0.

[0404] In certain embodiments, for the reaction of the first, second, or third method described above, or for 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 as described herein) is used in molar excess relative to the oxidative cell binder (e.g., an oxidative antibody or an oxidative antigen binding moiety). In certain embodiments, the ratio of the compound having an aldehyde-reactive group to the oxidative cell binder is about 10:1 to about 1.1:1, and about 5:1 to about 2:1. In one embodiment, the ratio is about 4:1.

[0405] In a third embodiment, the immune complex of the present invention comprises a CD123 / IL-3Rα binder (including an antibody, its antigen-binding fragment, or a polypeptide comprising an antibody or its antigen-binding fragment) as described in the first embodiment of the present invention, which is covalently bound to the cytotoxic agent described herein by thiol groups (-SH) of one or more cysteine ​​residues present in the CD123 binder.

[0406] In a particular first embodiment, the immune complex of the third embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy C1 A CD123 / IL-3Rα binder according to the first aspect of the present invention, which is covalently bound by a hecysteine ​​residue (for example, the target antibody or its antigen-binding fragment, or the target polypeptide described above), Wc is 1 or 2, Cy C1 The formula is as follows: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, however, 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 amine protecting moiety, Y is -OH or -SO3M, and M is H + or provided that it is a 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 In each occurrence, Q is independently -H, (C1-C3) alkyl, or a charged substituent or ionic group. W' is -NR e’ And, R e’ is -(CH2-CH2-O) n -R k And, n is an integer between 2 and 6. R k is -H or -Me, R x3 It is an (C1-C6) alkyl group, Lc is [ka] It is represented as, In the formula, s1 is the site that is covalently bonded to CBA, and s2 is Cy C1 It is a site that is covalently bonded to the -C(=O)- group, R 19 and R 20 In each occurrence, it is independently -H or (C1~C3)alkyl, m'' is an integer between 1 and 10. R h is -H or (C1~C3) alkyl.

[0407] In a second specific embodiment, for an immune complex of formula (C1), Cy C1This is represented by formula (C1a) or (C1a1), and the remaining variable elements are as described above in the first specific embodiment.

[0408] In a third specific embodiment, for an immune complex of formula (C1), Cy C1 This is represented by formula (C1b) or (C1b1), and the remaining variable elements are as described above in the first specific embodiment.

[0409] In a fourth specific embodiment, with respect to the immune complex of formula (C1), Cy C1 is expressed by equation (C1a) or (C1a1), R a and R b Both are H, R5 is either H or Me, and the remaining variable elements are as described above in the first or second specific embodiment.

[0410] In a fifth specific embodiment, for the immunocomplex of formula (C1), P is a peptide containing 2 to 5 amino acid residues, and the remaining variable elements are as described above in the first, second, or fourth specific embodiments. In further specific embodiments, P is Gly-Gly-Gly, Ala-Val, Val-Ala, Val-Cit, Val-Lys, Phe-L 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- The following are 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 further specific embodiment, P is Gly-Gly-Gly, Ala-Val, Ala-Ala, Ala-D-Ala, D-Ala-Ala, or D-Ala-D-Ala.

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

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

[0413] In the eighth specific embodiment, for the immunocomplex of formula (C1), -LL C - is expressed by the following formula: [ka] It is represented as, The remaining variable elements 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 specific ninth embodiment, the immune complex of the third embodiment is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] X represents a single bond or a double bond, however, when it is a double bond, X is absent and Y The condition is that when is -H and it is a single bond, X is -H and Y is -OH or -SO3M. In further specific embodiments, a double line between N and C. [ka] represents a double bond, where X is absent and Y is -H. In another further specific embodiment, the double line between N and C [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0415] In a specific tenth embodiment, the immune complex of the third embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy C2 A CD123 / IL-3Rα binder according to the first aspect of the present invention, which is covalently bound by a hecysteine ​​residue (for example, the target antibody or its antigen-binding fragment, or the target polypeptide described above), Wc is 1 or 2, Cy C2 The formula is as follows: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, Double line between N and C [ka] X represents a single or double bond, however, 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 amine protecting moiety, Y is -OH or -SO3M, and M is H + or provided that it is a cation, R x1 It is an (C1-C6) alkyl group, 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 between 2 and 6. R k is -H or -Me, R x2 It is an (C1-C6) alkyl group, Lc' is given by the following formula: [ka] It is represented as, During the ceremony, s1 is the site that is covalently bound to CBA, and s2 is Cy C2 It is a site that is covalently bonded to the -S- group, 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 In each occurrence, it is independently -H or (C1~C3)alkyl, q and r are independent integers between 0 and 10 in each occurrence. m and n are independent integers between 0 and 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 a further specific embodiment, q and r are each an integer from 1 to 6, more specifically an integer from 1 to 3. More specifically, R 10 , R 11 , R 12 and R 13 All of them are H.

[0417] In another further specific embodiment, m and n are each an integer from 1 to 6, more specifically an integer from 1 to 3. More specifically, R 19 , R 20 , R 21 and R 22 All of them are H.

[0418] In the 11th specific embodiment, for the immune complex of formula (C2), Cy C2 This is represented by formula (C2a) or (C2a1), and the remaining variable elements are as described above in the tenth specific embodiment or as in any further specific embodiment described herein.

[0419] In a 12th specific embodiment, for an immune complex of formula (C2), Cy C2 This is represented by formula (C2b) or (C2b1), and the remaining variable elements are as described above in the tenth specific embodiment.

[0420] In the 13th specific embodiment, for the immunocomplex of formula (C2), P' is a peptide containing 2 to 5 amino acid residues, and the remaining variable elements are as described in the 10th, 11th or 12th specific embodiment, or as in any further specific embodiment described herein. In further specific embodiments, P' is 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 further 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 14th specific embodiment, for the immune complex of formula (C2), -L C '- is represented by the following expression: [ka] It is represented as follows.

[0422] In a 15th specific embodiment, for an immune complex 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 Each is independently -H or (C1-C4)alkyl, p is 0, 1, 2, or 3, and the remaining variable elements are as described above in the 10th, 11th, 12th, 13th, or 14th specific embodiment. More specifically, R f and R g They are either identical or different, and are selected from -H and -Me.

[0423] In a specific sixteenth embodiment, the immune complex of the third embodiment is given by the following formula: [ka] [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula, a double line between N and C. [ka] The ∫ represents a single or double bond, provided 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 further specific embodiments, the double line between N and C [ka] represents a double bond, X is absent, and Y is -H. In another specific embodiment, N and C and Double line between them [ka] The symbol represents a single bond, where X is -H and Y is -SO3M.

[0424] In the 17th specific embodiment, the immune complex of the third embodiment is given by the following formula: [ka] It is represented as, During the ceremony, CBA is Cy C3 A CD123 / IL-3Rα binder according to the first aspect of the present invention, which is covalently bound by a hecysteine ​​residue (for example, the target antibody or its antigen-binding fragment, or the target polypeptide described above), Wc is 1 or 2, Cy C3 The formula is as follows: [ka] It is represented as, During the ceremony, m' is either 1 or 2. R1 and R2 are each independently -H or (C1~C3) alkyl. Lc' is given by the following formula: [ka] It is represented as, During the ceremony, s1 is the site that is covalently bound to CBA, and s2 is Cy C3 It is a site that is covalently bonded to the -S- group, 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 In each occurrence, it is independently -H or (C1~C3)alkyl, q and r are integers between 0 and 10 in each occurrence. m and n are independent integers between 0 and 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 a further specific embodiment, q and r are each an integer from 1 to 6, more specifically an integer from 1 to 3. More specifically, R 10 , R 11 , R 12 and R 13 All of them are H.

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

[0427] In the 18th specific embodiment, for the immunocomplex of formula (C3), P' is a peptide containing 2 to 5 amino acid residues, and the remaining variable elements are as described above in the 17th specific embodiment or in any further specific embodiment described herein. In further specific embodiments, P' is 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- The following are 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 further 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 specific embodiment of the 19th, for the immunocomplex of formula (C3), -L C '- is represented by the following expression: [ka] It is represented as, In the formula, M is H + Alternatively, the elements may be cations, and the remaining variable elements may be as described above in the 17th or 18th specific embodiment, or as in any further specific embodiment described herein.

[0429] In the 20th specific embodiment, for the immunocomplex of formula (C3), m' is 1, R1 and R2 are both H, and the remaining variable elements are as described above in the 17th, 18th or 19th specific embodiment, or as in any further specific embodiment described herein.

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

[0431] In a specific embodiment 22, the immune complex of the third embodiment is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where DM is given by the following formula: [ka] It is represented as follows.

[0432] In the 23rd specific embodiment, with respect to the immune complex of the third embodiment, M is H + na + or K + The remaining variable elements are as described in any one of the first to twenty-two specific embodiments, or as in any further specific embodiments described herein.

[0433] In any of the first to twenty-third specific embodiments described above, the target antibody or its antigen-binding fragment, or a polypeptide comprising the target antibody or its antigen-binding fragment, has a Cys residue at a position corresponding to the manipulated Cys in the heavy chain CH3 domain (i.e., the fifth residue from the end) of SEQ ID NO: 54 or 56. The target antibody or its antigen-binding fragment may include an immunoglobulin heavy chain region (HC) having the amino acid sequence described in SEQ ID NO: 54, and an immunoglobulin light chain variable region (LCVR) having the amino acid sequence described in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). The target antibody or its antigen-binding fragment may also include an Ig heavy chain region having the amino acid sequence described in SEQ ID NO: 56, and an Ig LCVR having the amino acid sequence described in SEQ ID NO: 33, 35, 37, or 41 (preferably SEQ ID NO: 35 or 37). In certain embodiments, the second residue from the N-terminus of SEQ ID NOs. 54 and 56 is Phe, and in certain other embodiments, the second residue from the N-terminus of SEQ ID NOs. 54 and 56 is Val.

[0434] The immune complex of the third embodiment described above (for example, the immune complex of any one of the first to twenty-third specific embodiments, or any further specific embodiment described herein) can be prepared by reacting a CBA having one or more free cysteine ​​with a cytotoxic agent having a thiol-reactive group as described herein.

[0435] In one embodiment, a cytotoxic agent having a thiol-reactive group is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, in the formula -L C C The formula is as follows: [ka] It is represented as, In the formula, the variable element is as described above in any one of the first to ninth and twenty-third specific embodiments, or as in any further specific embodiment described herein.

[0436] In another embodiment, a cytotoxic agent having a thiol-reactive group is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, where L C C ' is expressed by the following formula: [ka] It is represented as, In the formula, the variable element is as described above in any one of the 10th to 16th and 23rd specific embodiments, or as in any further specific embodiments described herein.

[0437] In yet another embodiment, a cytotoxic agent having a thiol-reactive group is given by the following formula: [ka] It is expressed as, or a pharmaceutically acceptable salt thereof, L C C ' is as described above, and the remaining variable elements are as described above in any one of the 17th to 23rd specific embodiments, or in any further specific embodiments described herein.

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

[0439] 4. Composition and Method of Use The present invention includes a composition (e.g., a pharmaceutical composition) comprising a target antibody or its antigen-binding fragment, or its immune complex (e.g., complexes of formulas (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), and a carrier (a pharmaceutically acceptable carrier). The present invention further includes a composition (e.g., a pharmaceutical composition) comprising a target antibody or its antigen-binding fragment, or complexes of formulas (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 composition is useful for inhibiting abnormal cell proliferation in mammals (e.g., humans) or for treating their proliferative disorders, including hematological cancers, leukemia, or lymphoma.

[0440] In particular, the present invention provides pharmaceutical compositions comprising one or more CD123 conjugates or their immune complexes as described herein. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable solvent. These pharmaceutical compositions find applications in inhibiting tumor growth and in treating cancers in human patients, including hematological malignancies, leukemia, or lymphoma.

[0441] In certain embodiments, formulations are prepared for storage and use by combining the purified antibody or its immunocomplex of the present invention with a pharmaceutically acceptable solvent (e.g., a carrier, an excipient) (Remington, The Science and Practice of Pharmacy 20th Edition, Mack Publishing, 2000). Suitable pharmaceutically acceptable solvents include non-toxic buffers (e.g., phosphates, citrates, 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; alkylparabens (e.g., methyl or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (e.g., less than 10 amino acid residues); proteins (e.g., serum albumin, gelatin, or immunoglobulin); hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamate). This includes, but is not limited to, mine, asparagine, histidine, arginine, or lysine; carbohydrates (e.g., monosaccharides, disaccharides, glucose, mannose, or dextrin); 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 nonionic surfactants (e.g., TWEEN or polyethylene glycol (PEG)).

[0442] The pharmaceutical compositions described herein may be administered in any number of ways for local or systemic treatment. Administration may be local (including 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 with a nebulizer; intratracheal, intranasal, epidermal, and transdermal); oral; or parenteral, including intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., subarachnoid or intraventricular) administration. In some specific embodiments, administration is intravenous. The pharmaceutical compositions described herein may also be used in vitro or ex vivo.

[0443] The antibody or immune complex of the present invention can be combined with a second compound (for example, one known to be effective in treating the disease or disorder of interest) in a drug combination formulation or administration regimen as a combination therapy. In some embodiments, the second compound is an anticancer agent. In some embodiments, the method involves the administration of the second compound and the immune complex of the present invention, which yields a better effect compared to the administration of the immune complex alone. The second compound can be administered by any number of methods, including, for example, localization, pulmonary, oral, parenteral, or intracranial administration. In some embodiments, the administration is oral. In some embodiments, the administration is intravenous. In some embodiments, the administration is both oral and intravenous.

[0444] Antibodies or immune complexes can also be combined with analgesics or other drugs in combination drug formulations or administration regimens as part of a combination therapy.

[0445] Antibodies or immune complexes can be combined with a second compound having anticancer properties in a combination drug formulation or administration regimen as a combination therapy. The second compound in the combination drug formulation or administration regimen may have complementary activity to the combined ADC so that they do not adversely affect each other. Pharmaceutical compositions comprising a CD123 conjugate and a second anticancer agent are also provided.

[0446] The present invention provides a method for inhibiting abnormal cell proliferation in a mammal (e.g., human) or for treating a proliferative disorder thereof, comprising administering to the mammal a therapeutically effective amount of a target antibody or its antigen-binding fragment, or an immune complex (e.g., a complex of formulas (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)), or a composition thereof, alone or in combination with a second therapeutic agent.

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

[0448] For example, cancer includes acute myeloid leukemia (AML including CD33-low AML, P-glycoprotein-positive AML, relapsed AML, or refractory AML), chronic myeloid leukemia (CML) including CML and acute transformation of Abelson's oncogene associated with CML (Bcr-ABL translocation), and myelodysplastic syndrome. The following can be selected from the group consisting of MDS, acute B-lymphoblastic leukemia or B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) including Richter syndrome or Richter transformation of CLL, pilocytic cell leukemia (HCL), acute promyelocytic leukemia (APL), B-cell chronic lymphoproliferative disorder (B-CLPD), atypical chronic lymphocytic leukemia (preferably with marked CD11c expression), blastocytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL) including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin lymphoma, systemic mastocytosis, and Burkitt lymphoma.

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

[0450] In certain embodiments, cancer has at least one negative prognostic factor (e.g., overexpression of P-glycoprotein, overexpression of EVI1, p53 alteration, DNMT3A mutation, or intracellular duplication of the FLT3 gene).

[0451] In certain embodiments, an effective therapeutic amount of the target antibody or its antigen-binding fragment, or immune complex (e.g., complexes of formulas (L1), (L2), (L3), (S1), (S2), (S3), (S4), (C1), (C2), and (C3)) or a composition thereof, either alone or in combination with a second therapeutic agent, selectively inhibits the proliferation of leukemic stem cells (LSCs), leukemic progenitor cells (LPs), and / or leukemic blasts, exceeding the proliferation of normal hematopoietic stem cells (HSCs). In certain embodiments, IC of the above-mentioned target agent inhibiting the proliferation of leukemic stem cells (LSCs), leukemic progenitor cells (LPs), and / or leukemic blasts 50 The value or semi-maximal concentration is at least 1 / 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 300, or 500th of that of normal hematopoietic stem cells (HSCs).

[0452] In certain embodiments, the anti-leukemia treatment of the present invention not only targets and kills leukemic blasts, but preferably also targets and kills leukemic progenitor cells (LPs) and leukemic stem cells (LSCs). In certain embodiments, the treatment is further less selective than normal HSCs. In certain embodiments, CD123 expression in LSCs, LPs, and leukemic blasts is much higher than that in normal lymphocytes (which may be nearly negative) (e.g., at least 20-25 times higher in LSCs, AML progenitor cells, and AML blasts). In certain embodiments, the CD123 expression levels in LPs and LSCs are at least the same as those in leukemic blasts.

[0453] Similarly, the present invention provides a method for inducing cell death in a selected cell population, comprising contacting target cells or tissue containing target cells with an effective amount of the target antibody of the present invention, its antigen-binding fragment, or an immune complex. The target cells are cells to which the cell-binding agent of the complex can bind.

[0454] If necessary, other activators (e.g., other antitumor agents) can be administered together with the complex.

[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 U.S. Physician's Desk Reference (PDR). The PDR discloses the dosages of drugs that have been used in the treatment of various cancers. The therapeutically effective drug regimens and dosages of these aforementioned chemotherapy drugs can be determined by the physician, depending on the specific cancer being treated, the extent of the disease, and other factors well known to physicians in the art. The contents of the PDR are expressly incorporated herein by reference in their entirety. Those skilled in the art will be able to determine the drug regimens and dosages of chemotherapy drugs and conjugates that can be used in accordance with the teachings of this invention. A PDR can be considered using one or more of the following parameters. These parameters include a general index, manufacturer, product (by company or trademarked drug name), classification index, general / chemical index (untrademarked common drug name), color image of the drug, product information matching FDA labeling, chemical information, function / action, efficacy and contraindications, clinical trials, side effects, and warnings.

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

[0457] The conditions for non-clinical in vitro use can be easily determined by those skilled in the art.

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

[0459] For clinical in vivo use, the cytotoxic compounds or complexes of the present invention are supplied as solutions or lyophilized powders for testing sterility and endotoxin concentration.

[0460] Suitable pharmaceutically acceptable carriers, diluents, and excipients are well known and can be determined by those skilled in the art as a guarantee of clinical status. Examples of suitable carriers, diluents, and / or excipients include (1) Dulbecco's phosphate-buffered saline with a pH of approximately 7.4 containing or not containing human serum albumin at a concentration of approximately 1 mg / mL to 25 mg / mL, (2) 0.9% saline (0.9% w / v NaCl), and (3) 5% (w / v) dextrose, which may also contain antioxidants such as tryptamine and stabilizers such as Tween20.

[0461] The present invention's methods for inducing cell death in a selected cell population, inhibiting cell proliferation, and / or treating cancer can be carried out in vitro, in vivo, or ex vivo. [Examples]

[0462] Example 1: Generation of mouse monoclonal antibodies against human and cynomolgus monkey CD123 antigens To produce a mouse anti-CD123 antibody (wild-type BALB / c female mouse) (Charles River Laboratory, Wilmington, MA), a human CD123-expressing stable 300-19 cell line (BALB / c-derived B progenitor cell line (MGReth et al. 1985, Nature, 317:353-355)) was administered in PBS every two weeks for five doses at a rate of 5 × 10⁶. 6 Cells / mouse doses were administered subcutaneously. Three days prior to sacrifice for hybridoma formation, immunized mice received intraperitoneal infusion of another dose of the antigen. Splenes from mice were harvested according to standard animal protocols, ground between two sterile frosted microscope slides, and placed in RPMI-1640 medium. A single-cell suspension was obtained. After lysing the erythrocytes with ACK lysis buffer, the spleen cells were mixed with mouse myeloma P3X63Ag8.653 cells (P3 cells) (JF Kearney et al., 1979, J. Immunol, 123:1548-1550) in a ratio of P3 cells to spleen cells of 1:3. The mixture of spleen cells and P3 cells 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 fusion cells were slowly added to RPMI-1640 selective medium (Sigma Aldrich) containing hypoxanthine-aminopterin-thymidine (HAT) and incubated at 37°C for 20 minutes, then seeded at 200 μL / well in flat-bottom 96-well plates. The plates were then incubated in a 5% CO2 incubator at 37°C until the hybridoma clones were ready for antibody screening. Other immunization and hybridoma creation techniques can also be used, including those described in 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 flow cytometry-coupled assays with human CD123-expressing stable 300-19 cell lines 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 in a 1:1 ratio, and incubated on ice for 2 hours in hybridoma supernatant. The cells were then washed, incubated with PE-labeled anti-mouse IgG (Jackson Immunoresearch), washed, fixed with formalin, and analyzed using FACS sequencing (BD Bioscience). Hybridomas exhibiting specific reactivity to the human CD123 antigen were grown, and the supernatants were re-screened using flow cytometry-coupled assays with three independent cell lines: human CD123-expressing stable 300-19 cell lines, cynomolgus monkey CD123-expressing stable 300-19 cell lines, and wild-type 300-19 cell lines. Hybridomas that exhibited positive binding to human and cynomolgus monkey CD123 antigens but were negative in wild-type 300-19 cells were further subcloned by limiting dilution. One subclone from each hybridoma that showed specific binding to human and cynomolgus monkey CD123 antigens was selected for subsequent analysis.

[0464] A total of six fusions were performed during this study. Approximately 6,000 hybridomas were screened to create 33 hybridomas specific to human and cynomolgus monkey CD123 antigens, and 18 hybridomas were subcloned. Stable subclones were cultured, and the isotype of the monoclonal antibody was identified using a commercially available mouse IgG isotyping reagent (e.g., IsoStrip Mouse Monoclonal Antibody Isotyping Kit, product no. 11493027001, Roche Diagnostics GmbH (Germany)).

[0465] Antibody purification The antibodies were purified from hybridoma subclonal supernatants using standard methods such as protein A or G chromatography (HiTrap protein A or G HP, 1 mL, Amersham Biosciences). In short, the supernatant was 1 The supernatant was prepared for chromatography by adding 1 / 10th of M 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 conjugation buffer (PBS, pH 7.3). The column was washed with conjugation buffer until a stable baseline with no absorption at 280 nm was obtained. The antibody was eluted with 0.1 M acetate buffer containing 0.15 M NaCl (pH 2.8) at a flow rate of 0.5 mL / min. Approximately 0.25 mL of the fraction was collected and neutralized by adding 1 / 10th of 1 M Tris / HCl (pH 8.0). The 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 A280.

[0466] The purified protein A fraction was further polished using ion-exchange chromatography (IEX) equipped with quaternary ammonium (Q) chromatography for mouse antibodies. Briefly, the sample from the purified protein A was buffer-exchanged to 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 Q fast flow resin (GE Lifesciences) equilibrated with binding buffer at a flow rate of 120 cm / hour. The column size was selected to have sufficient volume to bind all Mabs in the sample. The column was then washed with binding buffer until a stable baseline with no absorption at 280 nm was obtained. The antibody was eluted by initiating a gradient of 10 mM to 500 mM sodium chloride at 20 column volumes (CV). Peak fractions were collected based on 280 nm (A280) absorption measurements. Monomer content was assessed by size exclusion ...

Claims

1. The following formula: 【Chemistry 1】 An immune complex represented by or a pharmaceutically acceptable salt thereof, wherein, Wc is 1 or 2, Double line between N and C 【Chemistry 2】 The symbol represents a single bond, where X is -H and Y is -SO ３ M is H ＋ Na ＋ , or K ＋ And CBA is as follows: (a) an immunoglobulin heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 5, CDR2 having the amino acid sequence of SEQ ID NO: 8, and CDR3 having the amino acid sequence of SEQ ID NO: 11; and (b) Immunoglobulin light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 20, CDR2 having the amino acid sequence of SEQ ID NO: 21, and CDR3 having the amino acid sequence of SEQ ID NO: 22 It is an anti-CD123 antibody that includes The aforementioned immune complex.

2. M is Na ＋ The immune complex according to claim 1.

3. M is H ＋ The immune complex according to claim 1.

4. CBA states the following: a) A variable region of an immunoglobulin heavy chain having the amino acid sequence described in SEQ ID NO: 34; and b) Immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 35 The immune complex according to claim 2 or 3, comprising an anti-CD123 antibody.

5. The immune complex according to claim 4, wherein Xaa, the second residue from the N-terminus of sequence number 34, is Phe.

6. The immunocomplex according to claim 4, wherein Xaa, the second residue from the N-terminus of sequence number 34, is Val.

7. CBA states the following: a) A variable region of an immunoglobulin heavy chain having the amino acid sequence described in SEQ ID NO: 54; and b) Immunoglobulin light chain variable region having the amino acid sequence described in SEQ ID NO: 51 The immune complex according to claim 2 or 3, comprising an anti-CD123 antibody.

8. The immunocomplex according to claim 7, wherein Xaa, the second residue from the N-terminus of sequence number 54, is Phe.

9. The immunocomplex according to claim 7, wherein Xaa, the second residue from the N-terminus of sequence number 54, is Val.

10. A pharmaceutical composition comprising an immune complex according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier.

11. An immune complex according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10, for use in a method for treating a subject having cancer, or for use in a method for treating a cell proliferation disorder of a subject.

12. The immune complex or pharmaceutical composition according to claim 11: (i) The cancer is leukemia or lymphoma; (ii) The cancer is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); (iii) The cancer is acute myeloid leukemia (AML); (iv) The cancer is B-cell acute lymphoblastic leukemia (B-ALL); or (v) The cell proliferative disorder is selected from the group consisting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), pilocytic cell leukemia (HCL), myelodysplastic syndrome, blastic plasmacytoid dendritic cell neoplasm (BPDCN) leukemia, non-Hodgkin lymphoma (NHL), mantle cell lymphoma, and Hodgkin leukemia (HL). The aforementioned immune complex or pharmaceutical composition.

13. The immunocomplex or pharmaceutical composition according to claim 12, wherein the method is for treating blastic plasmacytoid dendritic cell neoplasm (BPDCN).

14. The immune complex or pharmaceutical composition according to claim 12, wherein the method is for treating acute myeloid leukemia (AML).

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