CD123 antibody-drug conjugates and methods of use thereof

Abstract

The present disclosure relates generally to antibodies, antigen-binding fragments, polypeptides, and immunoconjugates thereof that bind to CD123 antigen (the alpha chain of interleukin-3 receptor) and deliver a payload to CD123-expressing cells. The effective load is a topoisomerase inhibitor, and the topoisomerase inhibitor effectively kills tumor cells expressing CD123 and protects hematopoietic stem cells and mature hematopoietic cells at the same time.

Description

[0001] Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 516,678, filed July 31, 2023; No. 63 / 607,876, filed December 8, 2023; and No. 63 / 635,835, filed April 18, 2024, each of which is incorporated herein by reference.

[0003] References to sequence lists

[0004] The entire contents of the sequence list (name: CD123-100-WO-PCT_ST26.xml; size: 90.9KB; and creation date: April 11, 2024) filed electronically in this application are incorporated herein by reference. Background Technology

[0005] Antibody-drug conjugates (ADCs) have become a powerful tool in the fight against various types of diseases, including cancer. The antibody component of an ADC mediates selective targeting of specific cells, while the cytotoxic drug component allows for the selective killing of the targeted cells. However, the efficiency of target cell killing and the stability of ADCs remain unresolved issues.

[0006] CD123 is expressed in hematologic malignancies, but its expression is limited in normal hematopoietic cells. CD123 has been targeted by antibody-drug conjugates (ADCs) (e.g., CD123-alkylating agent ADCs, such as pivekimab sunirine), but low stability and high toxicity necessitate the use of low doses and low response rates. Therefore, there is a need for improved CD123-based therapies. Summary of the Invention

[0007] An antibody comprising an antigen-binding domain or an antigen-binding fragment thereof is provided, the antigen-binding domain comprising: (i) a variable heavy chain region (VH), the variable heavy chain region comprising: a VH complementarity-determining region (CDR)1 comprising an amino acid sequence selected from SEQ ID NO: 1 and 9, a VH-CDR2 comprising an amino acid sequence selected from SEQ ID NO: 2 and 10, and a VH-CDR3 comprising an amino acid sequence selected from SEQ ID NO: 3 and 11, and (ii) a variable light chain region (VL), the variable light chain region comprising: a VL-CDR1 comprising an amino acid sequence selected from SEQ ID NO: 4 and 12, a VL-CDR2 comprising an amino acid sequence selected from SEQ ID NO: 5 and 13, and a VL-CDR3 comprising an amino acid sequence selected from SEQ ID NO: 6 and 14.

[0008] In some respects, the antibodies or their antigen-binding fragments described herein specifically bind to CD123.

[0009] In some respects, the antigen-binding domain comprises Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof.

[0010] In some respects, the antigen-binding domain contains scFv.

[0011] In some aspects, the antigen-binding domain comprises: VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0012] In some aspects, the antigen-binding domain comprises: VH-CDR1 containing the amino acid sequence shown in SEQ ID NO: 9, VH-CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, VH-CDR3 containing the amino acid sequence shown in SEQ ID NO: 11, VL-CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, VL-CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and VL-CDR3 containing the amino acid sequence shown in SEQ ID NO: 14.

[0013] In some respects, the antigen-binding domain includes a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 7 and 15.

[0014] In some respects, the antigen-binding domain comprises: a VH containing an amino acid sequence selected from SEQ ID NO: 7 and 15.

[0015] In some respects, the antigen-binding domain includes a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 8 and 16.

[0016] In some respects, the antigen-binding domain comprises a VL containing an amino acid sequence selected from SEQ ID NO: 8 and 16.

[0017] In some aspects, the antigen-binding domain comprises: VH, which contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and VL, which contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0018] In some aspects, the antigen-binding domain comprises: VH, which comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; and VL, which comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16.

[0019] In some respects, the antigen-binding domain comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 7 and a VL containing the amino acid sequence shown in SEQ ID NO: 8.

[0020] In some respects, the antigen-binding domain comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 15 and a VL containing the amino acid sequence shown in SEQ ID NO: 16.

[0021] An antibody or an antigen-binding fragment thereof comprising an antigen-binding domain is also provided, the antigen-binding domain comprising:

[0022] VH, comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 64, 68, 70, 72, 74, 76, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114; and

[0023] VL, which contains an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with an amino acid sequence selected from SEQ ID NO: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, and 115.

[0024] In some respects, the antigen-binding domain of an antibody or antigen-binding fragment includes:

[0025] VH, which contains the amino acid sequence shown in SEQ ID NO: 64, and VL, which contains the amino acid sequence shown in SEQ ID NO: 65;

[0026] VH, which contains the amino acid sequence shown in SEQ ID NO: 68, and VL, which contains the amino acid sequence shown in SEQ ID NO: 69;

[0027] VH, which contains the amino acid sequence shown in SEQ ID NO: 70, and VL, which contains the amino acid sequence shown in SEQ ID NO: 71;

[0028] VH, which contains the amino acid sequence shown in SEQ ID NO: 72, and VL, which contains the amino acid sequence shown in SEQ ID NO: 73;

[0029] VH, which contains the amino acid sequence shown in SEQ ID NO: 74, and VL, which contains the amino acid sequence shown in SEQ ID NO: 75;

[0030] VH, which contains the amino acid sequence shown in SEQ ID NO: 76, and VL, which contains the amino acid sequence shown in SEQ ID NO: 77;

[0031] VH, which contains the amino acid sequence shown in SEQ ID NO: 80, and VL, which contains the amino acid sequence shown in SEQ ID NO: 81;

[0032] VH, which contains the amino acid sequence shown in SEQ ID NO: 82, and VL, which contains the amino acid sequence shown in SEQ ID NO: 83;

[0033] VH, which contains the amino acid sequence shown in SEQ ID NO: 88, and VL, which contains the amino acid sequence shown in SEQ ID NO: 89;

[0034] VH, which contains the amino acid sequence shown in SEQ ID NO: 90, and VL, which contains the amino acid sequence shown in SEQ ID NO: 91;

[0035] VH, which contains the amino acid sequence shown in SEQ ID NO: 92, and VL, which contains the amino acid sequence shown in SEQ ID NO: 93;

[0036] VH, which contains the amino acid sequence shown in SEQ ID NO: 94, and VL, which contains the amino acid sequence shown in SEQ ID NO: 95;

[0037] VH, which contains the amino acid sequence shown in SEQ ID NO: 96, and VL, which contains the amino acid sequence shown in SEQ ID NO: 97;

[0038] VH, which contains the amino acid sequence shown in SEQ ID NO: 98, and VL, which contains the amino acid sequence shown in SEQ ID NO: 99;

[0039] VH, which contains the amino acid sequence shown in SEQ ID NO: 100, and VL, which contains the amino acid sequence shown in SEQ ID NO: 101;

[0040] VH, which contains the amino acid sequence shown in SEQ ID NO: 102, and VL, which contains the amino acid sequence shown in SEQ ID NO: 103;

[0041] VH, which contains the amino acid sequence shown in SEQ ID NO: 104, and VL, which contains the amino acid sequence shown in SEQ ID NO: 105;

[0042] VH, which contains the amino acid sequence shown in SEQ ID NO: 106, and VL, which contains the amino acid sequence shown in SEQ ID NO: 107;

[0043] VH, which contains the amino acid sequence shown in SEQ ID NO: 108, and VL, which contains the amino acid sequence shown in SEQ ID NO: 109;

[0044] VH, which contains the amino acid sequence shown in SEQ ID NO: 110, and VL, which contains the amino acid sequence shown in SEQ ID NO: 111;

[0045] VH containing the amino acid sequence shown in SEQ ID NO: 112 and VL containing the amino acid sequence shown in SEQ ID NO: 113; or

[0046] VH contains the amino acid sequence shown in SEQ ID NO: 114 and VL contains the amino acid sequence shown in SEQ ID NO: 115.

[0047] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:64 and a VL containing the amino acid sequence shown in SEQ ID NO:65.

[0048] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:68 and a VL containing the amino acid sequence shown in SEQ ID NO:69.

[0049] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:70 and a VL containing the amino acid sequence shown in SEQ ID NO:71.

[0050] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:72 and a VL containing the amino acid sequence shown in SEQ ID NO:73.

[0051] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:74 and a VL containing the amino acid sequence shown in SEQ ID NO:75.

[0052] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:76 and a VL containing the amino acid sequence shown in SEQ ID NO:77.

[0053] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:80 and a VL containing the amino acid sequence shown in SEQ ID NO:81.

[0054] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:82 and a VL containing the amino acid sequence shown in SEQ ID NO:83.

[0055] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:88 and a VL containing the amino acid sequence shown in SEQ ID NO:89.

[0056] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 90 and a VL containing the amino acid sequence shown in SEQ ID NO: 91.

[0057] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:92 and a VL containing the amino acid sequence shown in SEQ ID NO:93.

[0058] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:94 and a VL containing the amino acid sequence shown in SEQ ID NO:95.

[0059] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:96 and a VL containing the amino acid sequence shown in SEQ ID NO:97.

[0060] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO:98 and a VL containing the amino acid sequence shown in SEQ ID NO:99.

[0061] In some aspects, the antibody-binding domain (e.g., an antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 100 and a VL containing the amino acid sequence shown in SEQ ID NO: 101.

[0062] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 102 and a VL containing the amino acid sequence shown in SEQ ID NO: 103.

[0063] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 104 and a VL containing the amino acid sequence shown in SEQ ID NO: 105.

[0064] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 106 and a VL containing the amino acid sequence shown in SEQ ID NO: 107.

[0065] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 108 and a VL containing the amino acid sequence shown in SEQ ID NO: 109.

[0066] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 110 and a VL containing the amino acid sequence shown in SEQ ID NO: 111.

[0067] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 112 and a VL containing the amino acid sequence shown in SEQ ID NO: 113.

[0068] In some aspects, the antibody-binding domain (e.g., antibody or antibody-binding fragment) comprises: a VH containing the amino acid sequence shown in SEQ ID NO: 114 and a VL containing the amino acid sequence shown in SEQ ID NO: 115.

[0069] A polynucleotide is also provided that encodes the antibody or antigen-binding fragment thereof described herein.

[0070] A cell comprising the polynucleotides described herein is also provided.

[0071] Another formula I is provided: A - (DL) p The conjugate or its pharmaceutically acceptable salt or solvate, wherein A is the antibody or its antigen-binding fragment described herein, and DL is a drug linker unit having Formula II:

[0072] .

[0073] In some respects, R L It is a connector used to attach to an antibody or its antigen-binding fragment, wherein R L Having formula IIa:

[0074] .

[0075] In some respects, Q of equation IIa is:

[0076] .

[0077] In some respects, Q's Q X As follows: Q is an amino acid residue, dipeptide residue, tripeptide residue, or tetrapeptide residue, where the superscript label is... C(=O) and NH Indicates the group to which the atom is bonded.

[0078] In some respects, X of equation IIa is:

[0079] .

[0080] In some respects, in X, a is 0 to 5, b1 is 0 to 16, b2 is 0 to 16, c1 is 0 or 1, and d is 0 to 5; and GL is a connector for linking to the antibody or its antigen-binding fragment described herein.

[0081] In some respects, Q in formula IIa is selected from the following amino acid residues: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0082] In some respects, Q in formula IIa is selected from the following dipeptide residues: NH-Phe-Lys-C=O, NH-Val-Ala-C=O, NH-Val-Lys-C=O, NH-Ala-Lys-C=O, NH-Val-Cit-C=O, NH-Phe-Cit-C=O, NH-Leu-Cit-C=O, NH-Ile-Cit-C=O, NH-Phe-Arg-C=O, NH-Trp-Cit-C=O, and NH-Gly-Val-C=O.

[0083] In some respects, Q in formula IIa is selected from the following tripeptide residues: NH-Glu-Val-Ala-C=O, NH-Glu-Val-Cit-C=O, NH-αGlu-Val-Ala-C=O and NH-αGlu-Val-Cit-C=O.

[0084] In some respects, Q in formula IIa is selected from the following tetrapeptide residues: NH-Gly-Gly-Phe-Gly-C=O; and NH-Gly-Phe-Gly-Gly-C=O, where NH represents the N-terminus of the residue and C=O represents the C-terminus of the residue.

[0085] In some respects, the a of X is 0 to 3; 0 or 1.

[0086] In some respects, b1 of X is 0 to 8; 0; 2; 3; 4; 5; or 8.

[0087] In some respects, the b2 of X is 0 to 8; 2; 3; 4; 5; or 8.

[0088] In some respects, c1 of X is 0; 1; or 2.

[0089] In some respects, the d of X is 0 to 3; 0, 1, or 2.

[0090] In some respects, a is 0, b1 is 0, c1 is 1, d is 2, and b2 is 0, 2, 3, 4, 5, or 8.

[0091] In some respects, a is 1, b2 is 0, c1 is 0, d is 0, and b1 is 0, 2, 3, 4, 5, or 8.

[0092] In some respects, a is 0, b1 is 0, c1 is 0, d is 1, and b2 is 0, 2, 3, 4, 5, or 8.

[0093] In some respects, b1 is 0, b2 is 0, c1 is 0, one of a and d is 0, and the other of a and d is 1 or 5.

[0094] In some respects, a is 1, b2 is 0, c1 is 0, d is 2, and b1 is 0, 2, 3, 4, 5, or 8.

[0095] In some respects, a is 0, b1 is 0, b2 is 8, c1 is 1, and d is 0.

[0096] In some respects, GL is

[0097] .

[0098] In some respects, Q is NH-Val-Ala-C=O.

[0099] In some aspects, X's a is 1. In some aspects, X's b1 is 8. In some aspects, X's c1 is 1. In some aspects, X's d is 1. In some aspects, a is 0. In some aspects, b1 is 0. In some aspects, c1 is 1. In some aspects, d is 0. In some aspects, a is 0, b1 is 0, b2 is 8, c1 is 1, and d is 0.

[0100] In some respects, equation IIa is:

[0101] .

[0102] In some respects, the CD123-binding antibody described in this article is an IgG antibody.

[0103] In some respects, at least one DL unit binds to the hinge region of the IgG antibody.

[0104] In some respects, one to three DL units bind to the heavy chain of the IgG antibody.

[0105] In some respects, a DL unit binds to the light chain of an IgG antibody.

[0106] In some respects, one to three DL units bind to each heavy chain of the IgG antibody, and one DL unit binds to each light chain of the IgG antibody.

[0107] In some respects, Formula II is a form of single enantiomer or enantiomer enrichment.

[0108] In some respects, p in Equation I is an integer from 1 to 20. In some respects, p is an integer from 1 to approximately 10. In some respects, p is 8.

[0109] In some respects, antibodies or their antigen-binding fragments are conjugated to...

[0110] (SG3932).

[0111] Mixtures of the conjugates described herein are also provided, wherein the average drug loading of each antibody or its antigen-binding fragment in the mixture of conjugates is from about 1 to about 10.

[0112] A pharmaceutical composition is provided comprising a conjugate or a mixture of conjugates and a pharmaceutically acceptable diluent, carrier, or excipient.

[0113] A polynucleotide sequence is also provided that encodes the antibody or antigen-binding fragment thereof described herein.

[0114] In some aspects, this disclosure provides a method for generating an antibody or antigen-binding fragment thereof that specifically binds to CD123, the method comprising culturing the cells described herein under suitable conditions.

[0115] In some aspects, this disclosure provides a method for producing the conjugates or mixtures of conjugates described herein, the method comprising conjugating an antibody or an antigen-binding fragment thereof to a drug linker unit.

[0116] In some aspects, this disclosure provides a method for treating proliferative diseases, the method comprising administering to a subject in need a therapeutically effective amount of the antibody or antigen-binding fragment thereof described herein, a polynucleotide, a carrier or a cell.

[0117] A method for treating proliferative disorders is also provided, comprising administering to a subject in need a therapeutically effective amount of the conjugate, mixture, or pharmaceutical composition described herein. In some respects, proliferative disorders are cancers.

[0118] In some respects, cancer is a hematologic cancer. In some respects, a hematologic cancer is leukemia or lymphoma. In some respects, cancer is acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, refractory anemia with excessive blasts, DLBCL non-Hodgkin lymphoma, marginal zone non-Hodgkin lymphoma, mantle zone non-Hodgkin lymphoma, follicular non-Hodgkin lymphoma, Hodgkin lymphoma, or minimal residual disease.

[0119] Also provided is the conjugate, mixture of conjugates, or pharmaceutical composition described herein for medical treatment.

[0120] Also provided is the conjugate, mixture, or pharmaceutical composition described herein for the treatment of proliferative diseases.

[0121] In some respects, conjugates, mixtures, or pharmaceutical compositions are used in the treatment of cancer.

[0122] The use of the conjugates, mixtures or pharmaceutical compositions described herein in the manufacture of medicaments for the treatment of proliferative diseases is also provided.

[0123] In some aspects, this disclosure provides an antibody-drug conjugate (ADC) comprising: (i) an antibody or antigen-binding fragment thereof that binds to CD123, the antibody or antigen-binding fragment comprising: HCDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; HCDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; HCDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; LCDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; LCDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and LCDR3 comprising the amino acid sequence shown in SEQ ID NO: 6;

[0124] (ii) One or more cysteine ​​residues of the antibody or antigen-binding fragment are covalently bound to the linker-payload via a succinimide thioether of the following formula:

[0125] ,

[0126] Furthermore, the ADC has a drug-to-antibody ratio (DAR) of approximately 8. In some aspects, the ADC comprises: a variable heavy (VH) chain comprising the amino acid sequence shown in SEQ ID NO: 7 and a variable light (VL) chain comprising the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the ADC comprises: a heavy chain (HC) comprising the amino acid sequence shown in SEQ ID NO: 116 and a light chain (LC) comprising the amino acid sequence shown in SEQ ID NO: 117. In some aspects, this disclosure provides a pharmaceutical composition comprising an ADC. In some aspects, this disclosure provides a method of treating cancer, the method comprising administering an ADC to a patient. In some aspects, the cancer is acute myeloid leukemia or myelodysplastic syndrome. In some aspects, this disclosure provides the ADC or pharmaceutical composition described herein for use in the treatment of cancer. In some aspects, the cancer is acute myeloid leukemia or myelodysplastic syndrome. In some aspects, the method herein further comprises administering venetoclax and / or a hypomethylating agent, optionally wherein said hypomethylating agent is 5-azacitidine or decitabine. Attached Figure Description

[0127] Figure 1A Flow cytometry analysis of bone marrow mononuclear cells (BMMCs) from live-gated healthy donors and BMMCs from AML patients is shown. Figure 1B The percentages of CD33 and CD123 expression are shown on healthy donor and AML BMMC cells. Figure 1C The binding capacity (ABC) of CD123 antibodies on hematopoietic cells from healthy donors and AML patients with leukemia is shown. Figure 1DThe study showed a high frequency of CD123+ cells in the bone marrow of AML patients compared to healthy donor bone marrow, as demonstrated by flow cytometry. Figure 1E This study shows increased CD123 expression in the bone marrow of AML patients, as indicated by Western blotting. Figure 1F The right panel shows the high prevalence of CD123 positivity in the bone marrow of AML patients compared to normal bone marrow (left panel).

[0128] Figure 2A This diagram illustrates the CD123 antibody topoisomerase inhibitor (Top1i) drug conjugate (ADC) and cysteine ​​conjugate site according to EU designation. Figure 2B The drug portion and linker-load are shown before and after the enzymatic release of the active Top1i payload from the linker-load.

[0129] Figure 3A The binding curves of the sensor-bound HT12-GL antibody and human IL3Ra (CD123) sample are shown. Figure 3B The binding curves of the sensor-bound HT12-GL antibody and human IL-5Ra sample are shown. Figure 3C Figure 3D shows the binding curves of the sensor-bound HT12-GL antibody and the GM-CSFRa sample. Figure 3E shows the binding curves of the sensor-bound J13 antibody and the human CD123 sample. Figure 3F shows the binding curves of the sensor-bound J13 antibody and the cynomolgus monkey CD123 sample. Figure 3G shows the binding curves of the sensor-bound HT12-GL antibody and the cynomolgus monkey CD123 sample.

[0130] Figure 4A The binding ability of HT12-GL antibody across AML cell lines is demonstrated. Figure 4B The binding affinity of the HT12-GL antibody to CD123 expressed in different species is shown. Figure 4C The results show that the HT12-GL antibody binds to EOL-1 cells compared to the control antibody NIP228. Figure 4D The results show that various HT antibody clones bind less or not at all to CD123-negative 293 cells. Figure 4E The binding of various HT antibody clones to 293 cells expressing human CD123 is shown. Figure 4F The binding of various HT antibody clones to 293 cells expressing cynomolgus monkey CD123 is shown. Figure 4G The binding of various HT antibody clones to Mv411 cells is shown. Figure 4H The binding of various HT antibody clones to OCI-AML5 cells is shown. Figure 4IThe lack of binding of various HT antibody clones to CD123-negative Jurkat cells was observed. Figure 4J The binding of HT and various 008Q antibody clones to 293 cells expressing human CD123 is shown. Figure 4K The binding of HT and various 008Q antibody clones to 293 cells expressing cynomolgus monkey CD123 is shown. Figure 4L The study showed a lack of binding between HT and various 008Q antibody clones and CD123-negative Jurkat cells.

[0131] Figure 5A The study showed that the HT12-GL antibody specifically binds to THP-1 cells with high CD123 expression induced by doxycycline (+dox), moderately binds to THP-1 cells with moderate CD123 expression, and does not bind to CD123-negative Hel92.1.7 cells. Figure 5B Microscopic images showing HT12-GL internalized in the lacrimal puncta (marked with arrows) into THP-1 cells expressing CD123. Figure 5C This study demonstrates that, using a flow-based approach, HT12-GL antibody and HT12-GL ADC (HT12-GL-SG3932) were mediated and lysosomal transported into CD123-expressing THP-1 cells over time, compared to control NIP228 antibody and NIP228 ADC. Figure 5D The results show that, compared to the control NIP228 antibody, the HT12-GL antibody undergoes time-dependent lysosomal transport into Molm13 cells. Figure 5E Various HT antibody clones were shown to be internalized into Molm13 cells.

[0132] Figure 6A This study demonstrated the ability of HT12-GL and some CD123 antibodies to inhibit IL-3-mediated cell proliferation. Figure 6B The results showed that GM-CSF did not affect TF-1 cell proliferation in the presence of CD123 antibody. Figure 6C The concentration of HT12-GL antibody required to inhibit IL-3-mediated cell proliferation is shown. Figure 6D The inhibition of IL-3-mediated STAT5 phosphorylation by HT12-GL antibody (CD123 MAb) and HT12-GL ADC ((HT12-GL-SG3932)) is shown.

[0133] Figure 7A The study demonstrated that HT12-GL ADC (HT12-GL-SG3932) caused cytotoxic killing of CD123-expressing cells, while the isotype NIP228 ADC did not cause killing of CD123-expressing cells. Figure 7BThe results showed that HT12-GL ADC (HT12-GL-SG3932) did not kill CD123-negative Hel92.1.7 cells. Figure 7C The study showed increased caspase 3 / 7 activity in CD123-expressing cells in the presence of HT12-GL ADC (HT12-GL-SG3932). Figure 7D The results showed that no caspase 3 / 7 activity was observed in CD123-negative cells in the presence of HT12-GL ADC (HT12-GL-SG3932). Figure 7E Flow cytometry images of THP-1 cells (CLEC GFP) with low CD123 expression are shown. Figure 7F Flow cytometry images of THP-1 cells (CLEC DOX) that highly express CD123 are shown. Figure 7G Flow cytometry images of co-cultures of THP-1 CLEC-GFP and THP-1 CLEC DOX cells are shown. Figure 7H This study demonstrates bystander killing of THP-1 cells (CLEC GFP) in the presence of HT12-GL ADC, with increasing levels of THP-1 cells (CLEC DOX) that highly express CD123. Figure 7I Western blot analysis of DNA damage response signaling proteins induced by HT12-GL ADC treatment is shown.

[0134] Figure 8A The results showed a relative lack of killing effect on CD123-negative Ad293 cells in the presence of various HT antibody ADCs (HT-SG3932). Figure 8B This demonstrates the killing effect of various HT antibody ADCs (HT-SG3932) on Ad293 cells ectopically expressing human CD123 in the presence of Ad293 cells compared to the control NIP228 ADC. Figure 8C This demonstrates the killing effect of various HT antibody ADCs on Ad293 cells ectopically expressing CD123 in cynomolgus monkeys in the presence of Ad293 cells, compared to the control NIP228 ADC. Figure 8D The results showed a relative lack of killing effect on CD123 low-THP-1 cells in the presence of various HT antibody ADCs and the control NIP228 ADC. Figure 8E This demonstrates the killing effect of various HT antibody ADCs on dox-induced THP-1 cell lines with high CD123 expression in the presence of THP-1 cells compared to the control NIP228 ADC.

[0135] Figure 9A The efficacy of HT12-GL ADC (HT12-GL-SG3932) compared with CD33 ADC Mylotarg and isotype ADC is shown in CFU assays of healthy donor bone marrow and bone marrow from AML patients. Figure 9B This demonstrates the treatment of healthy donor hematopoietic stem cells (HSCs: lineage-CD34) with HT12-GL ADC (HT12-GL-SG3932) in a bone marrow CD34+ BMMC amplification assay. + CD38 - CD90 + CD45RA - ), granulocyte-monocyte progenitor cells (GMP: lineage-CD34) + CD38 + CD135 + CD45RA + ) and differentiated cells (lineages) + The total cell count after 6 days. Figure 9C shows the relative lack of toxicity of HT12-GL ADC ((HT12-GL-SG3932)) to HUVECs compared to HT12-GL mAb, control ADC (NIP228 TOPO ADC), and comparative ADC-1. Figure 9D shows the relative lack of killing effect of HT12-GL ADC ((HT12-GL-SG3932)) on HPAECs compared to HT12-GL mAb, control ADC (NIP228 TOPO ADC), and comparative ADC-1. Figure 9E shows the relative lack of killing effect of HT12-GL ADC ((HT12-GL-SG3932)) on RPMI-8226. Figure 9F shows the relative lack of killing effect of HT12-GL ADC ((HT12-GL-SG3932)) on K562. Figure 9G shows the Western blot of CD123 measured in RPMI-8226, K562, HPAEC, and HUVEC cell lines. Figure 9H shows the surface expression of CD123 in RPMI-8226, HUVEC, HPAEC, and K562 cell lines as measured by flow cytometry.

[0136] Figure 10A Tumor volumes are shown in subcutaneous Mv4-11 xenograft models in untreated SCID mice and in SCID mice treated with increasing concentrations of HT12-GL ADC (HT12-GL-SG3932) or allotype antibody ADC. Figure 10B Tumor volumes in subcutaneous Mv4-11 xenograft models in untreated SCID mice and SCID mice treated with 2 mg / kg HT12-GL ADC ((HT12-GL-SG3932)), J13 ADC (J13-SG3932), or allotype control antibody ADC (NIP-228). Figure 10CTumor volumes are shown in subcutaneous EOL xenograft models in untreated nude mice and nude mice treated with HT12-GL antibody (CD123 mAb), HT12-GL ADC (HT12-GL-SG3932), or allotype antibody ADC. Figure 10D Tumor volumes are shown in subcutaneous EOL xenograft models in untreated nude mice and nude mice treated with HT12-GL antibody (CD123 mAb), HT12-GL ADC (HT12-GL-SG3932), or allotype antibody ADC.

[0137] Figure 11A Survival was shown in untreated mice and in a disseminated Mv4-11 xenograft model in mice treated with 5 mg / kg HT12-GL ADC (HT12) (HT12-GL-SG3932), J13 ADC (J13-SG3932), or allotype antibody ADC (NIP228). Figure 11B Survival was demonstrated in disseminated Mv4-11 xenograft models in untreated mice and in mice treated with a single or double dose of 5 mg / kg HT12-GL ADC or allotype antibody ADC.

[0138] Figure 12A Characterization of xenograft (PDX) models derived from AML patients is shown. Figure 12B The efficacy of 5 mg / kg HT12-GL ADC (HT12-GL-SG3932) in reducing CD45+ cells in whole blood of a disseminated AML PDX model was demonstrated. Figure 12C The efficacy of 5 mg / kg HT12-GL ADC (HT12-GL-SG3932) in reducing CD45+ cells in the bone marrow of a disseminated AML PDX model was demonstrated. Figure 12D The efficacy of 10 mg / kg HT12-GL ADC (HT12-GL-SG3932) in reducing CD45+ cells in whole blood of a disseminated AML PDX model was demonstrated. Figure 12E The efficacy of 10 mg / kg HT12-GL ADC (HT12-GL-SG3932) in reducing CD45+ cells in the bone marrow of a disseminated AML PDX model was demonstrated. Figure 12F The antileukemic efficacy of 5 mg / kg and 10 mg / kg HT12-GL ADC (HT12-GL-SG3932) in human CD123+ cells in the bone marrow of a reduced disseminated AML PDX model was demonstrated.

[0139] Figure 13A shows the total antibody concentration of HT12-GL ADC (HT12-GL-SG3932) as measured by ELISA versus LCMS. Figure 13B shows the antibody and payload stability of HT12-GL ADC (HT12-GL-SG3932) as measured by LCMS. Figure 13C shows the stability of the HT12-GL heavy and light chains. Figure 13D shows the payload stability of HT12-GL ADC.

[0140] Figure 14A The stability of HT12-GL ADC (HT12-GL-SG3932), J13 ADC (J13-SG3932) in mouse serum is shown. Figure 14B The stability of HT12-GL ADC (HT12-GL-SG3932) and J13 ADC (J13-SG3932) in cynomolgus monkey serum is shown. Figure 14C The stability of HT12-GL ADC (HT12-GL-SG3932) and J13 ADC (J13-SG3932) in rat serum is shown.

[0141] Figure 15A The cytotoxic synergistic effect of HT12-GL-SG3932 with venetoclax (VEN) is shown in vitro in both THP-1 and OCI-AML3 AML cell lines, expressed as a percentage of cell viability. Figure 15B The cytotoxic synergistic effect of HT12-GL-SG3932 with HMA (decitabine) is shown in vitro in both THP-1 and OCI-AML3 AML cell lines, expressed as a percentage of cell viability. Figure 15C The cytotoxic synergistic effect of HT12-GL-SG3932 with SoC (VEN and decitabine) in vitro is shown in both THP-1 and OCI-AML3 AML cell lines, expressed as a percentage of cell viability.

[0142] Figure 16 The cytotoxic synergistic effect of HT12-GL-SG3932 with cytarabine is shown in vitro in both THP-1 and Mv411 AML cell lines, expressed as a percentage of cell viability.

[0143] Figure 17A summary heatmap showing the synergistic scores of HT12-GL-SG3932 with VEN, HT12-GL-SG3932 with decitabine, HT12-GL-SG3932 with cytarabine, and HT12-GL-SG3932 with SoC (VEN and decitabine) in the eight AML cell lines tested. Data were analyzed in Rstudio, and the beneficial effects of the combinations were assessed using the Synergyfinder software package. A Bliss score ≥5 with <50% cell viability was interpreted as a meaningful synergistic effect.

[0144] Figure 18A The drug sensitivity analysis of 8 out of 11 AML patient samples identified as Top1i sensitive is shown at 4 days post-treatment, expressed as a percentage of blast cells normalized to the control (untreated). Figure 18B The drug sensitivity analysis of 3 AML patient samples identified as Top1i insensitive out of 11 AML patient samples is shown at 4 days post-treatment, expressed as a percentage of blast cells normalized to the control (untreated). Figure 18C The drug sensitivity analysis of 8 AML patient samples identified as Top1i sensitive out of 11 AML patient samples is shown at 7 days post-treatment. Figure 18D The drug sensitivity analysis of 3 AML patient samples identified as Top1i insensitive out of 11 AML patient samples is shown at 7 days post-treatment.

[0145] Figure 19 The table shows the survival of the SoC-insensitive AML PDX (DFAM-68555) model after processing according to groups 1-8 as shown in the table.

[0146] Figure 20 This shows the AML disease burden, as measured by %hCD45 in peripheral blood, in a SoC-insensitive AML PDX (DFAM-68555) model 16 days after the first dose treatment. + Cellular measurements.

[0147] Figure 21 The table shows the survival of the SoC-sensitive AML PDX (DFAM-49600) model after processing according to groups 1-8.

[0148] Figure 22 The disease burden of AML in a SoC-insensitive PDX (DFAM-68555) model, as shown by %hCD45 in peripheral blood, is illustrated at 16, 31, 44, 59, 73, 87, 100, and 146 days after the first dose treatment. + Cellular measurements. Detailed Implementation

[0149] This disclosure relates to antibodies that bind to the CD123 antigen (the α chain of the interleukin-3 receptor or IL3Rα), their antigen-binding fragments, and antibody-drug conjugates. The antibody-drug conjugates comprise a topoisomerase inhibitor derivative drug unit linked via a linker to the antibody or antigen-binding fragment described herein. In some aspects, the linker is cleavable and releases the drug unit in the cellular environment after the antibody-drug conjugate has been taken up into a target cell. The released topoisomerase inhibitor mediates cytotoxicity against the target cells and bystander killing of non-target cells. Also provided are polynucleotides encoding CD123-binding antibodies or antigen-binding fragments, vectors for expressing them in host cells, and methods for preparing and using CD123 antibody-drug conjugates for cancer therapy.

[0150] I. Definition

[0151] To facilitate understanding of this description, certain terms are first defined. Additional definitions are set forth throughout the specific implementation.

[0152] It should be noted that the terms "an" or "a" entity refer to one or more of that entity; for example, "nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "an" (or "a"), "one or more," and "at least one" are used interchangeably herein.

[0153] Furthermore, the term “and / or” as used herein is considered to refer to each of two specified features or components, whether or not they are specifically disclosed with the other. Therefore, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to include each of the following: 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).

[0154] It should be understood that wherever the term “comprising” is used to describe an aspect herein, other similar aspects described by the terms “consisting of” and / or “substantially consisting of” are also provided. As used herein, the terms “comprising” and “including” and their variations (e.g., “containing”, “having”) will be understood to indicate that a group of components, features, elements, or steps or components, features, elements, or steps are included, but do not exclude any other components, features, elements, or steps or groups of components, features, elements, or steps. Any of the terms “comprising,” “substantially consisting of,” and “consisting of” may be replaced by any of the other two terms while retaining their ordinary meaning.

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0156] Units, prefixes, and symbols are represented in their internationally recognized (SI) form. Numerical ranges include the values ​​that define that range. Unless otherwise indicated, nucleotide sequences are written from left to right with a 5' to 3' orientation. Amino acid sequences are written from left to right with an amino-to-carboxyl orientation. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the entire specification. Therefore, the terms that are immediately defined below are more fully defined by reference to the entire specification.

[0157] The term “approximately” is used in this document to mean approximately, roughly, about, or in the range of… When the term “approximately” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated numerical value. Generally, the term “approximately” can modify numerical values ​​above and below the stated value by varying upwards or downwards (higher or lower), for example, by 10%.

[0158] In some respects, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). In some antibodies (e.g., naturally occurring IgG antibodies), the heavy chain constant region consists of a hinge and three domains: CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (VL) and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs) and scattered with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of both the heavy and light chains contain binding domains that interact with antigens. The constant regions of an antibody mediate its binding to host cells, tissues, or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise stated herein, the amino acids in the variable regions are numbered using the Kabat numbering system.

[0159] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the “Kabat numbering system” is usually used (e.g., Kabat et al., Sequences of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0160] The amino acid position numbering in Kabat refers to the numbering system used by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991), for the heavy chain variable domain or light chain variable domain compiled with antibodies. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids that correspond to shortening or insertion of the FW or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (according to residue 52a in Kabat) and inserted residues after residue 82 of the heavy chain FW (e.g., residues 82a, 82b, and 82c, etc., according to Kabat).

[0161] For a given antibody, the Kabat numbering of the residues is determined by comparing the homology region of the antibody sequence with the “standard” Kabat numbering sequence. Chothia, on the other hand, refers to the position of the structural loop (Chothia and Lesk, J.Mol.Biol.196:901-917 (1987)). When using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34, depending on the loop length (this is because the Kabat numbering scheme places the insertion 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; 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 Diagnostics’ AbM antibody modeling software. The table below lists the positions of the amino acids in the variable regions of the antibody in each system.

[0162]

[0163] ImMunoGeneTics (IMGT) also provides a numbering system for immunoglobulin variable regions (including CDRs). See, for example, Lefranc, MP et al., Dev. Comp. Immunol. 27: 55-77 (2003). The IMGT numbering system is based on alignments, structural data, and characterization of hypervariable loops from over 5,000 sequences and allows for easy comparison of variable regions and CDRs across all species. According to the IMGT numbering scheme, VH-CDR1 is located at positions 26–35, VH-CDR2 at positions 51–57, VH-CDR3 at positions 93–102, VL-CDR1 at positions 27–32, VL-CDR2 at positions 50–52, and VL-CDR3 at positions 89–97.

[0164] As used throughout this manual, the described VH CDR sequences correspond to the classic Kabat numbering positions: Kabat VH-CDR1 is at positions 31-35, VH-CDR2 at positions 50-65, and VH-CDR3 at positions 95-102. VL-CDR1, VL-CDR2, and VL-CDR3 also correspond to the classic Kabat numbering positions, namely positions 24-34, 50-56, and 89-97, respectively.

[0165] Antibodies can be derived from any commonly known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG isotypes are subclassed in some species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some respects, the antibodies described herein are IgG1 subtypes. Antibodies (e.g., IgG1) exist in several allotypes, differing from each other by at most a few amino acids. The antibodies described herein include, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human and non-human antibodies; and fully synthetic antibodies.

[0166] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, meaning that the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, unlike polyclonal antibody formulations which comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies intended for use according to this disclosure may be produced by a hybridoma method first described by Kohler et al. (1975) Nature 256:495, or by a recombinant DNA method (see U.S. Patent 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described in the following literature (Clackson et al. (1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol., 222:581-597), or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459). Specifically, the monoclonal antibodies in this invention include chimeric antibodies, humanized antibodies, and human antibodies.

[0167] As used herein, the term “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen (e.g., human CD123). The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding fragment” of an antibody (e.g., the anti-CD123 antibody described herein) include (i) Fab fragments (fragments derived from papain cleavage) or fragments derived from V… L V H (ii) Similar monovalent fragments consisting of LC and CH1 domains; (iii) F(ab')2 fragments (fragments derived from pepsin cleavage) or similar divalent fragments comprising two Fab fragments connected by a disulfide bridge in the hinge region; H (iv) The Fd fragment composed of the CH1 domain; L and V H The Fv segment, composed of structural domains, (v) is composed of V HThe dAb fragment consists of domains (Ward et al., (1989) Nature 341:544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs optionally connected by a synthetic linker. Furthermore, although the two domains V of the Fv fragment... L and V H Encoded by individual genes, they can be linked together using recombination methods via synthetic adapters, which allow them to become a single protein chain, where V... L District and V H Regions pair to form monovalent molecules (called single-chain Fvs (scFvs); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibodies. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and screening for them in the same manner as intact antibodies is ineffective. Antigen-binding fragments can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0168] As used herein, the term "affinity" refers to a measure of the strength of binding between an antigen or target (such as an epitope) and its homologous binding domain (such as a complementary site). As used herein, the term "affinity" refers to the overall stability of the complex between a population of epitopes and complementary sites (i.e., antigens and antigen-binding domains).

[0169] The term "epitope" refers to a site on an antigen (e.g., CD123) where an antibody or antigen-binding fragment specifically binds, for example, as defined by a particular method used to identify the site. Epitopes can be formed from consecutive amino acids (typically linear epitopes) or from discontinuous amino acids juxtaposed through the ternary folding of a protein (typically conformational epitopes). Epitopes formed from consecutive amino acids are generally, but not always, retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation.

[0170] The term "binds to the same epitope" for two or more antigen-binding moieties means that the antigen-binding moieties bind to the same amino acid residue segment. An antigen-binding moiety that "competes with another antibody to bind to the target" refers to an antigen-binding moiety that inhibits (partially or completely) the binding of another antibody to the target.

[0171] The terms "specific binding," "selective binding," "selectively binding," and "specific binding" refer to the binding of an antigen-binding moiety (e.g., an antibody or antigen-binding fragment) to an epitope on a predetermined antigen. Typically, an antigen-binding moiety: (i) when, for example, via BIACORE... ® When using surface plasmon resonance (SPR) technology, or Scatchard assay of antibody-antigen-positive cell binding, with a predetermined antigen (e.g., human CD123) as the analyte and antibody as the ligand in the 2000 instrument, the result is approximately less than 10. -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower equilibrium dissociation constant (K) D (i) binding, and (ii) binding the predetermined antigen with an affinity at least twice that of an antigen other than the predetermined antigen or closely related antigen (e.g., BSA, casein). Therefore, the antigen-binding portion (e.g., antibody or antigen-binding fragment) of “specifically binding human CD123” refers to binding with an affinity of 10... -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or even lower K D It binds to the antigen-binding portion of human CD123.

[0172] "Immune response," as understood in the art and generally refers to a biological response in vertebrates against exogenous factors or abnormal cells (e.g., cancer cells) that protects the organism against these factors and the diseases they cause. An immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in the selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation within the vertebrate body. Immune responses include, for example, T cells (e.g., effector T cells, Th cells, CD4+). + Cells, CD8 + The activation or suppression of T cells (or Treg cells), or the activation or suppression of any other cell of the immune system (e.g., NK cells).

[0173] As used herein, the term "immunotherapy" refers to the treatment of a subject who has a disease or is at risk of contracting or relapsing into the disease by means of methods including inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0174] As used herein, the term "peptide" is intended to cover both the singular and plural "peptide" and any one or more chains comprising two or more amino acids. Therefore, as used herein, "peptide," "peptide subunit," "protein," "amino acid chain," "amino acid sequence," or any other term used to refer to one or more chains of two or more amino acids is included in the definition of "peptide," even though each of these terms may have a more specific meaning. The term "peptide" may be used in place of any of these terms or used interchangeably with any of these terms. The term also includes peptides that have undergone post-translational or post-synthetic modifications, such as palmitoyl group conjugation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, disulfide bond formation, proteolytic cleavage, or modification by non-naturally occurring amino acids. As used herein, the term "peptide" covers full-length peptides and their fragments, variants, or derivatives. As disclosed herein, a "peptide" can be a portion of a fusion peptide that includes additional components (such as, for example, albumin or PEG moieties) to increase its half-life. The peptides described herein can be derivatized in a variety of different ways. The peptides described herein may include modifications, including, for example, conjugations of palmitoyl groups. As disclosed herein, a “peptide” can also be part of a drug linker unit that covalently links an antibody or an antigen-binding fragment thereof to a drug to form an antibody-drug conjugate.

[0175] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, predicted non-essential amino acid residues in CD123-binding moieties (e.g., anti-CD123 antibodies or CD123-binding fragments) are substituted with another amino acid residue from the same side chain family.

[0176] As used herein, the terms “polynucleotide” and “nucleic acid molecule” are intended to include both DNA and RNA molecules. Polynucleotide or nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0177] As used herein, the term "promoter" refers to a DNA sequence recognized by cellular mechanisms or introduced synthetic mechanisms required for the specific transcription of a gene. The term "promoter" is also intended to encompass those nucleic acid elements sufficient for use in controlling cell-type-specific, tissue-specific, or inducible expression of promoter-dependent genes through external signals or agents; such elements may be located in the 5' or 3' region of a natural gene. In some respects, a promoter can be a constitutively active promoter, a cell-type-specific promoter, or an inducible promoter.

[0178] As used herein, the term “IRES” refers to an element that facilitates direct entry of the internal ribosome into the cistron (protein-coding region) of the start codon (such as ATG), thereby leading to cap-independent translation of the gene. See, for example, Jackson RJ et al., Trends Biochem Sci 15(12):477-83 (199); Jackson RJ and Kaminski, A. RNA 1(10):985-1000 (1995). Under the translational control of IRES, translation proceeds in a cap-independent manner.

[0179] As used herein, the term "termination signal sequence" can refer to any genetic element that causes RNA polymerase to terminate transcription, such as a polyadenylation signal sequence. The polyadenylation signal sequence is a recognition region essential for endonuclease cleavage of the RNA transcript, followed by the polyadenylation concordance sequence AATAAA. The polyadenylation signal sequence provides a "polyA site," a site on the RNA transcript where adenine residues are added post-transcriptionally via polyadenylation.

[0180] As used herein, the terms “operably linked,” “operably inserted,” “operably positioned,” “under control,” or “under transcriptional control” mean that the promoter is in the correct position and orientation relative to the nucleic acid to control RNA polymerase initiation and gene expression. The term “operably linked” means that the DNA sequence and the regulatory sequence are linked in such a way that gene expression is permitted when an appropriate molecule (e.g., a transcription-activating protein) binds to the regulatory sequence. The term “operably inserted” means that the DNA of interest introduced into the cell is located near the DNA sequence that guides the transcription and translation of the introduced DNA (i.e., promotes, for example, the production of a polypeptide encoded by the DNA of interest).

[0181] The percentage of identity between two sequences is a function of the number of common positions in the sequences (i.e., percentage of homology = number of common positions / total number of positions × 100), taking into account the number of gaps required for optimal alignment of the two sequences and the length of each gap. As described in the following non-restrictive example, mathematical algorithms can be used to compare sequences and determine the percentage of identity between two sequences.

[0182] The percentage of identity between two nucleotide sequences can be determined using several known algorithms, including the following: the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which has been incorporated into the alignment algorithm (version 2.0); and the algorithm of Needleman and Wunsch (J. Mol.Biol.(48):444-453 (1970)), which has been incorporated into the GAP program in the GCG software package.

[0183] The nucleic acid and protein sequences described herein can be further used as “query sequences” for searching public databases to, for example, identify relevant sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules described herein. For obtaining vacancy alignments for comparative purposes, vacancy BLAST, as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402, can be utilized. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0184] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is the "plasmid," which is a circular double-stranded DNA loop in which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells in which they are introduced (e.g., bacterial vectors with bacterial origins of replication and paraneoplastic mammalian vectors). Other vectors (e.g., non-paraneoplastic mammalian vectors) can integrate into the host cell genome after introduction into the host cell, thereby replicating along with the host genome. Moreover, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors that are practically useful in recombinant DNA technology are often in plasmid form. In this disclosure, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors that perform equivalent functions are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0185] As used herein, the terms “recombinant cell” and “recombinant host cell” (or simply “host cell”) refer to a cell containing nucleic acids that are not naturally present in cells, and may be a cell in which a recombinant expression vector has been introduced. It should be understood that these terms refer not only to the specific test cell, but also to the progeny of such cells. Because certain modifications may occur in the offspring due to mutations or environmental influences, such progeny may not actually be identical to the parent cells, but are still included within the scope of the terms “recombinant cell” or “host cell” as used herein.

[0186] As used herein, the term "antibody-drug conjugate" refers to a molecule that typically comprises three distinct elements: a cell binder (e.g., an antibody or antigen-binding fragment); a linker; and a cytotoxic drug moiety. In some aspects, the cytotoxic drug moiety is covalently attached to the antibody or antigen-binding fragment via one or more lysine residues, or covalently attached to one or more cysteine ​​residues on the antibody or antigen-binding fragment. In some aspects, two or more cysteine ​​residues can be obtained in the antibody or antigen-binding fragment by reducing interchain disulfide bonds. In some aspects, one or more cysteine ​​residues can be obtained in the antibody or antigen-binding fragment by introducing one or more mutations into the polynucleotide encoding the antibody or antigen-binding fragment. For example, a non-cysteine ​​amino acid can be mutated to a cysteine, or a cysteine ​​can be added to the amino acid sequence of the antibody or antigen-binding fragment. In some aspects, lysine can be chemically converted to a thiol group. The term "antibody-drug conjugate" includes heterogeneous mixtures of antibody or antigen-binding fragments containing different numbers of drugs attached, and antibody or antigen-binding fragments containing drugs attached at different positions on the antibody or antigen-binding fragment.

[0187] As used herein, the term "connector" refers to any chemical part capable of stably covalently linking a compound (e.g., a drug described herein) to a cell binding agent (e.g., a CD123 antibody or its antigen-binding fragment described herein). Connectors may be susceptible to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, or may be substantially resistant to these cleavages, provided that the drug, antibody, or antigen-binding fragment remains active. Suitable connectors are known in the art and include, for example, peptide connectors, disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups, and esterase-labile groups. Connectors also include charged connectors described herein and known in the art, and their hydrophilic forms.

[0188] As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids that covalently links the drug described herein, either directly or via an additional linker group, to the antibody or its antigen-binding fragment described herein. Peptide linkers may comprise dipeptides, tripeptides, tetrapeptides, or longer peptide chains.

[0189] As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0190] As used herein, the terms “subject,” “individual,” or “patient” mean any organism to which the compositions disclosed herein (e.g., antibodies, antigen-binding fragments, or antibody-drug conjugates (ADCs)) may be administered, for example, for experimental, diagnostic, preventative, and / or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). Subjects may be persons or animals who seek or have a need for treatment, require treatment, are currently receiving treatment, will receive treatment in the future, or are being cared for by a trained professional for a specific disease or symptom.

[0191] As used herein, the term “treatment” refers to therapeutic treatment and preventive or preventative measures aimed at preventing or mitigating (alleviating) an undesirable physical condition (e.g., cancer) or achieving a beneficial or desired clinical outcome. In some respects, treatment reduces or alleviates symptoms associated with, for example, cancer. In some respects, treatment produces a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, the reduction of symptoms; reduction of the severity of a symptom, condition, or disease; achieving a stable (i.e., non-worsening) state of a symptom, condition, or disease; delaying the onset of or slowing the progression of a symptom, condition, or disease; improving or alleviating a symptom, condition, or disease state (whether partial or complete), whether detectable or undetectable; improving at least one measurable bodily parameter, which is not necessarily identifiable to the patient; or enhancing or improving a symptom, condition, or disease. In some respects, treatment includes causing a clinically significant response without excessive levels of side effects. In some respects, treatment includes prolonging survival compared to expected survival without treatment. As used herein, the term “improvement” refers to reducing the severity of at least one indicator of a symptom or disease. As used in this article, the term "prevention" refers to delaying or stopping the onset, development, or progression of a symptom or disease for a period of time, including weeks, months, or years. Improving a disease or condition includes slowing its progression or reducing the severity of its symptoms.

[0192] Terms such as “effective amount,” “therapeutic effective amount,” and “sufficient amount” for antibodies, their antigen-binding fragments, antibody-drug conjugates, or compositions disclosed herein refer to amounts sufficient to produce a beneficial or desired outcome, including: relieving symptoms when administered to subjects, including humans; reducing the severity of symptoms, symptom, or disease; achieving a stable (i.e., non-worsening) state of symptoms, symptom, or disease; delaying the onset of or slowing the progression of symptoms, symptom, or disease; improving or alleviating the state of symptoms, symptom, or disease (whether partial or complete), whether detectable or undetectable; improving at least one measurable bodily parameter, which is not necessarily identifiable by the patient; or enhancing or improving symptoms, symptom, or disease. In some aspects, treatment includes causing a clinically significant response without excessive levels of side effects. Therefore, “therapeutic effective amount” or its synonyms depend on the context in which it is applied. In some aspects, a therapeutically effective amount of a drug (e.g., an antibody, antigen-binding fragment, antibody-drug conjugate, or composition described herein) is an amount that produces a beneficial or desired outcome in subjects compared to a control not receiving the drug. The amount of a given agent (e.g., an antibody-antigen binding fragment, an antibody-drug conjugate, or a composition) will vary depending on various factors, such as the given agent, the drug formulation, the route of administration, the type of disease or condition, and the identity of the subject being treated (e.g., age, sex, and / or weight).

[0193] As used herein, the term "preventive effective dose" refers to the amount of a drug (e.g., antibody, antigen-binding fragment, antibody-drug conjugate, or composition) that delays, prevents, or blocks the onset, development, or progression of symptoms or disease for a period of time (including weeks, months, or years). Preventive effective doses can vary depending on: the characteristics of the drug (e.g., antibody, antigen-binding fragment, antibody-drug conjugate, or composition); how the drug is administered; the degree of risk for the disease; and the subject's medical history, age, weight, family history, genetic makeup; the type of prior or concomitant treatment (if any); and other individual characteristics of the patient to be treated.

[0194] As used in this article, the terms “ug” and “uM” are used interchangeably with “μg” and “μM”, respectively.

[0195] As used herein, the term "topoisomerase inhibitor" refers to a compound that blocks the action of topoisomerases (topoisomerases I and II), which are enzymes that control changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of the DNA strand during the normal cell cycle.

[0196] As used in this article, the phrase "superscript label" C(=O) and NH For example, in the following formula

[0197] , refers to the group to which the atom is bonded. For example, the NH group in the above formula is shown as bonded to the carbonyl group (superscript). C(=O) ), which is not a part of the shown part; and the carbonyl group in the above formula is shown to be bound to the NH group (superscript). NH ), which is not a part of the part shown.

[0198] As used in this article, the term "chirality" refers to a molecule that has a non-superimposable property of a mirror-couple, while the term "chirality" refers to a molecule that can be superimposed on its mirror-couple.

[0199] As used in this article, the term "stereoisomer" refers to drugs that have the same chemical composition but different spatial arrangements of atoms or groups.

[0200] As used herein, the term "diastereomer" refers to a stereoisomer that has two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers possess different physical properties, such as melting point, boiling point, spectral properties, and reactivity. Mixtures of diastereomers can be separated using high-resolution analytical procedures such as electrophoresis and chromatography.

[0201] As used herein, the term “enantiomer” refers to two stereoisomers of a drug that are non-superimposable mirror images of each other. Stereochemical definitions and conventions used herein generally follow SP Parker, ed., McGraw-Hill Dictionary of Chemical Terms (1984), McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Drugs”, John Wiley & Sons, Inc., New York, 1994. Drugs disclosed herein may contain asymmetric or chiral centers and thus exist in different stereoisomeric forms. All stereoisomeric forms of drugs disclosed herein (including, but not limited to, diastereomers, enantiomers, and transisomers, as well as mixtures thereof, such as racemic mixtures) are contemplated to form part of this disclosure. Many organic drugs exist in an optically active form, i.e., they have the ability to rotate the plane of plane-polarized light. In describing optically active drugs, the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule around its chiral center. The prefixes d and l, or (+) and (-), are used to indicate that a drug rotates plane-polarized light; (-) or l indicates that the drug is levorotatory. Drugs with the prefix (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical, differing only in that they are mirror images of each other. Specific stereoisomers are also called enantiomers, and mixtures of such isomers are generally called enantiomeric mixtures. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which can occur in the absence of stereoselectivity or stereodirection in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two optically inactive enantiomers.

[0202] As used herein, the term "enantiomer-enriched form" refers to a sample of chiral material whose enantiomer ratio is greater than 50:50 but less than 100:0. Apart from the tautomerisms discussed below, structural (or compositional) isomers (i.e., isomers differing only in the spatial positions of atoms, but also in the connections between atoms) are explicitly excluded from the term "isomer" as used herein. For example, a reference to the methoxy group -OCH3 should not be interpreted as a reference to its structural isomer, the hydroxymethyl group -CH2OH. Similarly, a reference to the o-chlorophenyl should not be interpreted as a reference to its structural isomer, the m-chlorophenyl. However, a reference to a class of structures is likely to include structural isomers falling within that class (e.g., C...). 1-7Alkyl groups include n-propyl and isopropyl; butyl groups include n-butyl, isobutyl, sec-butyl, and tert-butyl; methoxyphenyl groups include o-methoxyphenyl, m-methoxyphenyl, and p-methoxyphenyl. The above exclusions do not apply to tautomer forms, such as ketone, enol, and enolate forms, as in, for example, the following tautomer pairs: ketone / enol (as shown below), imine / enamine, amide / imine alcohol, amidine / endiamine, nitroso / oxime, thionone / enthiol, N-nitroso / hydroxyazo, and nitro / acid nitro.

[0203]

[0204] As used herein, the term "tautomer" or "tautomer form" refers to structural isomers with different energies that can interconvert via low energy barriers. For example, proton tautomers (also known as proton-transfer tautomers) include interconversions via proton transfer, such as keto-enol and imine-enamine isomerization. Valence tautomers include interconversions through the rearrangement of some bonding electrons. The term "isomer" specifically includes drugs having one or more isotopic substitutions. For example, H can be any isotopic form, including 1H, 2H (deuterium, D), and 3H (tritium, T); C can be any isotopic form, including 12C, 13C, and 14C; O can be any isotopic form, including 16O and 18O; and so on. Examples of isotopes that can be incorporated into the pharmaceuticals of this disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as, but not limited to, 2H, 3H, 12C, 13C, 14C, 15N, 18F, 31P, 32P, 35S, 36Cl, and 125I. Various isotope-labeled pharmaceuticals of this disclosure include, for example, those incorporating radioactive isotopes such as 3H, 13C, and 14C. Such isotope-labeled pharmaceuticals can be used in metabolic studies, reaction kinetic studies, detection or imaging techniques such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or for radiotherapy of patients. The deuterium-labeled or substituted therapeutic pharmaceuticals of this disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties related to distribution, metabolism, and excretion (ADME). Substitution with a heavier isotope (such as deuterium) can provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life, reduced dose requirement, or improved therapeutic index. It should be understood that, in this context, deuterium is considered a substituent. 18F-labeled drugs can be used in PET or SPECT studies. The isotope-labeled drugs and prodrugs of this disclosure can generally be prepared by substituting non-isotope-labeled reagents with readily available isotope-labeled reagents. The concentration of such heavier isotope (particularly deuterium) can be defined by an isotope enrichment factor. In the drugs of this disclosure, any atom not specifically designated as a particular isotope is intended to represent any stable isotope of that atom.

[0205] Unless otherwise stated, references to a particular drug include all such isomers, including (in whole or in part) their racemic and other mixtures. Methods for preparing (e.g., asymmetric synthesis) and separating (e.g., fractional crystallization and chromatographic methods) such isomers are known in the art or readily available by adapting the methods taught in WO 2020 / 200880, which is incorporated herein in its entirety.

[0206] II. Antibodies and their antigen-binding fragments

[0207] An antibody or antigen-binding fragment thereof that specifically binds to human CD123 (IL3Rα) is provided. In some aspects, the antibody or antigen-binding fragment thereof comprises a variable heavy chain region (VH) and a variable light chain region (VL), wherein the VH comprises VH complementarity-determining region (CDR) 1, VH-CDR2, and VH-CDR3; and wherein the VL comprises VL-CDR1, VL-CDR2, and VL-CDR3.

[0208] In some aspects, the antibody or its antigen-binding fragment comprises: VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3 or 11. In some aspects, the antibody or its antigen-binding fragment comprises: VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2 or 10. In some aspects, the antibody or its antigen-binding fragment comprises: VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or 9.

[0209] In some aspects, the antibody or its antigen-binding fragment comprises: VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6 or 14. In some aspects, the antibody or its antigen-binding fragment comprises: VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5 or 13. In some aspects, the antibody or its antigen-binding fragment comprises: VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4 or 12.

[0210] In some aspects, the antibody or its antigen-binding fragment comprises: VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0211] In some aspects, the antibody or its antigen-binding fragment comprises: VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14.

[0212] In some aspects, the antibody or its antigen-binding fragment comprises VH-CDR1, VH-CDR2 and VH-CDR3 present in the VH region containing the amino acid sequence shown in SEQ ID NO: 7; and VL-CDR1, VL-CDR2 and VL-CDR3 present in the VL region containing the amino acid sequence shown in SEQ ID NO: 8.

[0213] In some aspects, the antibody or its antigen-binding fragment comprises VH-CDR1, VH-CDR2 and VH-CDR3 present in the VH region containing the amino acid sequence shown in SEQ ID NO: 15; and VL-CDR1, VL-CDR2 and VL-CDR3 present in the VL region containing the amino acid sequence shown in SEQ ID NO: 16.

[0214] In some aspects, the antibody or its antigen-binding fragment comprises a VH containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence comprising SEQ ID NO: 7. In some aspects, the antibody or its antigen-binding fragment comprises a VH containing the amino acid sequence comprising SEQ ID NO: 15.

[0215] In some aspects, the antibody or its antigen-binding fragment comprises a VL containing an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence comprising SEQ ID NO: 8. In some aspects, the antibody or its antigen-binding fragment comprises a VL containing the amino acid sequence comprising SEQ ID NO: 16.

[0216] In some aspects, the antibody or its antigen-binding fragment comprises: a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the antibody or its antigen-binding fragment comprises: a VH comprising the amino acid sequence shown in SEQ ID NO: 7 and a VL comprising the amino acid sequence shown in SEQ ID NO: 8.

[0217] In some aspects, the antibody or its antigen-binding fragment comprises: a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the antibody or its antigen-binding fragment comprises: a VH comprising the amino acid sequence shown in SEQ ID NO: 15 and a VL comprising the amino acid sequence shown in SEQ ID NO: 16.

[0218] In some respects, the antibody or its antigen-binding fragment cross-competes with the antibody or its antigen-binding fragment disclosed herein for binding to human CD123. In some respects, the antibody or its antigen-binding fragment binds to the same epitopes on human CD123 as the antibody or its antigen-binding fragment disclosed herein. In some respects, the antibody or its antigen-binding fragment binds to epitopes on human CD123 that overlap with the antibody or its antigen-binding fragment disclosed herein.

[0219] In some respects, the antibodies or antigen-binding fragments described herein bind to CD123 with high affinity. In some respects, the antibodies or antigen-binding fragments described herein bind to CD123 with approximately 10... -7 M or smaller K D Binding to CD123. In some respects, the antibodies or antigen-binding fragments described herein bind at approximately 10 -8 M, approximately 10 -9 M, approximately 10 -10 M, approximately 10 -11 M or lower K D Combined with CD123.

[0220] In some respects, the antibodies or antigen-binding fragments described herein exhibit approximately 5 × 10⁻⁶ in in vitro binding assays. -8 M, approximately 1×10 -8 M, approximately 5×10 -9 M, approximately 1×10 -9 M, approximately 5×10 -10 M, approximately 1×10 -10 M, approximately 5×10 -11 M or approximately 1×10 -11 K between M D Binding to CD123. In some respects, the antibodies or antigen-binding fragments described herein exhibit approximately 9 × 10⁻⁶ in in vitro binding assays. -9 M and approximately 1.7 × 10 -10 K between M D Binding to human CD123. In some respects, the antibodies or antigen-binding fragments described herein exhibit approximately 9 × 10⁻⁶ in in vitro binding assays. -9 M or approximately 1.7 × 10 -10 M of K D Binding to human CD123. In some respects, the antibodies or antigen-binding fragments described herein exhibit a binding rate of approximately 1.2 × 10⁻⁶ in in vitro binding assays. -8 M and approximately 6.5 × 10 -10 K between M D Binding to cynomolgus monkey CD123. In some respects, the antibodies or antigen-binding fragments described herein show a binding rate of approximately 1.2 × 10⁻⁶ in in vitro binding assays. -8 M or approximately 6.5 × 10 -10 M of KD Combined with cynomolgus monkey CD123.

[0221] In some respects, the antibodies or antigen-binding fragments described herein competitively bind to IL3Rα with IL3. In some respects, the antibodies or antigen-binding fragments described herein inhibit IL3-mediated cell proliferation. In some respects, the antibodies or antigen-binding fragments described herein inhibit IL3Rα-mediated cell signaling.

[0222] Antibody preparation

[0223] The antibodies disclosed herein can be obtained using conventional techniques known to those skilled in the art, and their efficacy has been demonstrated through conventional binding studies (exemplary methods are described in Example 4). For instance, a simple binding assay involves incubating cells expressing the antigen with the antibody. If the antibody is labeled with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

[0224] The antibodies disclosed herein can be used in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys, or horses. Antibodies can be generated after immunization with a single capsular polysaccharide or with multiple capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen. The antigen can also be injected into chickens to generate polyclonal antibodies in egg yolks. To obtain monoclonal antibodies specific to a single epitope of the antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells, called hybridomas, will continue to grow in a culture medium and secrete antibodies. Individual hybridoma cells are isolated by diluting the clone to generate cell clones that all produce the same antibody; these antibodies are called monoclonal antibodies. Methods used to generate monoclonal antibodies are conventional techniques known to those skilled in the art (see, for example, Making and Using Antibodies: A Practical Handbook. GCHoward. CRC Books. 2006. ISBN 0849335280). Polyclonal antibodies and monoclonal antibodies are typically purified using protein A / G or antigen affinity chromatography.

[0225] The antibodies or antigen-binding fragments of this disclosure can be prepared as monoclonal anti-CD123 antibodies, which can be prepared using hybridoma methods, such as those described in Kohler and Milstein, Nature 256:495 (1975). Using hybridoma methods, mice, hamsters, or other suitable host animals are immunized as described above to induce lymphocytes to produce antibodies that will specifically bind to the immunogenic antigen. Lymphocytes can also be immunized in vitro. After immunization, lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol to form hybridoma cells, which can then be selected from unfused lymphocytes and myeloma cells. The hybridomas can then be proliferated in vivo in vitro in culture media or as ascites tumors using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986), producing monoclonal antibodies specific to the selected antigen, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Monoclonal antibodies can then be purified from culture medium or ascites using known methods.

[0226] Alternatively, antibodies or antigen-binding fragments thereof (e.g., as monoclonal antibodies) may also be prepared using recombinant DNA methods as described in U.S. Patent 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using standard procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into a suitable expression vector, which, when transfected into host cells that do not additionally produce immunoglobulins (such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells), generates monoclonal antibodies from the host cells. In addition, recombinant monoclonal antibodies or antigen-binding fragments of the desired species can be isolated from phage display libraries expressing the desired species' CDR, as described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature, 352:624-628 (1991); and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0227] The polynucleotides encoding the antibodies or antigen-binding fragments thereof disclosed herein can be further modified in a variety of different ways using recombinant DNA technology to generate alternative antibodies. In some aspects, for example, the constant domains of the light and heavy chains of mouse monoclonal antibodies can be substituted (1) those regions, such as those of human antibodies, to generate chimeric antibodies or (2) non-immunoglobulin polypeptides to generate fusion antibodies. In some aspects, constant regions are truncated or removed to generate desired antibody fragments of monoclonal antibodies. Site-directed or high-density mutagenesis of variable regions can be used to optimize the specificity, affinity, etc., of monoclonal antibodies.

[0228] In one aspect, the antibody or its antigen-binding fragment is a human antibody or its antigen-binding fragment. Human antibodies can be prepared directly using a variety of techniques known in the art. Immortalized human B lymphocytes, either immunized in vitro or isolated from an immunized individual, can be generated, producing antibodies against the target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147 (1): 86-95 (1991); U.S. Patent 5,750,373.

[0229] In one aspect, the antibody or its antigen-binding fragment may be selected from a phage library expressing a human antibody, as described, for example, in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998); Hoogenboom 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 also described in U.S. Patents 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; and Rothe et al., J. Molec. Biol. 376:1182-1200 (2008), each of which is incorporated herein by reference.

[0230] Affinity maturation strategies and chain shuffling strategies are known in the art and can be used to generate high-affinity human antibodies or their antigen-binding fragments. See Marks et al., BioTechnology 10:779-783 (1992), the full text of which is incorporated herein by reference.

[0231] In one aspect, the antibody or its antigen-binding fragment (e.g., a monoclonal antibody) may be a humanized antibody. Methods for engineering, humanizing, or surface-reconstructing non-human or human antibodies, and such methods are well known in the art, may also be used. Humanized, surface-reconstructed, or similarly engineered antibodies may have one or more amino acid residues from a non-human source, such as, but not limited to, mice, rats, rabbits, non-human primates, or other mammals. These non-human amino acid residues are replaced with residues commonly referred to as “input” residues, which are typically derived from “input” variable, constant, or other domains of a known human sequence. Such input sequences can be used to reduce immunogenicity or to reduce, enhance, or modify binding, affinity, binding rate, dissociation rate, specificity, half-life, or any other suitable characteristic known in the art. Suitably, CDR residues may directly and most substantially participate in influencing CD123 binding. Therefore, it is preferable to retain some or all of the non-human or human CDR sequence, while the non-human sequences in the variable and constant regions may be replaced with human or other amino acids.

[0232] Antibodies may optionally be humanized, surface-reconstituted, engineered antibodies, or engineered human antibodies that retain high affinity for the antigen CD123 and other favorable biological properties. To achieve this, humanized (or human) or engineered anti-CD123 antibodies and surface-reconstituted antibodies may optionally be prepared by analyzing parental sequences and various conceptually humanized and engineered products using three-dimensional models of parental, engineered, and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs illustrating and displaying possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examination of these displays allows analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues affecting the ability of the candidate immunoglobulin to bind its antigen, such as CD123. In this way, FW residues can be selected and combined from common sequences and input sequences to obtain desired antibody characteristics, such as increased affinity for the target antigen.

[0233] Humanization, surface reconstruction, or engineering of the anti-CD123 antibody or its antigen-binding fragment disclosed herein can be performed using any known method, such as, but not limited to, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); US Patents 5,639,641, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539, 4,816,567, 7,5 57,189, 7,538,195 and 7,342,110; international applications PCT / US98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755; international patent applications published WO90 / 14443, WO90 / 14424, WO90 / 14430; and those described in European Patent Publication EP 229246; the entire text of each of these documents is incorporated herein by reference, including any references cited therein.

[0234] Humanized anti-CD123 antibodies and their antigen-binding fragments can also be prepared in transgenic mice containing human immunoglobulin loci, which, upon immunization, can generate a complete human antibody library 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.

[0235] In one aspect, fragments of antibodies (e.g., anti-CD123 antibodies) are provided. Various techniques for generating antibody fragments are known. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies, as described, for example, by Morimoto et al., J. Biochem.Biophys.Meth.24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some aspects, recombinant synthesis generates anti-CD123 antibody fragments. Fab, Fv, and scFv antibody fragments can all be expressed and secreted from *E. coli* or other host cells, allowing for the production of large quantities of these fragments. Such anti-CD123 antibody fragments can also be isolated from the aforementioned antibody phage libraries. Anti-CD123 antibody fragments can also be linear antibodies, as described in U.S. Patent 5,641,870. Other techniques for generating antibody fragments will be readily apparent to those skilled in the art.

[0236] According to this disclosure, the technology is applicable to the generation of single-chain antibodies specific to CD123. See, for example, U.S. Patent 4,946,778. Furthermore, the method is applicable to the construction of Fab expression libraries to allow for rapid and efficient identification of monoclonal Fab fragments or their derivatives, fragments, analogs, or homologs with desired specificity to CD123. See, for example, Huse et al., Science 246:1275-1281 (1989). Antibody fragments can be generated by techniques known in the art, including but not limited to: F(ab')2 fragments generated by pepsin digestion of antibody molecules; Fab fragments generated by reducing the disulfide bonds of F(ab')2 fragments; Fab fragments generated by treating antibody molecules with papain and a reducing agent; or Fv fragments.

[0237] In some aspects, the antibodies or antigen-binding fragments thereof disclosed herein can be modified to increase their serum half-life. This can be achieved, for example, by incorporating a rescue receptor-binding epitope into the antibody or antibody fragment, by mutating an appropriate region of the antibody or antibody fragment, or by incorporating the epitope into a peptide tag and then fusing it to the antibody or antibody fragment at either end or in the middle (e.g., via DNA or peptide synthesis), or by YTE mutation. Other methods for increasing the serum half-life of antibodies or antigen-binding fragments thereof are known in the art, such as conjugation with heterologous molecules (such as PEG).

[0238] The modified antibodies or antigen-binding fragments thereof provided herein may contain any type of variable region that enables association between the antibody or peptide and CD123. In this respect, the variable region may contain or be derived from any type of mammal capable of inducing a humoral response and generating immunoglobulins against the desired antigen. Thus, the variable region of an anti-CD123 antibody or antigen-binding fragment thereof may be derived, for example, from humans, mice, non-human primates (e.g., cynomolgus monkeys, macaques, etc.), or wolves. In some aspects, both the variable and constant regions of the modified antibody or antigen-binding fragment thereof are human. In some aspects, the variable region of a compatible antibody (typically derived from a non-human source) may be engineered or specifically tailored to improve binding properties or reduce the immunogenicity of the molecule. In this respect, the variable regions available in this disclosure may be humanized or otherwise modified by including an input amino acid sequence.

[0239] In some aspects, variable domains in both the heavy and light chains of an antibody or its antigen-binding fragment are altered by at least partial substitution of one or more CDRs and / or by partial frame region substitution and sequence changes. While CDRs may originate from antibodies of the same class or even the same subclass as the antibody from which the frame region originates, it is anticipated that CDRs will originate from antibodies of different classes, and in some embodiments, from antibodies of different species. It is not necessary to replace all CDRs with intact CDRs from the donor variable region to transfer the antigen-binding capacity of one variable domain to another. Instead, only those residues necessary to maintain the activity of the antigen-binding site need to be transferred. Given the interpretations described in U.S. Patents 5,585,089, 5,693,761, and 5,693,762, those skilled in the art are fully capable of performing routine experiments to obtain functional antibodies with reduced immunogenicity.

[0240] Despite the alterations to the variable regions, those skilled in the art will understand that the modified antibodies or antigen-binding fragments thereof of this disclosure will comprise antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as increased tumor localization or shortened serum half-life, when compared to antibodies containing native or unaltered constant regions and having substantially the same immunogenicity. In one aspect, the constant regions of the modified antibodies will include human constant regions. Modifications to constant regions compatible with this disclosure include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise alterations or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to the light chain constant domain (CL). In some aspects, the use of constant regions modified with partial or complete deletion of one or more of these domains is contemplated. In some aspects, the modified antibodies will comprise constructs or variants with domain deletions in which the entire CH2 domain has been removed (ΔCH2 construct). In some respects, the omitted constant region domains can be replaced by short amino acid spacer regions (e.g., 10 residues), which provide some of the molecular flexibility that is usually conferred by the absence of constant regions.

[0241] In addition to their conformation, constant regions are known in the art to mediate several effector functions. For example, antibodies bind to cells via Fc regions, where Fc receptor sites on the antibody's Fc region bind to Fc receptors (FcRs) on the cell. Many Fc receptors are specific to different classes of antibodies, including IgG (γ receptor), IgE (η receptor), IgA (α receptor), and IgM (μ receptor). Binding of antibodies to Fc receptors on the cell surface triggers many important and diverse biological responses, including engulfing and disrupting antibody-coated particles, clearing immune complexes, killing cells that lyse antibody-coated target cells (a phenomenon known as antibody-dependent cell-mediated cytotoxicity, or ADCC), releasing inflammatory mediators, placental transfer, and controlling immunoglobulin production.

[0242] In some respects, antibodies or their antigen-binding fragments provide altered effector functions, which in turn affect the biological characteristics of the administered antibody or its antigen-binding fragment. For example, deletion or inactivation of the constant region domain (through point mutation or otherwise) can reduce Fc receptor binding of circulating modified antibodies. In other cases, constant region modifications consistent with this disclosure can mitigate complement binding and thus reduce serum half-life and non-specific binding of conjugated cytotoxins. Other modifications to the constant region can be used to eliminate disulfide bonds or oligosaccharide moieties, which allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant regions according to this disclosure can be readily performed using well-known biochemical or molecular engineering techniques within the scope known to those skilled in the art.

[0243] In some respects, the antibody or its antigen-binding fragment does not have one or more effector functions. For example, in some respects, the antibody or its antigen-binding fragment does not have antibody-dependent cytotoxic (ADCC) activity and / or complement-dependent cytotoxic (CDC) activity. In some respects, the antibody or its antigen-binding fragment does not bind to Fc receptors and / or complement factors. In some antibodies or their antigen-binding fragments, no effector function is present.

[0244] In some respects, antibodies or their antigen-binding fragments can be engineered to directly fuse the CH3 domain with the hinge region of the corresponding modified antibody or fragment. In other constructs, a peptide spacer region can be inserted between the hinge region and the modified CH2 and / or CH3 domains. For example, compatible constructs can be expressed where the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a 5-20 amino acid spacer region. For example, such spacers can be added to ensure that the regulatory elements of constant domains remain free and accessible, or that the hinge region remains flexible. In some cases, the amino acid spacer region can prove to be immunogenic and elicit an unwanted immune response against the construct. In some respects, any spacer region added to the construct can be relatively non-immunogenic, or even omitted entirely, to maintain the desired biochemical quality of the modified antibody.

[0245] In addition to the deletion of the entire constant region domain, the antibodies or antigen-binding fragments described herein can also be modified by partially deleting or substituting several or even a single amino acid in the constant region. For example, a mutation of a single amino acid in a selected region of the CH2 domain can be sufficient to significantly reduce Fc binding, thereby increasing tumor localization. Similarly, one or more constant region domains controlling effector functions (e.g., complement C1Q binding) can be completely or partially deleted. Such partial deletions of constant regions can improve selected characteristics of the antibody or antigen-binding fragment (e.g., serum half-life) while keeping other desired functions associated with the subject constant region domain intact. Furthermore, the constant regions of antibodies and their antigen-binding fragments can be modified by mutations or substitutions of one or more amino acids that enhance the characteristics of the resulting construct. In this respect, it is possible to disrupt the activity provided by conserved binding sites (e.g., Fc binding) while substantially maintaining the conformational and immunogenic characteristics of the modified antibody or its antigen-binding fragment. In some aspects, one or more amino acids can be added to the constant region to enhance desired characteristics, such as reducing or increasing effector function, or providing more cytotoxic or carbohydrate attachment. In some respects, it may be necessary to insert or copy a specific sequence derived from the selected constant region structural domain.

[0246] This disclosure further includes variants and equivalents that are substantially homologous to the antibody or antigen-binding fragments of this disclosure (e.g., mouse antibodies, chimeric antibodies, humanized antibodies, or human antibodies or their antigen-binding fragments). These may contain, for example, conserved substitution mutations, where one or more amino acids are substituted with similar amino acids. For example, a conserved substitution refers to the substitution of an amino acid with another amino acid from the same class, such as, for example, one acidic amino acid being substituted with another acidic amino acid, one basic amino acid being substituted with another basic amino acid, or one neutral amino acid being substituted with another neutral amino acid. The purpose of conserved amino acid substitution is well known in the art.

[0247] In some respects, antibodies or their antigen-binding fragments can be further modified to include additional chemical parts that are not normally part of a protein. These derived parts can improve protein solubility, biological half-life, or absorption. They can also reduce or eliminate any desired side effects of proteins, etc. An overview of these parts can be described in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012).

[0248] III. Polynucleotides

[0249] A polynucleotide comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof as described herein is provided. In some aspects, the polynucleotide includes sequences that have been removed from their natural environment, recombinant or cloned (e.g., DNA) isolates, and chemically synthesized analogs or analogs biosynthesized via heterologous systems.

[0250] In some aspects, the polynucleotide sequence is prepared by any method known in the art. For example, a large number of polynucleotide sequences can be generated by replication and / or expression in suitable host cells. In some aspects, a natural or synthetic DNA fragment encoding an antibody or antigen-binding fragment as described herein is incorporated into a recombinant nucleic acid construct, which is typically a DNA construct capable of being introduced into and replicated in prokaryotic or eukaryotic cells. Typically, the DNA construct will be adapted for autonomous replication in a single-celled host such as yeast or bacteria, but may also be intended for introduction and integration into the genome of cultured bacteria, insects, mammals, plants, or other eukaryotic cell lines.

[0251] In some respects, polynucleotide sequences are prepared by chemical synthesis, such as by phosphoramide or triester methods, and can be performed on commercially available automated oligonucleotide synthesizers. Double-stranded (e.g., DNA) fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary strands and annealing them together under appropriate conditions, or by adding complementary strands using a DNA polymerase with appropriate primer sequences.

[0252] In some respects, polynucleotide sequences are isolated, which means that the sequence has been removed from its natural genetic environment and therefore does not contain other foreign or unwanted coding sequences (but may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for use in genetically engineered protein production systems. Such isolated polynucleotides are those molecules isolated from their natural environment.

[0253] In some respects, variants of the aforementioned polynucleotides are provided. In some respects, polynucleotide variants contain alterations in coding regions, non-coding regions, or both. In some respects, polynucleotide variants contain alterations that produce silent substitutions, additions, or deletions but do not change the properties or activity of the encoded polypeptide. In some respects, polynucleotide variants are produced through silent substitutions due to the degeneracy of the genetic code. In some respects, polynucleotide variants are produced for a variety of reasons, such as to optimize codon expression in a particular host (e.g., changing codons in mammalian sequences to those preferred by prokaryotic cells).

[0254] In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising an antigen-binding domain comprising: VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0255] In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising an antigen-binding domain comprising: VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 9, VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 10, VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 11, VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, and VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 14.

[0256] In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising: a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising: a VH comprising the amino acid sequence shown in SEQ ID NO: 7 and a VL comprising the amino acid sequence shown in SEQ ID NO: 8.

[0257] In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising: a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the polynucleotide comprises a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof, the antibody or antigen-binding fragment comprising: a VH comprising the amino acid sequence shown in SEQ ID NO: 15 and a VL comprising the amino acid sequence shown in SEQ ID NO: 16.

[0258] In some respects, polynucleotides are contained within cells.

[0259] In some respects, the cell contains the polynucleotide SEQ ID NO: 118.

[0260] In some respects, the polynucleotides SEQ ID NO:118 and SEQ ID NO:119 are present on separate polynucleotides. In other respects, the polynucleotides SEQ ID NO:118 and SEQ ID NO:119 are present on the same polynucleotide.

[0261] In some aspects, the polynucleotides described herein are present in a vector. Therefore, vectors comprising the polynucleotides of this disclosure are provided herein. In some aspects, vectors or a set of vectors comprising polynucleotides encoding the antibodies described herein or antigen-binding fragments thereof are provided. In some aspects, vectors or a set of vectors comprising polynucleotides encoding the antibodies disclosed herein that bind to CD123 or antigen-binding fragments thereof are provided.

[0262] In some aspects, the polynucleotides described herein comprise regulatory elements that initiate the expression of an antibody or an antigen-binding fragment thereof. In some aspects, the polynucleotides comprise a promoter operably linked to a nucleic acid encoding an antibody or an antigen-binding fragment thereof. In some aspects, the polynucleotides comprise a first promoter operably linked to a first nucleic acid encoding a first polypeptide encoding an antibody or an antigen-binding fragment thereof. In some aspects, the polynucleotides comprise a second promoter operably linked to a second nucleic acid encoding a second polypeptide encoding an antibody or an antigen-binding fragment thereof.

[0263] In some respects, the first promoter and the second promoter are the same. In some respects, the first promoter and the second promoter are different promoters. In some respects, the promoter is an inductive promoter. In some respects, the promoter is a constitutive promoter.

[0264] In some respects, polynucleotides also contain polyadenylation signal sequences. In some respects, the polyadenylation signal sequence is the SV40 polyadenylation signal sequence, the human growth hormone polyadenylation signal sequence, or the bovine growth hormone polyadenylation signal sequence.

[0265] In some respects, polynucleotides also include IRES.

[0266] In some respects, the polynucleotide comprises, in the 5' to 3' orientation, a first nucleic acid encoding a first polypeptide of an antibody or an antigen-binding fragment thereof, an IRES, and a second nucleic acid encoding a second polypeptide of an antibody or an antigen-binding fragment thereof.

[0267] In some aspects, this set of vectors comprises a first vector and a second vector, wherein the first vector contains a nucleic acid sequence encoding a polypeptide chain of the antibody or antigen-binding fragment described herein, and the second vector contains a nucleic acid sequence encoding a second polypeptide chain of the antibody or antigen-binding fragment described herein. For example, the first vector contains a nucleic acid sequence encoding a heavy chain of the antibody or antigen-binding fragment described herein, and the second vector contains a nucleic acid sequence encoding a light chain of the antibody or antigen-binding fragment described herein, or vice versa.

[0268] Any vector known in the art is suitable for this disclosure. In some aspects, the vector is a viral vector. In other aspects, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0269] In some respects, polynucleotides are present in cells. In some respects, cells contain the polynucleotides or carriers described herein. In some respects, cells contain polynucleotides encoding antibodies or antigen-binding fragments thereof described herein. In some respects, cells contain carriers that contain polynucleotides encoding antibodies or antigen-binding fragments thereof described herein. In some respects, cells contain carriers that contain polynucleotides encoding antibodies or antigen-binding fragments thereof that specifically bind CD123 as described herein. In some respects, cells containing the polypeptides described herein are used to produce antibodies or antigen-binding fragments thereof that specifically bind CD123.

[0270] Any cell can be used as the host cell for the polynucleotides, vectors, or polypeptides disclosed herein. In some respects, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia (e.g., E. coli); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella (e.g., Salmonella typhimurium); Serratia (e.g., Serratia marcescans and Shigella); Bacilli (e.g., B. subtilis and B. licheniformis); Pseudomonas (e.g., P. aeruginosa); and Streptomyces. In some respects, the cell is a human cell.

[0271] Table 1. Sequences

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] IV. Conjugates

[0281] This disclosure also provides conjugates comprising the antibodies or antibody-binding fragments described herein. In some aspects, the conjugates of this disclosure comprise the antibodies or antibody fragments described herein and a drug via a linker.

[0282] Medicinal material (projectile) and connector

[0283] In some respects, the drug in the antibody-drug conjugates described herein is a topoisomerase inhibitor. In some respects, the drug inhibits topoisomerases I and II.

[0284] In some respects, the drug contains formula (II):

[0285] ,

[0286] Where R L It is a connector used to attach a drug to a connector unit. In some respects, a connector unit comprises a connector and an antibody or its antigen-binding fragment.

[0287] The synthesis of Formula II drugs has been described in detail in WO2020 / 200880A1, the full text of which is incorporated herein by reference.

[0288] In some respects, drug molecules of Formula II pass through the linker R L Binds to the antibody or its antigen-binding fragment described in this article.

[0289] In some respects, connector R L Including IIa

[0290] .

[0291] In some respects, connector R L The “Q” in the text is And Q X As follows: Connector R L The Q is an amino acid residue, dipeptide residue, tripeptide residue, or tetrapeptide residue. In some respects, amino acid residues, dipeptide residues, tripeptide residues, or tetrapeptide residues bind to, as indicated by a superscript tag (…). C(=O) and NH The adjacent group indicated by ) For example, the "NH" of "Q" is combined with the C(=O) of formula IIa, and the C=O of "Q" is combined with the "NH" of formula II.

[0292] In some respects, the "X" of the connector in Formula IIa is

[0293] , where C(=O) on the left is C=O of equation IIa, and GL on the right is GL of equation IIa.

[0294] In some respects, Q is selected from the following amino acid residues: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

[0295] In some respects, Q is a dipeptide. In some respects, a dipeptide is any combination of natural and non-natural amino acids. In some respects, a dipeptide contains natural amino acids.

[0296] In some respects, the linker is an unstable linker for cathepsins. In other respects, the dipeptide is a recognition site for cathepsins and a site of action for cathepsin-mediated cleavage.

[0297] In some respects, Q is selected from: NH-Phe-Lys-C=O, NH-Val-Ala-C=O, NH-Val-Lys-C=O, NH-Ala-Lys-C=O, NH-Val-Cit-C=O, NH-Phe-Cit-C=O, NH-Leu-Cit-C=O, NH-Ile-Cit-C=O, NH-Phe-Arg-C=O, NH-Trp-Cit-C=O, and NH-Gly-Val-C=O; wherein NH represents the N-terminus and C=O represents the C-terminus of the residue.

[0298] In some respects, Q is a tripeptide. In some respects, the tripeptide is selected from: NH-Glu-Val-Ala-C=O, NH-Glu-Val-Cit-C=O, NH-αGlu-Val-Ala-C=O, and NH-αGlu-Val-Cit-C=O; wherein NH represents the N-terminus, and C=O represents the C-terminus of the residue. Glu represents a glutamic acid residue, and αGlu represents a glutamic acid residue when linked via an α-chain.

[0299] In some respects, Q is a tetrapeptide. In some respects, the tetrapeptide is selected from: NH-Gly-Gly-Phe-Gly C=O; and NH-Gly-Phe-Gly-Gly-C=O, where NH represents the N-terminus of the residue and C=O represents the C-terminus of the residue.

[0300] In some respects, connector R L It is equation IIa, and X is:

[0301] GL is a connector used to attach to an antibody or its antigen-binding fragment.

[0302] In some respects, the elements a, b1, b2, c1, and d of X include numbers between 0 and 16. In other respects, X contains a from 0 to 5, b1 from 0 to 16, b2 from 0 to 16, c1 from 0 to 1, and d from 0 to 5.

[0303] In some respects, X contains 0 to 3; or 0 or 1 of a.

[0304] In some respects, X contains 0 to 8; or b1 of 0, 2, 3, 4, 5 or 8.

[0305] In some respects, X contains 0 to 8; or b2 of 0, 2, 3, 4, 5 or 8.

[0306] In some respects, X contains c1 of 0, 1, or 2.

[0307] In some respects, X includes 0 to 3; or d of 1 or 2.

[0308] In some aspects, a is 1, c1 is 1, d is 2, and b1 is 2, 3, 4, 5, 6, 7, or 8. In some aspects, a is 2, c1 is 1, d is 1, and b1 is 2, 3, 4, 5, 6, 7, or 8. In some aspects, a is 1, c1 is 2, d is 1, and b1 is 2, 3, 4, 5, 6, 7, or 8. In some aspects, a is 1, c1 is 1, d is 1, and b1 is 2, 3, 4, 5, 6, 7, or 8. In some aspects, a is 0, b1 is 0, c1 is 1, d is 2, and b2 is 0, 2, 3, 4, 5, or 8. In some aspects, a is 1, b2 is 0, c1 is 0, and b1 is 0, 2, 3, 4, 5, or 8. In some cases, a is 0, b1 is 0, c1 is 0, d is 1, and b2 is 0, 2, 3, 4, 5, or 8. In some cases, b1 is 0, b2 is 0, c1 is 0, one of a and d is 0, and the other of a and d is 1 or 5. In some cases, a is 1, b2 is 0, c1 is 0, d is 2, and b1 is 0, 2, 3, 4, 5, or 8. In some cases, a is 0, b1 is 0, b2 is 8, c1 is 1, and d is 0.

[0309] In some respects, GL is:

[0310] .

[0311] In some respects, connector R L yes:

[0312] .

[0313] In some respects, Formula II is

[0314] (SG3932).

[0315] To avoid ambiguity, the number "8" specifies that the structure within the square brackets is repeated eight times. Therefore, another representation of SG3932 is:

[0316]

[0317] (1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide).

[0318] In some respects, antibody-drug conjugates comprise drugs of formula II; a linker of formula IIa, wherein Q comprises an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, and X comprises element a (0 to 5), element b1 (0 to 16), element b2 (0 to 16), element c1 (0 to 2), and element d (0 to 5); and GL of the following formula

[0319]

[0320] And the CD123 antibody or antigen-binding fragment described herein that binds to GL.

[0321] In some aspects, a drug of Formula II and a linker bind to the antibody or antigen-binding fragment described herein to form an antibody-drug conjugate. In some aspects, more than one drug of Formula II and more than one linker bind to the antibody or antigen-binding fragment described herein to form an antibody-drug conjugate.

[0322] In some aspects, a drug of formula II, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and a linker are conjugated to the antibody or antigen-binding fragment described herein to form an antibody-drug conjugate. In some aspects, a drug of formula II, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, is conjugated to the antibody or antigen-binding fragment described herein to form an antibody-drug conjugate.

[0323] In some respects, approximately eight Formula II drugs and linkers are conjugated to the antibodies described herein to form antibody-drug conjugates. In other respects, approximately eight SG3932 drugs are conjugated to the antibodies described herein to form antibody-drug conjugates.

[0324] In some respects, the antibodies described herein, via reductive conjugation of reduced cysteine ​​residues to the maleimide of SG3932, produce, as Figure 2B The antibody-drug conjugate is represented in the image.

[0325] In some aspects, this disclosure provides an anti-CD123 antibody comprising: (a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6; wherein the antibody is conjugated to one or more compounds represented by the following formula:

[0326] .

[0327] In some aspects, this disclosure provides an anti-CD123 antibody comprising: (a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; wherein the antibody is conjugated to 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionamidyl)-N-((S)-1-(((S) ...(((S)-1-yl)propionamidyl)-N-((S)-1-(((S)-1-yl)-1-yl)- (((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide.

[0328] In some aspects, this disclosure provides an anti-CD123 antibody comprising: heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively; wherein the antibody is conjugated to about eight compounds represented by the following formula:

[0329] .

[0330] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8, wherein the antibody is conjugated to one or more compounds represented by the following formula:

[0331] .

[0332] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8, wherein the antibody is conjugated to about eight compounds represented by the following formula:

[0333] .

[0334] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7 and a region comprising SEQ ID NO: The light chain variable region of the amino acid sequence shown in 8, wherein the antibody is conjugated to 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propamido)-N-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)amino)-1-oxoprop-2-yl)amino)-3-methyl-1-oxobut-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide.

[0335] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 116 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 117, wherein the antibody is conjugated to one or more compounds represented by the following formula:

[0336] .

[0337] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 116 and a light chain comprising the amino acid sequence shown in SEQ ID NO: 117, wherein the antibody is conjugated to about eight compounds represented by the following formula:

[0338] .

[0339] In some respects, using the EU numbering system, compounds are conjugated to heavy chain cysteine ​​220, heavy chain cysteine ​​226, heavy chain cysteine ​​229 and light chain cysteine ​​214.

[0340] In some aspects, this disclosure provides an anti-CD123 antibody comprising: a CDR of a heavy chain comprising the amino acid sequence shown in SEQ ID NO: 116 and a CDR of a light chain comprising the amino acid sequence shown in SEQ ID NO: 117, wherein the CDR is defined by EU, Kabat, Clobia and / or IMGT, and wherein the antibody is conjugated to about eight compounds represented by the following formula:

[0341] .

[0342] In some respects, the antibodies disclosed herein (e.g., HT12-GL) are conjugated to compounds of the following formula.

[0343] ,

[0344] It produces one or more of the following heavy-chain cysteine ​​220, heavy-chain cysteine ​​226, heavy-chain cysteine ​​229, and light-chain cysteine ​​214 (EU numbering system), forming a succinimide sulfide of the following formula.

[0345] .

[0346] Synthesis of topoisomerase I inhibitors

[0347] To complete this, certain general synthetic routes for the preparation of preferred topoisomerase I inhibitors will now be described. Further details are available in the Examples section.

[0348] Where R L Compounds of formula Ia can be synthesized from compounds of formula 2:

[0349] ;

[0350] in It is -QH, through the connection of compounds of formula 3:

[0351] ,

[0352] Or its activated form.

[0353] This reaction can be carried out under amide coupling conditions.

[0354] Compound 2 can be synthesized by deprotecting compound 4:

[0355] ;

[0356] in Yes -Q-Prot N Among them, Prot N It is an amine protecting group.

[0357] Compound 4 can be obtained by combining compound 5:

[0358] ;

[0359] It was synthesized by coupling with compound A3 using the Friedlander reaction.

[0360] Compound (5) can be derived from compound (6):

[0361] ;

[0362] It is synthesized by removing the protecting group of trifluoroacetamide.

[0363] Compound of Formula 6 can be obtained by... It is synthesized by coupling with compound I7.

[0364] Where R L Compounds of formula Ia or Ib can be obtained by adding compound R. L The compound I11 is synthesized by coupling -OH or its activated form.

[0365] amine protecting group :

[0366] Amine protecting groups are well known to those skilled in the art. In particular, refer to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, 4th Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.

[0367] Drug conjugation

[0368] In some respects, the antibodies or antigen fragments thereof described herein are conjugated to heterologous agents (e.g., drugs) using site-specific or non-site-specific conjugation methods. In some respects, more than one drug is conjugated to an antibody or antigen-binding fragment thereof. In some respects, one, two, three, four, five, six, seven, eight, or more drug molecules are conjugated to an antibody or antigen-binding fragment thereof. In some respects, all conjugated drug molecules have the same structure. In some respects, drug conjugation to the antibody or antigen-binding fragment described herein is achieved by using a predetermined ratio of drug molecules to antibody or antibody fragment molecules during the drug conjugation procedure described herein.

[0369] In some respects, conventional conjugation strategies, including, for example, random conjugation of lysine, cysteine, or non-natural amino acids, are used to conjugate the drugs described herein to the antibody or antigen-binding fragments described herein.

[0370] In some respects, a drug is randomly conjugated to an antibody or its antigen-binding fragment by reducing the antibody or its antigen-binding fragment and then reacting it with the drug, with or without a connector to which the drug is attached.

[0371] In some respects, more than one drug molecule (e.g., a drug molecule of formula II) is randomly conjugated to an antibody or its antigen-binding fragment.

[0372] In some respects, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP), or similar reducing agents are used to reduce antibodies or antigen-binding fragments. In some respects, in the presence of DMSO, a drug molecule, with or without a linker, is added in a molar excess to the reduced antibody or its antigen-binding fragment. In some respects, the molar excess is adjusted such that more than one drug molecule (e.g., a drug molecule of formula II) is conjugated to each reduced antibody or its antigen-binding fragment.

[0373] In some cases, excess free cysteine ​​is added after conjugation to quench unreacted reagents. In other cases, the reaction mixture is purified and buffer-exchanged into PBS.

[0374] In some respects, drug molecules are conjugated to antibodies or their antigen-binding fragments via site-specific conjugation. In some respects, more than one drug molecule is conjugated to an antibody or its antigen-binding fragment via site-specific conjugation. In some respects, site-specific conjugation of a drug molecule to an antibody or its antigen-binding fragment is achieved using reactive amino acid residues at specific sites within the antibody or its antigen-binding fragment. In some respects, site-specific conjugation produces homogeneous formulations of antibody-drug conjugates with homogeneous stoichiometry.

[0375] In some respects, more than one drug molecule is conjugated to an antibody or its antigen-binding fragment via site-specific conjugation. In some respects, more than one drug molecule conjugated to an antibody or its antigen-binding fragment via site-specific conjugation is a drug molecule of Formula II.

[0376] In some respects, drug molecules, with or without connectors, are added in molar excess to antibodies or their antigen-binding fragments for site-specific conjugation. In other respects, molar proximity is adjusted such that more than one drug molecule is site-specifically conjugated to each antibody or its antigen-binding fragment.

[0377] In some respects, drug molecules conjugate to amino acid side chains. In some respects, drug molecules conjugate to amino acid side chains in the Fc region of an antibody. In some respects, drug molecules conjugate to amino acid side chains in the CH3 constant region of the heavy chain of an antibody. In some respects, drug molecules conjugate to amino acid side chains in the CH2 constant region of the heavy chain of an antibody or its antigen-binding fragment.

[0378] In some respects, drug molecules are conjugated to amino acid side chains in the hinge region of an antibody or its antigen-binding fragment. In other respects, drug molecules are conjugated to amino acid side chains in the CH1 constant region of the heavy chain of an antibody or its antigen-binding fragment.

[0379] In some respects, drug molecules are conjugated to amino acid side chains in the Cκ region of the light chain of an antibody or its antigen-binding fragment. In other respects, drug molecules are conjugated to amino acid side chains in the Cλ region of the light chain of an antibody or its antigen-binding fragment.

[0380] In some respects, the drug molecule is conjugated to an amino acid side chain of at least two of the heavy chain CH1 constant region, heavy chain CH2 constant region, heavy chain CH3 constant region, hinge region, light chain Cκ region, or light chain Cλ region of the antibody or its antigen-binding fragment.

[0381] In some respects, the drug molecule is conjugated to an amino acid side chain of at least three of the following: the heavy chain CH1 constant region, the heavy chain CH2 constant region, the heavy chain CH3 constant region, the hinge region, the light chain Cκ region, or the light chain Cλ region of the antibody or its antigen-binding fragment.

[0382] In some respects, the drug molecule is conjugated to an amino acid side chain in at least four of the following: the heavy chain CH1 constant region, the heavy chain CH2 constant region, the heavy chain CH3 constant region, the hinge region, the light chain Cκ region, or the light chain Cλ region of the antibody or its antigen-binding fragment.

[0383] In some respects, the drug molecule is conjugated to an amino acid side chain of at least five of the following: the heavy chain CH1 constant region, the heavy chain CH2 constant region, the heavy chain CH3 constant region, the hinge region, the light chain Cκ region, or the light chain Cλ region of the antibody or its antigen-binding fragment.

[0384] In some respects, drug molecules are conjugated to amino acid side chains in the heavy chain CH1 constant region, heavy chain CH2 constant region, heavy chain CH3 constant region, hinge region, light chain Cκ region, and light chain Cλ region of the antibody or its antigen-binding fragment.

[0385] In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the CH1 constant region of the heavy chain. In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the CH2 constant region of the heavy chain. In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the CH3 constant region of the heavy chain. In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the hinge region. In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the Cκ region of the light chain. In some aspects, the drug molecule is conjugated with at most one amino acid side chain in the Cλ region of the light chain.

[0386] In some respects, the drug molecule is conjugated to an amino acid side chain in at least two heavy chain CH1 constant regions, at least two heavy chain CH2 constant regions, at least two hinge regions, and at least two light chain constant domain regions (Cκ or Cλ) of the antibody or its antigen-binding fragment.

[0387] In some respects, drug molecules are conjugated to amino acid side chains in the two heavy chain CH1 constant regions, the two heavy chain CH2 constant regions, the two hinge regions, and the two light chain constant domain regions (Cκ or Cλ) of the antibody or its antigen-binding fragment.

[0388] In some aspects, the drug molecule is conjugated to Cys220 of the heavy chain of the antibody or antigen-binding fragment described herein. In some aspects, the drug molecule is conjugated to Cys226 (EU numbering system) of the heavy chain of the antibody or antigen-binding fragment described herein. In some aspects, the drug molecule is conjugated to Cys229 (EU numbering system) of the heavy chain of the antibody or antigen-binding fragment described herein. In some embodiments, the drug molecule is conjugated to Cys214 (EU numbering system) of the light chain of the antibody or antigen-binding fragment described herein. In some aspects, the drug molecule is conjugated to heavy chain Cys220, heavy chain Cys226, heavy chain Cys229, and light chain Cys214 (EU numbering system).

[0389] In some respects, drug molecules are conjugated to antibodies or their antigen-binding fragments via thiol-maleimide bonds.

[0390] In some respects, where a drug molecule is conjugated to the side chain of cysteine ​​but only one or a few thiol groups have sufficient conjugation reactivity, additional nucleophilic groups can be introduced into the antibody or its antigen-binding fragment described herein by means of the reaction of lysine with 2-iminothiacyclopentane (Traut reagent), resulting in the conversion of the amine to a thiol.

[0391] In some respects, the drug molecule is conjugated to the cysteine ​​side chain of the antibody or its antigen-binding fragment, wherein the cysteine ​​is part of the disulfide bridging portion, and prior to conjugation, the antibody or its antigen-binding fragment is treated with a reducing agent (such as DTT or TCEP) under partial or complete reducing conditions to reduce the thiol group of the antibody or its antigen-binding fragment.

[0392] In some respects, reactive thiol groups can be introduced into antibodies or their antigen-binding fragments by engineering one, two, three, four, or more cysteine ​​residues (e.g., to prepare mutant antibodies containing one or more non-natural cysteine ​​residues). In some respects, cysteine ​​amino acids can be engineered at reactive sites in antibodies. In some respects, cysteine ​​amino acids can be engineered at sites where intrachain or intermolecular disulfide bonds do not form.

[0393] In some aspects, the drug molecule is conjugated to an amino acid side chain of VH containing the amino acid sequence shown in SEQ ID NO: 7. In some aspects, the drug molecule is conjugated to an amino acid side chain of VL containing the amino acid sequence shown in SEQ ID NO: 8. In some aspects, the drug molecule is conjugated to an amino acid side chain of the heavy chain containing the amino acid sequence shown in SEQ ID NO: 116. In some aspects, the drug molecule is conjugated to an amino acid side chain of both the heavy chain containing the amino acid sequence shown in SEQ ID NO: 116 and the light chain containing the amino acid sequence shown in SEQ ID NO: 117.

[0394] In some aspects, the drug molecule is conjugated to the amino acid side chain of VH, which contains the amino acid sequence shown in SEQ ID NO: 15. In some aspects, the drug molecule is conjugated to the amino acid side chain of VL, which contains the amino acid sequence shown in SEQ ID NO: 16.

[0395] In some respects, the loading (drug / antibody ratio) of antibody-drug conjugates is controlled in several different ways, including: (i) limiting the molar excess of the drug linker relative to the antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reduction conditions for cysteine ​​thiol modification.

[0396] In some respects, more than one nucleophilic or electrophilic group of an antibody reacts with the drug molecule described herein, and the resulting product may be a mixture of antibody-drug conjugates, wherein the distribution of drug molecules is attached to an antibody or antigen-binding fragment, for example, 1, 2, 3, 4, 5, 6, 7, or 8 drug molecules are conjugated to each antibody or its antigen-binding fragment.

[0397] Drug / drug load (p) is the average amount of drug per antibody or antigen-binding fragment. In some aspects, the average amount of drug per antibody or antigen-binding fragment ranges from about 1 to about 20, where, in the context of drug load (p), "about 1" means and includes 0 and 2. In some aspects, the range is selected from about 1 to about 10, about 2 to about 10, about 2 to about 8, about 2 to about 6, and about 4 to about 10. In some aspects, the drug load (p) is about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0398] In some respects, the average amount of drug in each antibody in an antibody-drug conjugate formulation derived from a conjugation reaction can be characterized by conventional methods such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assay, and electrophoresis.

[0399] In some respects, the separation, purification, and characterization of homogeneous antibody-drug conjugates, where p is a certain value, are achieved through methods including liquid chromatography (such as polymer reversed-phase (PLRP) and hydrophobic interaction (HIC)). In some respects, formulations of antibody-drug conjugates with a single drug loading value (p) are isolated. In some respects, antibody-drug conjugates with a single loading value can still be heterogeneous mixtures because the drug can attach to different sites on the antibody or antigen-binding fragment via a linker.

[0400] V. Methods using antibodies, antigen-binding fragments, and antibody-drug conjugates

[0401] In some aspects, the antibodies or antigen-binding fragments thereof described herein deliver a payload (e.g., a cytotoxic drug of Formula II) to cells and inhibit or suppress cell (e.g., tumor cells) proliferation by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% relative to the level of inhibition or suppression in the absence of the antibody-drug conjugate. In some aspects, cell proliferation is reduced after delivery of the antibody-drug conjugate, as determined using techniques recognized in the art, which measure the rate of cell division, the fraction of cells undergoing cell division within the cell population, and / or the rate of cell loss from the cell population due to terminal differentiation or cell death.

[0402] In some respects, the antibodies, antigen-binding fragments, or antibody-drug conjugates (e.g., CD123ADCs) described herein are in the form of 10 -7 M or smaller, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M of KD Binding to CD123 on the surface. In some respects, the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein (e.g., CD123 ADCs) bind at approximately 9 × 10⁻⁶. -9 M to approximately 1.7 × 10 -10 M of K D Binding to human CD123 on the surface. In some respects, the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein (e.g., CD123 ADCs) have a binding rate of approximately 9.01 × 10⁻⁶. -9 M or approximately 1.69 × 10 -10 M of K D Combined with the surface-level human CD123.

[0403] In some respects, the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein (e.g., CD123ADC) have a concentration of approximately 1.2 × 10⁻⁶. -8 M to approximately 6.6 × 10 -10 M of K D Binding to cynomolgus monkey CD123 on the surface. In some respects, the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein (e.g., CD123 ADCs) at approximately 1.17 × 10⁻⁶. -8 M or approximately 6.55 × 10 -10 M of K D Combined with the surface of the cynomolgus monkey CD123.

[0404] In some aspects, the antibodies, antigen-binding fragments, or antibody-drug conjugates described herein (e.g., CD123ADCs) bind to CD123 on the cell surface and are internalized into the cell. In some aspects, the antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into the cell, wherein the IC50 at 10 minutes is about 100 ng / ml to about 1 μg / ml, about 100 ng / ml to about 500 ng / ml, about 100 ng / ml to about 250 ng / ml, about 250 ng / ml to about 500 ng / ml, about 350 ng / ml to about 450 ng / ml, about 500 ng / ml to about 1 μg / ml, about 500 ng / ml to about 750 ng / ml, about 750 ng / ml to about 850 ng / ml, or about 900 ng / ml to about 1 μg / ml.

[0405] In some respects, antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into cells, wherein the IC50 at 30 minutes is about 100 ng / ml to about 1 μg / ml, about 100 ng / ml to about 500 ng / ml, about 100 ng / ml to about 250 ng / ml, about 250 ng / ml to about 500 ng / ml, about 250 ng / ml to about 350 ng / ml, about 350 ng / ml to about 450 ng / ml, about 500 ng / ml to about 1 μg / ml, about 500 ng / ml to about 750 ng / ml, about 750 ng / ml to about 850 ng / ml, or about 900 ng / ml to about 1 μg / ml.

[0406] In some respects, antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into cells, with IC50 values ​​of about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 100 ng / ml, about 100 ng / ml to about 200 ng / ml, about 200 ng / ml to about 300 ng / ml, about 300 ng / ml to about 400 ng / ml, or about 400 ng / ml to about 500 ng / ml at 120 minutes.

[0407] In some respects, antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into cells, with IC50 values ​​of about 5 ng / ml to about 250 ng / ml, about 10 ng / ml to about 25 ng / ml, about 25 ng / ml to about 50 ng / ml, about 50 ng / ml to about 100 ng / ml, about 100 ng / ml to about 150 ng / ml, about 150 ng / ml to about 200 ng / ml, or about 200 ng / ml to about 250 ng / ml at 8 hours.

[0408] In some aspects, antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into the endosomes of the cell within about one hour of cell exposure to the antibody, antigen-binding fragment, or antibody-drug conjugate. In some aspects, a method for delivering a payload (e.g., a drug of Formula II) into the endosomes of a cell is provided, the method comprising contacting the cell with the antibody-drug conjugate to deliver the payload into the endosomes of the cell within about one hour of contacting the cell with the antibody-drug conjugate.

[0409] In some aspects, antibodies, antigen-binding fragments, or antibody-drug conjugates are internalized into the lysosomes of cells within about 0.5 hours to about 5 hours after cells are exposed to the antibody, antigen-binding fragment, or antibody-drug conjugate. In some aspects, a method for delivering a payload (e.g., a drug of Formula II) to the lysosomes of cells is provided, the method comprising contacting cells with an antibody-drug conjugate to deliver the payload to the lysosomes of cells within about 0.5 hours to about 5 hours after contacting the cells with the antibody-drug conjugate.

[0410] In some aspects, a method for increasing caspase activity in cells is provided, the method comprising contacting CD123-expressing cells (e.g., CD123-expressing leukemia cells) with the antibody-drug conjugate described herein.

[0411] In some aspects, methods for delivering cytotoxic drugs to cells are provided. In some aspects, the method includes contacting cells expressing CD123 (e.g., leukemia cells expressing CD123) with the antibody-drug conjugate described herein.

[0412] In some aspects, methods are provided for delivering a cytotoxic drug to cells that do not express CD123. In some aspects, the method includes contacting cells expressing CD123 with an antibody-drug conjugate as described herein, wherein the drug is released in the CD123-expressing cells and exerts a cytotoxic effect by bystander killing of cells that do not express CD123. In some aspects, the method includes delivering a cytotoxic drug to a tissue of a subject, wherein the tissue contains cells expressing CD123 and cells that do not express CD123, wherein the drug is released in the CD123-expressing cells and exerts a cytotoxic effect by bystander killing of cells in the tissue that does not express CD123. In some aspects, optimal bystander cell killing is achieved in the tissue when the ratio of CD123-expressing cells to non-CD123-expressing cells is between about 50:50 and about 90:10.

[0413] In some respects, a method for treating a disease before or after its exacerbation is provided, the method comprising administering a therapeutically effective amount of the pharmaceutical conjugate described herein to a subject in need of treatment.

[0414] In some aspects, a method for treating leukemia is provided, comprising administering to a subject requiring treatment a therapeutically effective amount of the antibody-drug conjugate described herein, which effectively inhibits the activity of bone marrow colony-forming units (CFUs) in leukemic bone marrow cells. In some aspects, when administered to a subject suffering from leukemia, the therapeutically effective amount of the antibody-drug conjugate described herein is an amount that reduces the number of leukemic cells in the subject's whole blood and bone marrow.

[0415] Advantageously, the antibody-drug conjugates described herein do not affect the viability of healthy hematopoietic stem cells, healthy granulocyte-monocyte progenitor cells, healthy differentiated hematopoietic cells, or the CFU activity of normal bone marrow cells.

[0416] In some respects, the antibody-drug conjugates described herein are used in cellular or tissue environments in which endothelial cell damage must be avoided and / or where tight binding integrity must be maintained.

[0417] In some respects, the antibody-drug conjugates described herein are administered to subjects with tumors expressing CD123, wherein the CD123 antibody-drug conjugates reduce tumor volume.

[0418] In some respects, the antibody-drug conjugates described herein are administered to subjects with proliferative symptoms, including pre-exacerbation or worsening proliferative symptoms, wherein the pre-exacerbation or worsening cells express CD123. Pre-exacerbation or worsening symptoms include, but are not limited to, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), blastic plasmacytoid dendritic cell leukemia, piloblastic leukemia, systemic mastocytosis, Hodgkin's lymphoma, large B-cell lymphoma, chronic myeloid monocytic leukemia, chronic lymphocytic leukemia (CLL), and myelodysplastic syndromes.

[0419] In some respects, the antibody-drug conjugates described herein may be administered to the subject, alone or in combination with other treatments, simultaneously or sequentially. Examples of other treatments and therapies include, but are not limited to, chemotherapy (administration of an active agent, including, for example, a drug); surgery; and radiation therapy.

[0420] In some respects, alternative treatments are alternative cancer therapies, including, for example, alkylating agents, antimetabolites, spindle toxin plant alkaloids, cytotoxic / antitumor antibiotics, photosensitizers, and / or kinase inhibitors.

[0421] In some respects, treatment methods also include the administration of, for example, protein kinase inhibitors, such as MEK inhibitors; lipid kinase inhibitors; antisense oligonucleotides, particularly oligonucleotides that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, and H-Ras; vaccines; or anti-angiogenic agents.

[0422] III. Compositions, Formulations, and Kits

[0423] In some respects, the antibody-drug conjugates described herein are present in the pharmaceutical composition. In some respects, in addition to the antibody-drug conjugates, the pharmaceutical composition may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other substances known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the antibody or its antigen-binding fragment, or the pharmaceutical activity of the antibody-drug conjugate. The precise properties of the carrier or other substances will depend on the route of administration.

[0424] In some respects, the pharmaceuticals disclosed herein are salts. It may be convenient or desirable to prepare, purify, and / or dispose of corresponding salts (e.g., pharmaceutically acceptable salts). Examples of pharmaceutically acceptable salts are discussed in Berge et al., J. Pharm. Sci., 66, 1-19 (1977).

[0425] In some aspects, the composition comprises the antibody conjugate and solvate described herein. It may be convenient or desirable to prepare, purify, and / or dispose of the corresponding solvate of the active pharmaceutical ingredient. In some aspects, a solvate refers to a complex of a solute (e.g., the active pharmaceutical ingredient, a salt of the active pharmaceutical ingredient) and a solvent.

[0426] Kits according to this disclosure are also provided, comprising the antibody-drug conjugate or antigen-binding fragment drug conjugate described herein and optionally instructions for use, such as instructions for administration. In some aspects, a kit comprises multiple packages of a single-dose pharmaceutical composition, each package containing an effective amount of the antibody-drug conjugate or antigen-binding fragment drug conjugate for single administration according to the instructions for use. In some aspects, the kit also includes one or more additional cancer therapeutic agents. In some aspects, the kit includes an instrument or device for administering the pharmaceutical composition.

[0427] Example

[0428] Example 1 - CD123 expression in bone marrow mononuclear cells (BMMCs) from healthy donors and BMMCs from AML patients

[0429] Immunophenotypic analysis was performed on primary healthy donor samples (AllCells) and AML patient samples (Discovery Life Sciences and ABS) using multiparameter flow cytometry. Cell subgroups were identified using the parameters defined in Table 2. Antibody binding capacity for CD123 was measured using Bangs Laboratories Quantum Simply cell beads (Cat # 816).

[0430] CD123 is preferentially expressed in the BMMCs of AML patients compared to healthy donors. Figure 1ACompared to CD33, a significant difference in CD123 expression levels was observed between healthy donor BMMCs and BMMCs from AML patients, making CD123 a promising candidate for AML cell targeting. Figure 1B ).

[0431] HT12-GL antibody binding capacity (ABC) was assessed in healthy donor BMMCs and AML patient leukemia populations. Cell subgroups were further defined as shown in Table 2. CD123 expression was lower in healthy bone marrow cells and higher in AML patient bone marrow cells. Figure 1C ).

[0432] Table 2.

[0433]

[0434] Primary AML and normal bone marrow samples were available from Proteogenix. Cells were thawed at 37°C and washed twice with ice-cold FACS buffer (phosphate buffer + 2% fetal bovine serum). CD123+ cells were detected using a CD123 antibody conjugated to BV605 (Biolegend, clone 6H6). Primary bone marrow cells were incubated on ice for 30 minutes in 100 μL of staining solution (FACS buffer + 5 μL antibody). After incubation, cells were washed three times with FACS buffer and resuspended in 200 μL of FACS buffer for flow cytometry analysis on an LSRII (BD Bioscience) instrument. Data collected from flow cytometry were analyzed using Flowjo software (BD Bioscience).

[0435] A higher percentage of CD123+ cells were observed in bone marrow samples from AML patients compared to healthy bone marrow. Figure 1D ).

[0436] CD123 (IL3Rα) in cell lysates was assessed by Western blotting. Whole-cell lysates from Ad293 cells expressing human IL3Rα were prepared by washing cells once with ice-cold phosphate-buffered saline (PBS) followed by lysing cells with Laemmli reduction buffer (BP-111R; Boston BioProducts). Cell pellets from normal and AML bone marrow patient samples were directly lysed by adding Laemmli reduction buffer followed by vortexing. After a brief incubation, cell lysates were collected and loaded into 10-20 ml Bis NuPAGE Novex Bis-Tris gels (Invitrogen), and proteins were transferred to PVDF membranes (Invitrogen). The membranes were blocked with 5% skim milk powder and 0.1% Tween 20 (Sigma) (TBST) in TBS (pH 7.4) and incubated overnight at 4°C with antibodies against IL3Rα (NCL-L-CD123, Leica) and actin (A1978, Sigma). The membrane was washed in TBST and then incubated for 1 hour with horseradish peroxidase (HRP)-conjugated streptavidin secondary antibody (GE Healthcare). After washing, protein bands were captured and analyzed using SuperSignal West Femto chemiluminescent substrate and SuperSignal West Pico chemiluminescent substrate (Pierce / Thermo Scientific) on an ImageQuant LAS4000 instrument (GE Healthcare).

[0437] Western blot analysis revealed increased expression of CD123 (IL3Rα) in the bone marrow of AML patients compared to healthy bone marrow. Figure 1E ).

[0438] CD123 (IL3α) was assessed by immunohistochemistry (IHC). The anti-IL3Rα (CD123) mouse monoclonal antibody (clone [BR4MS]) used for IHC was available from Leica Biosystems (catalog number NCL-L-CD123) and diluted in antibody diluent with background reducing agent (Agilent, catalog number S3022). IHC was performed on a Leica Bond Autostainer instrument (Leica Biosystems, Buffalo Grove, IL, USA). Sections of formalin-fixed paraffin-embedded (FFPE) tissue were cut into 4 μm pieces and placed in a StarFrost display. ®The slides were mounted on microscope slides and placed on an automated staining machine. Antigen retrieval was performed on the slides using Bond Epitope Retrieval Solution 2 (Leica Biosystems, catalog number AR9640). HRP was then blocked with a peroxide blocking agent (Leica Biosystems, catalog number DS9800) and incubated with the primary antibody. Next, the samples were incubated with rabbit anti-mouse IgG secondary followed by primary (Leica Biosystems, catalog number DS9800), followed by anti-rabbit IgG HRP secondary polymer (Leica Biosystems, catalog number DS9800). Staining was visualized using the BOND Polymer Refine assay kit (Leica Biosystems, catalog number DS9800) with brown 3,3'-diaminobenzidine (DAB). The sections were counterstained with hematoxylin (Leica Biosystems, catalog number DS9800). Finally, the sections were dehydrated in graded ethanol, clarified in xylene, and covered with a slide.

[0439] Immunohistochemical images showed a high incidence of CD123 positivity in the bone marrow of AML patients compared to healthy bone marrow. Figure 1F ).

[0440] Example 2 - Production of CD123 Antibody

[0441] The anti-CD123 antibody disclosed in this paper was discovered using humanized transgenic Abelixis and Del-1 mouse strains (engineered to express fully human antibody variable domains) via hybridoma and immunoreplication technology. Mice were immunized with HEK293 cells overexpressing the human CD123 extracellular domain (ECD) using a repeated immunomodulatory multisite (RIMMS) strategy, and then boosted with recombinant human and cynomolgus monkey CD123 extracellular domain (ECD). B cells were harvested from draining lymph nodes and spleen. One million B cells from each mouse strain were transferred to immunoreplication activity, a method for antibody selection adapted from that described by Rajan, S., Kierny, MR, Mercer, A., et al. The recombinant human B cell library was able to screen for rare, specific, and naturally paired antibodies. Commun Biol 1, 5 (2018). The remaining B cells were mixed with SP2 / 0 myeloma cells and melted using an electrofusion apparatus to generate 6252 viable hybridoma clones. To identify cross-reactive CD123-specific binders in humans and cynomolgus monkeys, supernatants from hybridoma clones were screened for binding to huCD123 ECD antigen, CynoCD123 ECD antigen, and cells overexpressing human and cynomolgus monkey HEK293 CD123. Clones binding to parental HEK293 or Jurkat cells were considered non-specific binders. Of the screened hybridomas, 992 clones specifically bound to huCD123 ECD, CynoCD123 ECD, HEK-huCD123, and cynoCD123-HEK cells, but not to parental HEK-293 cells or Flt3 antigen, serving as negative controls. The ability of antibodies from the 992 hybridomas to induce hCD123 internalization on HEK293-overexpressing cells was screened, with approximately 90 showing internalization within 12 hours. Clamping of clones was performed using a competitive ELISA against two previously generated anti-CD123 antibodies; at least four clampings were identified. To confirm binding to CD123 parental cells, the supernatant was screened for binding to Molm13 (highly CD123-expressing) and E02-1 Pb (lowly CD123-expressing) cell lines. Ninety-one of the 992 clones bound to the CD123 parental cells Molm-13 and EOL-1, but not to Jurkat cells (CD123-negative). To identify heavy and light chain variable region sequences, cDNA from hybridomas was generated, and nGen sequencing was used to retrieve gene sequences. Twenty-four of the 91 clones were initially analyzed, and any repetitive antibody sequences were merged or antibodies with sequence biases that could not be fixed were eliminated. Sixteen useful and unique antibody genes were successfully retrieved. Sixteen parental antibody sequences were analyzed, and germlined if necessary, with any sequence biases removed. This process yielded 31 clones with optimized or parental sequences.In some cases, germplasmization leads to changes in affinity. Twenty-six mAbs were prepared, expressed, and purified for screening. These 26 mAbs were characterized in the various cell binding, internalization, and cytotoxicity assays described herein.

[0442] Example 3 - CD123 Antibody-Drug Conjugation

[0443] A 50 mM solution of tris(2-carboxyethyl)phosphine (TCEP) in phosphate-buffered saline (PBS) at pH 7.4 was added (6 molar equivalents / antibody, 320 μmol, 6400 µL) to a 1000 mL solution of antibody (e.g., HT12-GL, J13, various HT clones, antibodies described herein) in PBS containing 1 mM EDTA, resulting in a final antibody concentration of 8 mg / mL. The reduction mixture was then reacted at 37 °C on an orbital shaker with gentle shaking (60 rpm) for 2 hours (or until complete reduction was observed by UHPLC).

[0444] The drug SG3932 was added in DMSO solution (10 mol equivalents / antibody, 533 μmol, in 100 mL DMSO) to 1000 mL of this reducing antibody solution (8 g, 53.3 μmol) to bring the final DMSO concentration to 10% (v / v). The solution was mixed at room temperature for 2 hours, and then the conjugation was quenched by adding N-acetylcysteine ​​(134.4 μmol, 1.34 mL, at 100 mM).

[0445] Macromolecular aggregates and conjugating agents (including cysteine-quenched drugs) were removed using ceramic hydroxyapatite type II chromatography (CHT) as previously described (Thompson et al., J. Control Release, 236: 100-116 (2016)). Site-specific ADCs were formulated in 25 mM histidine-HCl, 7% sucrose, and 0.02% polysorbate-80 (pH 6).

[0446] To determine monomer content, aggregates, and fragments, analytical size exclusion chromatography (SEC-HPLC) was performed using 100 μg (100 μL volume) of antibody or ADC loaded onto a TSKgel G3000WXL column (Tosoh Bioscience, Tokyo, Japan). The mobile phase consisted of 0.1 M sodium sulfate, 0.1 M sodium phosphate, and 10% isopropanol at pH 6.8. The flow rate was 1 mL / min, and each analysis was performed for 20 minutes at room temperature. Hydrophobic interaction chromatography (HIC-HPLC) was used to assess conjugation and drug loading distribution, using a butyl nonporous resin (NPR) column (4.6 μm ID × 3.5 cm, 2.5 μm, Tosoh Bioscience). Mobile phase A consisted of 25 mM Tris-HCl and 1.5 M (NH4)2SO4 (pH 8.0); and mobile phase B consisted of 25 mM Tris-HCl and 5% isopropanol (pH 8.0). 100 μL of antibody or ADC at a concentration of 1 mg / mL was loaded and eluted for 13 min at a flow rate of 1 mL / min using a gradient from 5% B to 100% B. Chain-specific conjugation was confirmed using reductive reversed-phase chromatography (rRP-HPLC). The antibody and ADC were reduced at 37 °C for 20 min using 42 mM dithiothreitol (DTT) in PBS (pH 7.2). 10 μg of the reduced antibody or ADC was loaded onto a polymer reversed-phase medium (PLRP-S) 1000 A column (2.1 × 50 mm) (Agilent Technologies, Santa Clara, Calif.) and eluted for 25 min at 80 °C at a flow rate of 1 mL / min using a gradient from 5% B to 100% B (mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: 0.1% trifluoroacetic acid in acetonitrile).

[0447] Conjugation at the heavy and light chains and the drug:antibody ratio (DAR) were determined by reductive liquid chromatography-mass spectrometry (rLCMS) performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF (Agilent Technologies, Santa Clara, Calif.). 2 μg of reduced antibody or ADC was loaded onto a ZORBAX Fast Resolution High Definition (RRHD) 300-diphenyl column (2.1 × 50 mm, 1.8 μm) (Agilent Technologies, Santa Clara, Calif.) and eluted at a flow rate of 0.5 mL / min using a step gradient of 80% B (mobile phase A: 0.1% formic acid in water, and mobile phase B: 0.1% formic acid in acetonitrile) after 2.1 min. Positive time-of-flight MS scans were obtained, and data were collected and processed using MassHunter software (Agilent Technologies, Santa Clara, Calif.). The DAR was calculated using rLCMS data as described above by Thompson et al. The HT12-GL ADC was obtained using DAR 8. Figure 2A and Figure 2B .

[0448] Example 4 - CD123 antibody affinity

[0449] The affinity of the CD123 antibody described herein was determined using biomembrane interferometry.

[0450] Biomembrane interferometry binding experiments were performed using an Octet RED384 instrument, with all measurements conducted at 30°C. The AHC biosensor probe was immersed in kinetic buffer (PBS pH 7.2 + 0.02% Tween-20, 0.1% BSA, 0.05% sodium azide) for 10 minutes, followed by a 60-second baseline signal measurement in the kinetic buffer. The antibody HT12-GL was loaded onto the AHC biosensor from a 2 μg / mL solution for 120 seconds. To bind hIL3-Rα, the antibody-loaded biosensor tip was immersed in binding buffer for 60 seconds (baseline), then in a solution containing hIL3-Rα dilution (10⁻⁶ μg / mL) for 300 seconds (association), followed by immersion in kinetic buffer for 400 seconds (dissociation). For binding to hIL5-Rα and GM-CSFRα, all steps were identical, except for increasing the antigen concentration (50–1.56 μg / mL) and reducing the dissociation step to 300 seconds. Signals from buffer-only reference samples were subtracted, and all curves were fitted using a 1:1 binding model to determine KB. DThe value. HT12-GL showed an affinity of 0.31 nM for hIL3Rα and no measurable binding to the related proteins hIL5-Rα and GM-CSFRα. Figures 3A to 3C (and Figure 3E). HT12-GL also binds to cynomolgus monkey IL3Rα (Figure 3G). Antibody J13 also showed affinity for both human IL3Rα and cynomolgus monkey IL3Rα (Figures 3D and 3F).

[0451] Example 5 - CD123 antibody HT12-GL cell binding ability

[0452] The binding of HT12-GL to AD293 cells expressing CD123 (IL3Rα) from different species was evaluated. AD293 cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) containing 10% heat-inactivated fetal bovine serum (FBS) and cultured at 37°C under a humid atmosphere of 5% CO2. On the day of plating, cells were centrifuged at 1500 rpm for 3 minutes, counted, and resuspended to 0.3 × 10⁻⁶ cells / mL. 6 A final concentration of cells / ml was achieved. 200 μl of cells were seeded into the wells of a 96-well microtiter plate. The plate was centrifuged at 1500 rpm for 3 minutes and tapped to remove the culture medium. 100 μl of FACS buffer (PBS [phosphate-buffered saline] + 2% FBS) was added to the wells. The plate was then centrifuged again as described above, and then the primary antibody in 100 μl of FACS buffer was added. The primary antibody was HT12-GL labeled with Alexa 647. The antibody concentration was started at 12 μg / ml, followed by six 1:3 dilutions (no antibody control was performed). The plate was incubated at 4°C for 30 minutes. After incubation, the plate was washed three times by centrifugation at 1500 rpm for 3 minutes, tapped, and 100 μl of FACS buffer was added. After the final wash, 100 μl of FACS buffer containing unused / exhausted DAPI (diamidinyl-2-phenylindole) staining agent was added to the plate. Cells were collected and analyzed on a BD Bioscience LSRFortessa. Flow data were analyzed in FlowJo software.

[0453] Compared to other cell lines tested, the CD123 antibody HT12-GL showed high binding affinity in cell lines transduced with the CD123 / IL3Rα construct (AD293cIL3Rα and AD293hIL3Rα) and in cells transduced with the dox-inducible CD123 / IL3Rα construct and treated with doxycycline (THP-1 inducible + Dox). Figure 4A The CD123 antibody HT12-GL binds to human and cynomolgus monkey IL3Ra expressed on 293 cells, but not to rat or mouse IL3Ra. Figure 4B ).

[0454] The binding ability of the CD123 antibody HT12-GL was assessed across AML cell lines. Mv411, KG-1, OCI-AML5, and EOL-1 cells were maintained in their respective media and cultured at 37°C under a humid atmosphere of 5% CO2. On the day of plating, cells were centrifuged at 1500 rpm for 3 minutes, counted, and resuspended to 1×10⁻⁶ cells / mL. 6 A final concentration of cells / ml was achieved. 200 μl of cells were seeded into the wells of a 96-well microtiter plate. The plate was centrifuged at 1500 rpm for 3 minutes and gently tapped to remove the culture medium. 100 μl of FACS buffer (PBS + 2% FBS) was added to the wells. The plate was then centrifuged again as described above, and then HT12-GL primary antibody in 100 μl of FACS buffer was added. The antibody concentration was started at 10 µg / ml, followed by six 1:3 dilutions (no antibody control was performed). The plate was incubated at 4°C for 30 minutes. After incubation, the plate was washed three times by centrifugation at 1500 rpm for 3 minutes, gently tapped, and 100 μl of FACS buffer was added. Alexa 647 secondary antibody (goat anti-human IgG (H+L)) was added to 100 μl at a 1:1000 dilution, and the plate was incubated at 4°C for 30 minutes. The plate was washed as described above. After the final wash, 100 μl of FACS buffer containing unused / exhausted DAPI (diamidinyl-2-phenylindole) staining agent was added to the plate. Cells were analyzed on a BD Bioscience FACSSYMPHONY A3 by collecting 10,000 cells. Flow data were analyzed in FlowJo software.

[0455] Compared to NIP228 antibody, HT12-GL antibody binds to EOL-1 cells ( Figure 4C Various HT antibody clones bind to and express human IL3Ra ( ). Figure 4E ) and cynomolgus monkey IL3Ra ( Figure 4F ) 293 cells, but not bound to parental 293 cells ( Figure 4D Various HT antibody clones also bind to Mv4-11 cells ( ). Figure 4G ), OCI-AML5 cells ( Figure 4H ), but does not bind to IL3Ra-negative Jurkat cells ( Figure 4I ). HT antibody clones and various 008Q antibody clones are combined to express human IL3Ra ( Figure 4J ) and cynomolgus monkey IL3Ra ( Figure 4K 293 cells, but not IL3Ra-negative Jurkat cells ( Figure 4L ).

[0456] THP-1 cells were transfected with an IL3Rα expression construct under the control of a doxycycline inducible promoter. THP-1 cells were maintained in DMEM containing 10% heat-inactivated fetal bovine serum (FBS) and cultured at 37°C in a humid atmosphere of 5% CO2. Doxorubicin (Dox)-induced cells were stimulated overnight with 1 μg / ml Dox before use. Under these conditions, THP-1 cells were highly CD123-expressing cells. On the day of plating, cells were centrifuged at 1500 rpm for 3 minutes, counted, and resuspended to 1×10⁶ cells / mL. 6 The final concentration was determined by cells / ml. Two hundred μl of cells were seeded into the wells of a 96-well microtiter plate. The plate was centrifuged at 1500 rpm for 3 minutes and gently tapped to remove the culture medium. One hundred μl of FACS buffer (PBS + 2% FBS) was added to each well. The plate was centrifuged again as described above, and then 100 μl of primary antibody HT12-GL or HT12-GL ADC in FACS buffer was added. The antibody concentration was started at 12 μg / ml, followed by six 1:3 dilutions (no antibody control was performed). The plate was incubated at 4°C for 30 minutes. After incubation, the plate was washed three times by centrifugation at 1500 rpm for 3 minutes, gently tapped, and 100 μl of FACS buffer was added. Alexa 647 secondary antibody (goat anti-human IgG (H+L)) was added to 100 μl at a 1:1000 dilution, and the plate was incubated at 4°C for 30 minutes. The plate was washed as described above. After the final wash, 100 μl of FACS buffer containing unused / exhausted DAPI (diamidinyl-2-phenylindole) staining agent was added to the plate. Cells were analyzed on BD Bioscience LSRII by collecting 10,000 cells. Flow data were analyzed in FlowJo software.

[0457] Dox-treated THP-1 cells effectively bound HT12-GL antibody and HT12-GL ADC, while untreated THP-1 cells bound less HT12-GL antibody and HT12-GL ADC, and no binding was observed in control cells (Hel92.1.7-ve cells). Figure 5A ).

[0458] Example 6 - CD123 antibody internalization

[0459] CD123 antibody internalization was assessed using immunofluorescence microscopy with EEA1 endosome marker antibodies.

[0460] THP-1 cells expressing Dox-inducible CD123 (IL3Rα) were induced overnight with 1 μg / ml doxycycline to overexpress IL3Rα. Cells were incubated with 10 μg / ml HT12 antibody on ice to allow binding, followed by incubation at 37°C for 1 hour to allow internalization. After internalization, cells were fixed with 4% formaldehyde and stained with mouse anti-EEA1 antibody, followed by secondary staining with Alexa Flour 488 goat anti-mouse antibody and 546 goat anti-human antibody. Cells were evaluated at 60X magnification using a Zeiss 880 confocal microscope. Co-localization of the internalized HT12 antibody and the early endosome marker EEA1 showed the localization of HT12 antibody in early endosomes 1 hour after HT12 antibody addition. Figure 5B (white spots).

[0461] Internalization was also measured by conjugating a pH-sensitive anti-human Fc deep red dye with HT12-GL antibody, HT12-GL ADC (HT12-GL-SG3932), an allotype control NIP228 antibody, or a control NIP228 ADC. This dye fluoresces only when the dye reaches the lysosomes after internalization. Cells were internalized with 10 μg / ml of antibody and ADC for 5 hours, and fluorescence signals were measured by flow at the end of each time point. Each internalization time point represents the geometric mean of the signal subtracted from the T0 time point. HT12-GL antibody and HT12-GL ADC were effectively internalized into dox-treated THP-1 cells (…). Figure 5C and Figure 5D ).

[0462] Internalization of various HT antibody clones was also tested in Molm13 cells. Figure 5E ).

[0463] Example 7 - CD123 antibody inhibits IL3-mediated signal transduction and proliferation

[0464] The TF-1 erythroleukemia cell line, which proliferates only in the presence of one of the following growth factors (IL-3 or GM-CSF), was used. TF-1 cells were washed with RPMI and cultured overnight in complete RPMI medium (RPMI-1640, 10% fetal bovine serum) without any growth factors. To block Fc receptors on the cell surface, cells were incubated with a human Fc blocking agent at room temperature for 5 to 10 min. Cells were centrifuged at 1300 rpm for 5 min and seeded at 8,000 cells / well in complete medium with or without 10 μg / ml anti-CD123 antibody (HT12 and J13) or control antibody (NIP228, 9F5). Growth factor IL-3 (1 ng / ml) or GM-CSF (2 ng / ml) was added to the cells to initiate proliferation. Cells were incubated at 37°C in a humidified 5% CO2 incubator for 3 days. Cells were then analyzed using CellTiter-Globe. ® Cell viability was measured using a (CTG) luminescent cell viability assay. CTG reagent was added to the wells in equal volumes, and the plate was shaken at room temperature for 10 minutes. Absorbance was then measured using an Envision luminescent reader. The relative number of cells in each well was calculated by dividing the value of each antibody-treated sample by the average value of the wells containing untreated cells (controls). The 9F5 control antibody did not inhibit IL-3-dependent proliferation. The HT12-GL antibody and a smaller amount of the J13 antibody inhibited IL-3-dependent proliferation of TF-1 cells. Figure 6A However, it does not inhibit GM-CSF-mediated proliferation. Figure 6B Allotype antibodies have no effect on cell proliferation.

[0465] To measure the antibody concentration required for IL3 inhibition, TF-1 cells were seeded at 8,000 cells / well and antibody was added in 10-point dilutions starting at 10 μg / ml. IL-3 was then added at a concentration of 1 ng / ml. The final antibody concentration ranged from 66 M to 0.03 M. Cells were incubated at 37°C for 3 days. The relative cell number in each well was determined using Cell Titer Glo as described above. The relative cell number plotted against antibody concentration showed that the HT12-GL antibody dose-dependently inhibited TH-1 cell proliferation in the presence of IL-3. Figure 6C ).

[0466] The effects of CD123 antibody and CD123 ADC (HT12-GL-SG3932) on the IL3-mediated JAK-STAT5 signaling pathway were measured. TF-1 cells were cultured in RPMI and then in complete RPMI medium (RPMI-1640, 10% FBS) with 2 ng / ml GM-CSF. Cells were washed in Stem Span H3000 serum-free medium and treated for 2 h at 37°C with the same type of ADC, CD123 ADC (HT12-GL-SG3932), or CD123 antibody (pre-prepared in Stem Span H3000 SFM medium). Cells were washed and seeded at 2,000 cells / well in 96-well U-shaped plates. Cells were stimulated for 15 min with 10 ng / ml or 100 ng / ml GM-CSF or IL3, or untreated (cytokine-free). Following stimulation, cells were immediately fixed by adding pre-warmed BD Cytofix fixation buffer to the same well. Fixation was performed at 37°C for 12 min. After fixation, cells were permeabilized by adding cooled Perm buffer III on ice for 30 min. Cells were washed with staining buffer and stained overnight at 4°C with pStat5-Bv421 antibody. pSTAT5 activation was measured by flow cytometry. The geometric mean of pSTAT5-positive viable cells was plotted as the pSTAT5 activity RFU. The results showed that both GM-CSF and IL3 increased pSTAT5 activation after 15 min of stimulation when pretreated with the same type of ADC alone. Consistent with earlier findings, both the CD123 antibody and the CD123 ADC (HT12-GL-SG3932) inhibited the STAT5 signaling pathway mediated by IL3, but not GM-CSF. Figure 6D ).

[0467] Example 8 - CD123 ADC-mediated cytotoxicity

[0468] THP-1 cells expressing CD123 under the control of the dox-inducible promoter were grown in the presence of 1 μg / ml doxycycline. High CD123-expressing THP-1 cells and CD123-non-expressing Hel92.1.7 cells were seeded at 2500 cells per well (30 μl per well) in 384-well plates for 24, 48, 72, and 144 hours, with each time point repeated. Cells were treated with HT12-GL ADC (HT12-GL-SG3932) or the same type of NIP228 ADC. The drug treatment was prepared at 240 μg / ml and serially diluted 1:4. Triplets of 10 μl of the treatment were added to achieve a concentration of 60 μg / ml to 0.00001 μg / ml across all plates and time points. Baselines were established by treatment with complete growth medium without ADC. CellTiter-Glo from Promega, as recommended by the supplier, was used. ® (CTG) and Caspase Glo ® 3 / 7 reagents were used to read the cytotoxicity and caspase activity of the plates at each time point. The data were then normalized to baseline.

[0469] HT12-GL ADC (HT12-GL-SG3932) showed effective killing of CD123+dox THP-1 cells. Figure 7A However, it did not kill CD123-negative Hel92.1.7 cells. Figure 7B Furthermore, HT12-GL ADC (HT12-GL-SG3932) showed increased caspase 3 / 7 activity in CD123-positive dox THP-1 cells. Figure 7C ), but not in CD123-negative Hel92.1.7 cells ( Figure 7D ).

[0470] Cell-killing effects of various HT-SG3932 antibody clones were also tested. Although toxicity of all tested HT antibody clone ADCs in 293 cells was observed only at the highest tested concentrations ( Figure 8A However, in 293 cells expressing human IL3Ra ( Figure 8B ) and 293 cells expressing cynomolgus monkey IL3Ra ( Figure 8C In this study, cytotoxicity of various HT antibody clone ADCs was observed at lower concentrations. Furthermore, the HT antibody clone ADC (HT-SG3932) only showed toxicity in IL3Ra-negative THP-1 cells at the highest tested concentration. Figure 8D However, at lower concentrations, it did not show cytotoxicity in THP-1 cells treated with dox expressing IL3Ra. Figure 8E ).

[0471] Example 9: A-CD123 antibody-drug conjugate induces bystander killing.

[0472] THP-1 cells were maintained in DMEM medium containing 10% heat-inactivated FBS and cultured in a humid atmosphere of 5% CO2 at 37°C. Cells were stimulated overnight with 1 μg / ml dox or maintained in the absence of dox before use. Unstimulated cells contained GFP markers. Cells were seeded in 24-well plates at stimulated / unstimulated ratios of 100 / 0, 90 / 10, 75 / 25, 50 / 50, 25 / 75, 10 / 90, and 0 / 100%. 10 × 10⁶ cells were cultured in each well. 4 Add 250 μl of cells / ml along with 250 μl of culture medium to each well. Add HT12-GL ADC (HT12-GL-SG3932) or NIP228 (isotype control; NIP228-SG3932) ADC to the wells to a final concentration of 100 ng / ml. Incubate the plate as described above for 72 hours. After incubation, transfer all cells / culture medium to a deep-well 96-well plate. Centrifuge the plate at 1500 rpm for 3 minutes, then tap to remove the culture medium. Add 100 μl of FACS buffer (PBS + 2% FBS) to the plate and resuspend the cells. Then transfer the cells to a microtiter plate. Centrifuge the plate at 1500 rpm for 3 minutes, then tap to remove the FACS buffer. After a final wash, add 100 μl of FACS buffer containing unused / exhausted DAPI (diamidinyl-2-phenylindole) staining agent to the plate. Cells were analyzed on a BD Bioscience FACSSYMPHONY A3 sample, starting with a 90µl sample. All cells were counted starting from the 90µl sample. Flow data were analyzed in FlowJo software.

[0473] Although HT12-GL ADC (HT12-GL-SG3932) does not affect the viability of CD123-negative THP-1 CLEC GFP cells ( Figure 7E However, it does kill CD123-positive THP-1 CLEC DOX cells ( Figure 7F Co-culture of CD123-negative and CD123-positive THP-1 cells resulted in bystander killing of CD123-negative cells. Figure 7G Furthermore, increasing the ratio of CD123-positive doxTHP-1 cells increased bystander killing of CD123-negative THP-1 cells. Figure 7H ).

[0474] Example 9: B-CD123 antibody-drug conjugate induces double-strand breaks

[0475] THP1 cells were maintained in RPMI containing 10% heat-inactivated FBS and cultured at 37°C under a humidified atmosphere of 5% CO2. Doxorubicin-induced cells were stimulated overnight with 1 μg / ml dox. On the day of plating, cells were centrifuged at 1500 rpm for 3 minutes, counted, and resuspended to 3 × 10⁶ cells / mL. 6 A final concentration of cells / ml was determined. One ml of cells was seeded into 6-well plates with 4 ml of culture medium. HT12-GL ADC (e.g., HT12-GL-SG3932) or NIP228-SG3932 (isotype) ADC was added to the cells at a final concentration of 0.05 μg / ml. The cells were incubated with the ADC for 24, 48, and 72 hours. Cells were collected at appropriate time points and centrifuged at 1500 rpm for 3 minutes. Cells were then lysed with 500 μl of MPER (Mammalian Protein Extraction Reagent) containing Halt protease and phosphatase inhibitors. The lysates were stored at -80°C until Western blotting was performed.

[0476] Thaw the lysate and perform BCA to determine protein concentration. Adjust the protein concentration and load approximately 10 μg of protein into the gel. Run the gel until an appropriate molecular weight can be detected. Transfer the gel to a nitrocellulose membrane using an Iblot system. Block the blot with SuperBlock for at least 1 hour. Add the primary antibody (see table below) at a 1:1000 dilution to the blocking buffer, except for the actin loading control (1:10000 dilution), and incubate overnight at 4°C with shaking. Then wash the blot and add the secondary HRP-labeled antibody to the superblocking agent at room temperature for 1 hour. After incubation, wash the blot and add SuperSignal West Coast Pico to the blot. Image the blot on an ImageQuant 4000 imager. The primary antibodies are pRPA Cell Signaling 4124, pTIF1B Cell Signaling 4127S, pCHK2 Cell Signaling 2197, pCHK1 Cell Signaling 12302S, cCaspase Cell Signaling 9662S, PARPCell Signaling 9542, and actin Sigma A1978.

[0477] Treatment with CD123 ADC (HT12-GL-SG3932) induced an increase in double-strand break-related proteins. Figure 7I ).

[0478] Example 10 - Efficacy of CD123 antibody-drug conjugates compared to CD33 Mylotarg ADC

[0479] The efficacy of HT12-GL ADC (HT12-GL-SG3932) was compared with that of CD33Mylotarg ADC and allotype ADC in a colony-forming unit (CFU) assay. For the CFU assay, primary healthy donor samples and AML patient samples were titrated with HT12-GL ADC (HT12-GL-SG3932), allotype ADC, or Mylotarg for 24 hours. Cells were then mixed in Methodcult (StemCell) and seeded in 6-well SmartDish (StemCell). Colonies were counted using a STEMvision instrument (StemCell) 14 days post-seeding. EC50 values ​​were calculated based on the reduction in colonies after drug treatment compared to the control group.

[0480] Compared to healthy BMMCs, HT12-GL ADC (HT12-GL-SG3932) showed similar efficacy to CD33 Mylotarg ADC in reducing CFU levels in AML BMMCs, but HT12-GL ADC (HT12-GL-SG3932) lacked CFU toxicity in healthy donor BMMCs, while Mylotarg was toxic in healthy BMMCs. Figure 9A ).

[0481] Example 11 - CD123 ADC showed cytotoxicity against HSCs, GMP cells, and differentiated cells in CD34+ BMMC amplification assay. sex

[0482] For CD34 + BMMC amplification assay involves seeding healthy donor BMMCs into 24-well plates containing dose-titrated HT12-GL ADC (HT12-GL-SG3932) or an isotype ADC supplemented with CD34. + StemSpan SFEMII (StemCell) was supplemented with cell expansion supplement (StemCell), 1 μM UM729 (StemCell), and 500 nM Stemregenin (StemCell). Cells were cultured for 6 days, followed by immunophenotypic analysis using flow cytometry. Healthy donor HSCs (lineage) - CD34 + CD38 - CD90 + CD45RA - GMPs (GMP: lineage) - CD34 + CD38 + CD135 + CD45RA + ) and differentiated cells (lineages)+ The table marking is retained for the reference hole.

[0483] Compared with the same type of ADC, HT12-GL ADC (HT12-GL-SG3932) has no cytotoxic effect on HSCs, GMP cells, or differentiated cells. Figure 9B ).

[0484] Example 12 - CD123 ADC lacks cytotoxicity against HUVECs and HPAECs expressing CD123.

[0485] Endothelial cell lines HUVEC and HPAEC expressing CD123, along with control cell lines RPMI-8226 (high CD123 expression) and K562 (low expression or CD123 negativity), were cultured and co-incubated for 3 days with comparative compound-1 ADC, NIP228-SG3932 (an isotype control of HT12-GL-SG3932), HT12-GL-SG3932, or CD123 mAb (HT12-GL) at concentrations ranging from 0.0017 to 520 μg / mL (for CD123 mAb, HT12-GL-SG3932, or NIP228-SG3932) or from 0.0010 to 320 μg / mL (for IMGN-IGN). Cell viability was assessed using a CellTiter-Glo cell viability assay, and the IC50 for each compound against each cell line was calculated. 50 (Where possible) Assess CD123 expression by surface expression via Western blot or by flow cytometry (Figs. 9C to 9H).

[0486] The results are shown in Table 3. Compared with the isotype control, HT12-ADC did not show enhanced cytotoxicity against CD123-expressing endothelial cell lines. Compared with the CD123-independent HT12-GL-SG3932, Comparative-1 ADC showed higher cytotoxicity against all cell lines.

[0487] Table 3. Compared with Comparative-1 ADC, the HT12-GL TOPO ADC (e.g., HT12-GL-SG3932) targets the expression cytotoxicity of CD123 endothelial cell lines

[0488]

[0489] Comparative compound-1 comprises a CD123-ADC of pivekimab sunirine, described in U.S. Patents 10,919,969 and 11,332,535. In mice carrying subcutaneous EOL-1 tumors, comparative compound-1 ADC induced complete tumor regression with a single dose of 0.24 mg / kg, consistent with published data. See Kovtun et al., Blood Adv (2018) 2(8):848-858.

[0490] Example 13 - Study on in vivo subcutaneous xenografts

[0491] Mv4-11 cells, each expressing approximately 48,000 CD123 / IL3Rα, were maintained in IMDM (Iscove's Modified Dulbecco's Medium) containing 10% heat-inactivated FBS and cultured at 37°C under a humidified atmosphere of 5% CO2. On the day of engraftment, cells were centrifuged at 1500 rpm for 5 minutes, counted, and cultured in a 1:1 PBS:Matrigel solution. ® Resuspended in the (Corning) mixture to 25 × 10⁻⁶ 6 The final concentration was calculated as cells / mL. Each female CB-17SCID mouse was subcutaneously injected with 200 μL of 5 × 10⁻⁶ cells / mL in the right flank. 6 Each cell.

[0492] When the average tumor volume reached approximately 150 mm on day 21 3 -250mm 3 Mice were randomly assigned to treatment groups using a matching distribution method built into the research guidance software. Table 3 presents the group names, dosage levels, and number of mice in each group.

[0493] The test and control ADCs were diluted from the stock solution with mediator buffer (20 mM histidine, 240 mM sucrose, 0.02% polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection based on body weight. Group targets and dose levels for the Mv4-11 subcutaneous xenograft study are shown in Table 4.

[0494] Table 4

[0495]

[0496] Tumor volume and body weight were measured 1 to 2 times weekly from day 15 to day 98 after Mv4-11 implantation. The tumor was measured using calipers, and the tumor volume was calculated using the following formula:

[0497] Tumor volume = [length (mm) × width (mm)] 2 ] / 2; where the length and width are the longest and shortest diameters of the tumor, respectively.

[0498] HT12-GL ADC (HT12-GL-SG3932) induced a dose-dependent antitumor response in a dose range of 0.5 mg / kg to 2 mg / kg. Figure 10AAt a dose level of 2 mg / kg, HT12-GL ADC (HT12-GL-SG3932) induced complete tumor regression, while the control isotype ADC (NIP-228-SG3932 ADC) induced minimal antitumor activity. Figure 10A ).

[0499] At a dose level of 2 mg / kg, HT12-GL ADC (HT12-GL-SG3932) induced complete tumor regression, while other antibody ADCs (J13-SG3932) induced partial tumor growth inhibition, and the control isotype ADC (NIP-228-SG3932) induced minimal antitumor activity. Figure 10B ).

[0500] EOL-1 cells expressing approximately 2,000 CD123 / IL3Rα per cell were maintained in RPMI-1640 medium containing 10% heat-inactivated FBS and cultured at 37°C under a humidified atmosphere of 5% CO2. On the day of implantation, cells were centrifuged at 1500 rpm for 5 minutes, counted, and resuspended in PBS to a final volume of 50 × 10⁻⁶ cells / mL. 6 The final concentration was [number of cells / mL]. Each female nude mouse was subcutaneously injected with 200 μL of 10 × 10 [cells / mL]. 6 1 cell. When the average tumor volume reached approximately 150 mm on day 9. 3 -250mm 3 Mice were randomly assigned to treatment groups using a matching distribution method built into the research guidance software. The group names, dosage levels, and number of mice in each group are presented in the table below.

[0501] The test and control ADCs were diluted from the stock solution with media buffer (20 mM histidine, 240 mM sucrose, 0.02% polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection based on body weight. Group targets and dose levels in the EOL-1 subcutaneous xenograft studies are shown in Table 5.

[0502] Table 5

[0503]

[0504] Tumor volume and body weight were measured twice weekly from day 6 to day 60 post-EOL-1 implantation. Tumor volume was measured using calipers and calculated using the following formula:

[0505] Tumor volume = [length (mm) × width (mm)] 2 ] / 2; where the length and width are the longest and shortest diameters of the tumor, respectively.

[0506] HT12-GL ADC induced complete tumor regression at 3 mg / kg and partial tumor growth inhibition at 2 mg / kg, while the control isotype ADC (NIP-228 ADC) and naked antibody HT12-GL mAb induced minimal antitumor activity. Figure 10C and Figure 10D ).

[0507] Example 14 - Study on in vivo disseminated xenografts

[0508] Mv4-11 cells were maintained in IMDM (Iscove's Modified Dulbecco's Medium) containing 10% heat-inactivated FBS and cultured at 37°C under a humidified atmosphere of 5% CO2. On the day of implantation, cells were centrifuged at 1500 rpm for 5 minutes, counted, and resuspended in PBS to a final volume of 25 × 10⁶ cells / mL. 6 The final concentration was calculated as cells / mL. Each female NSG mouse was injected intravenously via the tail vein with 200 μL of 5 × 10⁻⁶ cells / mL. 6 Each cell.

[0509] Body weight was measured on day 5 after Mv4-11 cell implantation and day 4 after Molm-13 cell implantation. Mice were randomly assigned to treatment groups based on body weight using a matched distribution method built into the study guidance software. The group names, dose levels, and number of mice in each group are presented in the table below.

[0510] Test and control products were diluted from the stock solution with media buffer (20 mM histidine, 240 mM sucrose, 0.02% polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection based on body weight. Group targets and dose levels in the Mv4-11 disseminated survival study are shown in Table 6.

[0511] Table 6

[0512]

[0513] Monitor mice for signs of rough skin / fur and weak hind limbs. Euthanize mice immediately when they show hind limb paralysis or inability to eat / drink, and record survival days. Fit Weibull regression to survival time and use hazard ratio (better survival probability) and median survival to compare between groups.

[0514] Compared with the untreated group, all lead compounds induced a beneficial effect on survival in terms of intermediate survival. Figure 11A ).

[0515] Mv4-11 cell implants were also tested with single and two doses of HT12-GL ADC. As described, Mv4-11 cells were in 200 μL volumes of 5 × 10⁻⁶ cells. 6 Mv4-11 cells were intravenously injected into the tail vein of female NSG mice. Body weight was measured on day 5 post-implantation. Mice were randomly assigned to treatment groups based on body weight using a matched distribution method built into the study guidance software. The group names, dose levels, and number of mice in each group are presented in the table below.

[0516] The test and control ADCs were diluted from the stock solution with mediator buffer (20 mM histidine, 240 mM sucrose, 0.02% polysorbate 80, pH 6.0) and administered as a single intravenous (IV) injection or two IV injections (Q1Wx2) based on body weight. Group targets and dose levels in the MV4-11 disseminated survival study are shown in Table 7.

[0517] Table 7

[0518]

[0519] Monitor mice for signs of rough skin / fur and weak hind limbs. Euthanize mice immediately if they show signs of hind limb paralysis or inability to eat / drink and record the number of days they survive.

[0520] We fitted Weibull regression to survival time and used hazard ratio (better survival probability) and median survival to compare between groups.

[0521] In terms of risk, compared with the same type of ADC treatment group, both single administration of HT12-GL ADC and the Q1Wx2 dosing regimen induced a moderate survival benefit. Figure 11B ).

[0522] Example 15 - In vivo patient xenograft

[0523] AML patient samples are used for patient-derived graft models (PDX). Figure 12A The figure shows the characterization of AML PDX models, including the number of CD123 receptors per cell for each AML-PDX model (68555, 62736, 33766, 49600, 13086 and 40365).

[0524] For each AML-PDX model (68555, 49600, 33766, 62736, 13086, and 40365), cells were thawed and counted. 730,000 to 1,400,000 cells were intravenously implanted into NSG / NSG-SGM3 mice. Peripheral blood graft implantation (% of huCD45) was sporadically examined from the entire mouse cohort or subgroups by flow cytometry analysis to determine the dosing stage. Mice were randomized using peripheral blood graft implantation or body weight using a matching distribution method built into the StudyLog software. Group names, dose levels, and number of mice per group are presented in Tables 7 and 8.

[0525] The test and control ADCs were diluted from the stock solution with media buffer (20 mM histidine, 240 mM sucrose, 0.02% polysorbate 80, pH 6.0). All treated mice were administered two intravenous (IV) injections according to body weight. Group names and dose levels in AML-PDX dissemination endpoint study models 68555, 62736, 49600, and 33766 are shown in Table 8.

[0526] Table 8

[0527]

[0528] The day of the first dose was defined as day 0 of the study phase. Mice were weighed at least once a week. On day 14, mice from groups 1, 2, 3, 4, and 5 were euthanized. On day 28, mice from groups 6, 7, 8, 9, and 10 were euthanized.

[0529] Group names and dose levels in AML-PDX dissemination endpoint study models 13086 and 40365 are shown in Table 9.

[0530] Table 9

[0531]

[0532] The day of the first dose was defined as day 0 of the study phase. Body weight was measured at least once a week. For models 13086 and 40365, mice from all groups were euthanized on day 28.

[0533] For all PDX models, whole blood and bone marrow samples were collected from individual mice, processed, and stained for flow cytometry analysis. The percentage of huCD45 was used to define antileukemic efficacy. CD123 receptor density in bone marrow samples was also assessed using flow cytometry analysis. Percentage changes in each treatment group were calculated by normalization to a time-point matched untreated group, except for model AML-PDX 68555, which was calculated by normalization to the untreated group at day 14. Waterfall plots were generated for all AML-PDX models based on tissue matrix and dose levels.

[0534] In whole blood at a dose level of 5 mg / kg, HT12-GL ADC induced significant antileukemic efficacy in the 4 / 4 model on day 14 and the 3 / 6 model on day 28, compared with the untreated model. Figure 12B In bone marrow, compared with the untreated model, HT12-GL ADC induced significant antileukemic efficacy at 5 mg / kg in 3 / 4 models on day 14 and 2 / 6 models on day 28. Figure 12C ).

[0535] In whole blood at a dose level of 10 mg / kg, HT12-GL ADC induced significant antileukemic efficacy in the 4 / 4 model on day 14 and the 6 / 6 model on day 28, compared with the untreated model. Figure 12D In bone marrow, compared with the untreated model, HT12-GL ADC induced significant antileukemic efficacy at 10 mg / kg in the 4 / 4 model on day 14 and the 4 / 6 model on day 28. Figure 12E ).

[0536] HT12-GL ADC also induced a robust decrease in CD123 receptor density on bone marrow samples at 5 mg / kg and 10 mg / kg on day 14, indicating a strong antigen-targeting mechanism. Figure 12F On day 28, CD123 receptor density was restored at a lower dose of 5 mg / kg instead of 10 mg / kg. Figure 12F ).

[0537] These results demonstrate the robust antileukemic efficacy of HT12-GL ADC across various AML-PDX models with different genetic mutations, treatment statuses, and cytogenetic risk levels.

[0538] Example 16: Payload stability in HT12-GL ADC antibody and CB17-SCID mice

[0539] CB17-SCID mice were administered HT12-GL ADC at 5 mg / kg, and plasma was analyzed by immune capture antibody. Subsequently, the heavy chain, light chain, and warhead components of the ADC were measured by ELISA-based human antibody detection or LCMS.

[0540] Exposure over time demonstrated the antibody-like behavior of HT12-GL ADC, with the following p-values: based on ELISA results, AUCinf was 821 (μg·day / mL), clearance was 6.1 mL / day / kg, and half-life was 14 days (Figure 13A). Immunocapture of HT12-GL ADC, followed by trypsin digestion to generate heavy chain peptides (HC) and light chain peptides (LC), and papain digestion to release the warhead (WH), enabled targeted LC-MS / MS assessment of antibody and payload stability (Figure 13B). Comparison of exposures between alternative analytes indicated that HT12-GL ADC is stable in vivo (Figures 13C and 13D). HT12-GL ADC in mouse serum (… Figure 14A ), cynomolgus monkey serum ( Figure 14B ) and rat serum ( Figure 14C It is also stable in ).

[0541] Example 17 - Synthesis of Topoisomerase I Inhibitor

[0542] General information

[0543] Using Biotage ® Isolera ™ Rapid chromatography was performed, and the purity of the fractions was checked using thin-layer chromatography (TLC). TLC was performed on an aluminum plate using Merck Kieselgel 60 F254 silica gel and a fluorescent indicator. Visualization of the TLC was achieved using UV light.

[0544] Extraction and chromatography solvents are purchased from VWR UK and used directly without further purification.

[0545] Unless otherwise stated, all fine chemicals were purchased from Sigma-Aldrich. Polyethylene glycol reagents were available from Quanta biodesign US via Stratech UK.

[0546] LC / MS conditions

[0547] Method A

[0548] Positive mode electrospray ionization mass spectrometry was performed using a Waters Aquity H-class SQD2. The mobile phases used were solvent A (water containing 0.1% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The initial composition of 5% B was held for 25 seconds, then increased from 5% B to 100% B over a 1 min 35 sec time interval. The composition was held at 100% B for 50 seconds, then returned to 5% B over 5 seconds and held for 5 seconds. The total duration of the gradient run was 3.0 min. The flow rate was 0.8 mL / min. Detection was performed at 254 nm. Column: Waters Acquity UPLC at 50 °C. ® BEH Shield RP18 1.7µm 2.1×50mm, equipped with WatersAcquity UPLC ® BEH Shield RP18 VanGuard front pillar, 130A, 1.7µm, 2.1mm×5mm.

[0549] Method B

[0550] HPLC (WatersAlliance 2695) was run using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid). The initial composition of 5% B was held for 25 seconds, then increased from 5% B to 100% B over a 1 minute 35 second period. The composition was held at 100% B for 50 seconds, then returned to 5% B over 5 seconds and held for 5 seconds. The total duration of the gradient run was 3.0 minutes. The flow rate was 0.8 mL / min. The detection range was 190 nm to 800 nm. The column was a Waters Acquity UPLC at 50 °C. ® BEH Shield RP18 1.7µm 2.1×50mm, equipped with Waters AcquityUPLC ® BEH Shield RP18 VanGuard front pillar, 130A, 1.7µm, 2.1mm×5mm.

[0551] Method C

[0552] The HPLC was run using a mobile phase of water (A) (0.1% formic acid) and acetonitrile (B) (0.1% formic acid) (WatersAlliance 2695).

[0553] The initial composition was maintained at 5% B for 1 minute, then increased to 100% B over a 9-minute period. The composition was held at 100% B for 2 minutes, then returned to 5% B over 0.10 minutes and held for 3 minutes. The total gradient run time was 15 minutes. Flow rate: 0.6 mL / min. Wavelength detection range: 190 nm to 800 nm. Oven temperature: 50 °C. Column: ACE Excel 2 C18-AR, 2 μm, 3.0 × 100 mm.

[0554] HPLC conditions

[0555] Reversed-phase ultra-fast high-performance liquid chromatography (UFLC) was developed at Shimadzu Prominence. ™ The machine uses Phenomenex sensors with dimensions of 150×21.2mm. ™ The experiments were performed using a Gemini NX 5µ C18 column (at 50°C). The eluents used were solvent A (H₂O containing 0.1% formic acid) and solvent B (CH₃CN containing 0.1% formic acid). All UFLC experiments were performed under gradient conditions: an initial composition of 13% B was increased to 30% B over a 3-minute interval, then to 45% B over 8 minutes, and then to 100% B again over 6 minutes, before returning to 13% and holding for 1 minute over 2 minutes. The total duration of the gradient run was 20.0 minutes. The flow rate was 20.0 mL / min, and detection was performed at 254 nm and 223 nm.

[0556] NMR method

[0557] Proton NMR chemical shifts were measured at 400 MHz on a delta scale using a Bruker AV400. The following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; quintet; m, multiplet; br, broad peak. Coupling constants are reported in Hz.

[0558] Synthesis of key intermediates:

[0559]

[0560] a) N-(5,6,7,8-tetrahydronaphth-1-yl)acetamide (I2)

[0561] 5,6,7,8-Tetrahydronaphthyl-1-amine I1 (8.54 g, 58.0 mmol) was dissolved in dichloromethane (80 mL). Triethylamine (18 mL, 129 mmol) was added, and the mixture was cooled to 0 °C. Acetic anhydride (11.5 mL, 122 mmol) was added dropwise. After the addition was complete, the reaction mixture was warmed to room temperature and stirred for 45 min, at which point LC / MS indicated the reaction was complete. The mixture was diluted with CH2Cl2, washed with H2O-saturated NaHCO3 and 10% citric acid, and the organic phase was dried over MgSO4 and concentrated under vacuum. The off-white solid was ground with 1:3 Et2O / isohexane to give a white solid I2 (10.8 g, 57.1 mmol, 98% yield), which was used directly without further purification. LC / MS (Method A): Retention time 1.44 min (ES+) m / z 190 [M + H] +

[0562] b) N-(4-nitro-5,6,7,8-tetrahydronaphth-1-yl)acetamide (I3)

[0563] N-(5,6,7,8-tetrahydronaphthyl-1-yl)acetamide I2 (1.00 g, 5.2840 mmol) was added in portions to sulfuric acid (15 mL, 281 mmol) at -5 °C. Sodium nitrate (450 mg, 5.2945 mmol) was added in portions to the reaction mixture, and the mixture was stirred at -5 °C for 30 min, at which point LCMS indicated no further reaction progress. The reaction mixture was poured onto ice with external cooling, and the aqueous mixture was extracted with CH2Cl2. The organic phase was dried over MgSO4 and purified by Isolera (10-80% EtOAc in isohexane) to give a mixture of N-(4-nitro-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide I3 and N-(2-nitro-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (956 mg, 4.0811 mmol, 77% yield) as a white / yellow solid. LC / MS (Method A): Retention time 1.53 min (ES+) m / z 235 [M + H] + .

[0564] c) N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (I4)

[0565] N-(4-nitro-5,6,7,8-tetrahydronaphth-1-yl)acetamide I3 (1.01 g, 4.31 mmol) was dissolved in acetone (30 mL). Magnesium sulfate (3.9 mL, 5.9 mmol, 1.5 mol / L) was added to water, and the mixture was cooled to 0 °C. Potassium permanganate (2.07 g, 13.0 mmol) was added in portions to the reaction mixture, and the mixture was warmed to room temperature and stirred for 50 min, at which point TLC indicated the reaction was complete. The reaction mixture was filtered through diatomaceous earth, the solid was washed with CHCl3, and the resulting organic mixture was washed with H2O and brine, dried over MgSO4, and purified by isolatora (20-50% EtOAc in isohexane) to give a mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide as a white / yellow solid (709 mg, 2.86 mmol, 66%). LC / MS (Method A): Retention time 1.44 min (ES+) m / z 190 [M + H] +

[0566] d) 8-Amino-5-nitro-3,4-dihydronaphthyl-1(2H)-one (I5)

[0567] A mixture of N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide I4 and N-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide (709 mg, 2.8561 mmol) and 6N hydrochloric acid (7 mL) was stirred at 80 °C for 2.5 h, at which point LCMS indicated the reaction was complete. The reaction mixture was cooled in an ice bath, and 6N NaOH solution was added until the pH was alkaline. The aqueous mixture was extracted with CH2Cl2, and the organic phase was dried over MgSO4 and concentrated under vacuum. Isolera (0-50% EtOAc in isohexane) provided 8-amino-5-nitro-3,4-dihydronaphthyl-1(2H)-one I5 (320 mg, 1.552 mmol, 54% yield) as a yellow / orange solid. LC / MS (Method A): Retention time 1.54 min (ES+) m / z 207 [M + H] +

[0568] e) 2,2,2-Trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (I6)

[0569] 8-Amino-5-nitro-3,4-dihydronaphthyl-1(2H)-one I5 (430 mg, 2.0854 mmol) was dissolved in dichloromethane (20 mL). Pyridine (340 μL, 4.20 mmol) was added, and the mixture was cooled to 0 °C. Trifluoroacetic anhydride (590 μL, 4.197 mmol) was added, and the mixture was stirred for 30 min, at which point LCMS indicated the reaction was complete. The mixture was diluted with CH2Cl2, washed with H2O, dried over MgSO4, and concentrated under vacuum to give 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)acetamide I6 (630 mg, 2.0846 mmol, >99% yield) as a yellow solid, which was used without further purification. LC / MS (Method A): Retention time 1.86 min (ES+) m / z 30 1X [M - H] -

[0570] f) N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)-2,2,2-trifluoroacetamide (I7)

[0571] Zinc (2.73 g, 41.7 mmol) was suspended in methanol (80 mL), formic acid (4 mL), and water (4 mL), and the mixture was cooled to 0 °C. 2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide I6 (568 mg, 2.0865 mmol) was added in portions, and the mixture was stirred at 0 °C for 30 min, at which point LCMS indicated the reaction was complete. The reaction mixture was filtered, the filtrate was diluted with EtOAc, and washed with saturated NaHCO3. The organic phase was dried over MgSO4 and concentrated under vacuum to give N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)-2,2,2-trifluoroacetamide I7 (568 mg, 2.0865 mmol, >99% yield) as a yellow solid, which was used directly without further purification. LC / MS (Method A): Retention time 1.65 min (ES+) m / z 273 [M + H] +

[0572] g)N-(4-acetamido-8-oxo-5,6,7,8-tetrahydronaphth-1-yl)-2,2,2-trifluoroacetamide (I8)

[0573] N-(8-amino-4-oxo-tetrahydronaphthyl-5-yl)-2,2,2-trifluoro-acetamide I7 (568 mg, 2.0865 mmol) was dissolved in dichloromethane (20 mL). Triethylamine (580 μL, 4.16 mmol) was added, followed by acetyl chloride (297 μL, 4.173 mmol), and the mixture was stirred for 30 min, at which point LC / MS indicated the reaction was complete. The reaction mixture was diluted with CH2Cl2, washed with H2O, dried over MgSO4, and concentrated under vacuum to give N-(8-acetamido-4-oxo-tetrahydronaphthyl-5-yl)-2,2,2-trifluoro-acetamide I8 (655 mg, 2.084 mmol, >99% yield) as a yellow solid, which was used directly without further purification. LC / MS (Method A): Retention time 1.55 min (ES+) m / z 315 [M + H] + .

[0574] h)N-(4-amino-5-oxo-5,6,7,8-tetrahydronaphth-1-yl)acetamide (I9)

[0575] N-(8-acetamido-4-oxo-tetrahydronaphthyl-5-yl)-2,2,2-trifluoro-acetamide I8 (2.77 g, 8.81 mmol) was dissolved in methanol (240 mL) and water (17 mL). Potassium carbonate (4.88 g, 35.3 mmol) was added, and the mixture was stirred at 50 °C for 1.5 h, at which point LCMS indicated the reaction was complete. The reaction mixture was cooled, concentrated under vacuum, dissolved in 10% MeOH in CH2Cl2, and washed with H2O. The organic phase was dried over MgSO4 and purified by isolatora chromatography (2%–15% MeOH in CH2Cl2) to give N-(8-amino-1-oxo-tetrahydronaphthyl-5-yl)acetamide I9 (1.20 g, 5.50 mmol, 62.3% yield) as a yellow solid. LC / MS (Method A): Retention time 0.98 min (ES+) m / z 219 [M + H] + .

[0576]

[0577] i)(S)-N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo) [de]pyrano[3',4':6,7]inzazo[1,2-b]quinoline-4-yl)acetamide (I10)

[0578] N-(8-amino-1-oxo-tetrahydronaphthyl-5-yl)acetamide I9 (641 mg, 2.94 mmol, 1.0 equivalent), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indazine-3,6,10(4H)-trione A3 (840 mg, 3.19 mmol, 1.1 equivalent), and PPTS (740 mg, 2.95 mmol, 1.0 equivalent) were dissolved in toluene (60 mL) and stirred under reflux for 3 h, at which point LCMS indicated that I9 had been consumed. The reaction mixture was cooled and concentrated under vacuum. The resulting solid was ground with acetonitrile and then with acetone to give I10 (1.26 g, 96%) as a brown solid with a small amount of TsOH contamination. LC / MS (Method A): Retention time 1.32 min (ES+) m / z 447 [M+H] + .

[0579] j)(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano [3',4':6,7]Indazano[1,2-b]quinoline-10,13-dione (I11)

[0580] (S)-N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)acetamide (I10) (1.26 g, 2.83 mmol, 1.0 equivalent) was dissolved in hydrochloric acid (6 mol / L) in H2O (12 mL), and the mixture was stirred at 80 °C for 5 h, at which point LCMS indicated that I10 had been consumed. The reaction mixture was diluted with H₂O and concentrated under vacuum to provide (S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-10,13-dione I11 (1.51 g, 2.85 mmol, 90% by mass, 101% yield) as a red crystalline solid. LC / MS (Method A): Retention time 1.36 min (ES+) m / z 405 [M + H] + .

[0581]

[0582] a) Allyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((5-oxo-4-(2,2,2-trifluoroethyl) Amide group)-5,6,7,8-tetrahydronaphth-1-yl)amino)propyl-2-yl)amino)but-2-yl)carbamate (A1)

[0583] At 25 °C, DCC (6.54 g, 31.7 mmol) and HOPO (3.36 g, 30.2 mmol) were added to a solution of alloc-Val-Ala-OH (9.09 g, 31.7 mmol) and I7 (7.85 g, 28.8 mmol) in CH2Cl2 (300 mL). The resulting mixture was stirred overnight. The white solid formed during the reaction was filtered off and washed with cold CH2Cl2. The filtrate was washed with water (150 mL) and brine (150 mL). The organic layer was dried over MgSO4, filtered, and evaporated. The crude product was purified by silica gel chromatography (Hex / EtOAc, 60:40). The isolated product A1 was co-eluted with DCU contamination (21.1 g, 140% yield). LC / MS (Method B): ES + = 1.81 min, m / z 527.6 [M + H] .+ 。

[0584] b) Allyl((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthyl-1-yl)amino)-1-oxo (A2)propyl-2-yl)amino)-3-methyl-1-oxobut-2-yl)carbamate

[0585] Protected aniline A1 (18 g, 34.19 mmol) was solubilized in a 10:1 mixture of MeOH and H₂O (165 mL), and K₂CO₃ (10 g, 72.36 mmol) was added. The mixture was stirred at 50 °C until complete. The mixture was evacuated to near dryness, and the residue was absorbed with CH₂Cl₂, washed with H₂O and brine, dried over MgSO₄, filtered, and evaporated. The crude product was purified by silica gel chromatography (CHCl₃ / MeOH, 100% to 7:3). The isolated product A2 was contaminated with co-eluted impurities (10.71 g, 73% yield). LC / MS (Method B): ES + = 1.46 min, m / z 431.7 [M + H] .+ 。

[0586] c) Allyl((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13, 15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)amino)-1-oxo Propyl-2-yl)amino)-3-methylbut-2-yl)carbamate (A4)

[0587] Aniline A2 (450 mg, 1.045 mmol), lactone A3 (280 mg, 1.064 mmol), and pyridinium p-toluenesulfonate (273 mg, 1.086 mmol) were solubilized in toluene (20 mL), and the mixture was heated to 130 °C (high reflux). A few drops of MeOH were added from time to time to aid solubilization. After 7 hours, the crude reaction mixture was dried under vacuum. The crude product was purified by silica gel chromatography (CHCl3 / MeOH, 100% to 95:5) to give product A4 (360 mg, 52.3% yield). LC / MS (Method B): ES + = 1.51 min, m / z 658.8 [M + H] .+ 。

[0588] d) Allyl (S)-2-amino-N-((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9, 10,13,15-Hexahydro-1H,12H-Benzo[de]pyrano[3',4':6,7]inzizo[1,2-b]quinoline-4-yl)amino)-1- (Oxopropyl-2-yl)-3-methylbutyramide (A5)

[0589] Excess piperidine (642 μL) was added to a solution of A4 (543 mg, 0.82 mmol) and PdP(Ph3)4 (89 mg, 0.08 mmol) in CH2Cl2 (15 mL). The mixture was stirred at room temperature for 20 min, at which point the reaction was complete (as monitored by LC / MS). The reaction mixture was diluted with CH2Cl2 (25 mL), and the organic phase was washed with H2O (25 mL) and brine (25 mL). The organic phase was dried over MgSO4, filtered, and excess solvent was removed by rotary evaporation under reduced pressure to give crude product A5, which was used as is in the next step. LC / MS (Method B): ES + = 1.15min, m / z 574.6 [M + H] .+ 。

[0590] e)1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrolo-1-yl)propionamid)-N-((S)-1-(((S)-1- (((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano) [3',4':6,7]Indazin[1,2-b]quinolin-4-yl)amino)-1-oxopropyl-2-yl)amino)-3-methyl-1-oxobut- 2-yl)-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-amide (1)

[0591] Under an argon atmosphere, pyridine (83 μL, 1.03 mmol) and Mal-dPEG8-OTFP (767 mg, 1.03 mmol) were added to a solution of crude A5 (assumed 1.03 mmol) in dry CH2Cl2 (50 mL). The reaction was stirred overnight, and 0.5 equivalents of Mal-dPEG8-OTFP were added to attempt to advance the reaction due to incomplete reaction. The reaction was diluted with CH2Cl2 (25 mL), and the organic phase was washed with H2O (2 × 50 mL) and brine, dried over MgSO4, filtered, and excess solvent was removed by rotary evaporation under reduced pressure. The crude product was purified by reversed-phase HPLC (gradient of H2O / CH3CN + 0.05% FA) and lyophilized, giving 1 (1.189 g, 31% yield after 2 steps). LC / MS (Method B): ES + =1.43min, m / z 1149.3 [M + H] .+ LC / MS (Method C): ES + =5.37 min, m / z 1149.4 [M + H] .+ .

[0592] Example 18: Stability of HT12-GL-SG3932 in human serum

[0593] It is known that ADCs containing drugs linked to cysteine ​​via thiosuccinimide exhibit some drug loss in the physiological environment due to the reverse Michael reaction. This process utilizes the regeneration of the conjugated cysteine ​​and the maleimide-carrying drug, thereby reducing the ADC's DAR over time. This deconjugation process is a known property of ADCs containing drugs linked to antibodies via thiosuccinimide.

[0594] Serum stability was determined by incubating HT12-GL-SG3932 in human serum at 37°C for 15 days to approximate physiological conditions. Following serum incubation, all antibody species, including conjugated and unconjugated ADCs, were recovered by immunocapture using anti-human Fc agarose resin. The captured species were eluted from the resin and analyzed by rLC / MS to determine the relative amounts of conjugated and unconjugated species based on peak heights in the mass spectra, as previously described (Wiggens et al., 2015; Vallier-Douglas et al., 2012; Xu et al., 2011). The amount of conjugated drug at a given time point was converted to a total DAR value by combining the amounts of drug on the antibody light and heavy chain species.

[0595] HT12-GL-SG3932 (187 μL of 3.2 mg / mL stock solution) was added to 3 mL of serum and sterilized by passing the mixture through a 0.2 μm filter. An aliquot (600 μL) of this solution was removed and frozen as the time=0 sample. The remaining ADC-serum solution was sealed in vials and gently incubated at 37 °C. Subsequently, aliquots (600 μL) were removed on days 1, 4, 7, and 15 and frozen at -80 °C until MS analysis. The results were obtained using Dynabeads... ™ MyOne ™ Immunocapture of streptavidin T1 conjugated with 5F12G3 (anti-HT12 anti-complementary site antibody) recovers ADC from serum.

[0596] 5F12G3 anti-complementation site antibodies were generated by immunizing Balb C mice with the Fab region of the HT12 antibody and generating hybridoma clones from mouse B cells. These clones were screened for specific binding to HT12-GL and HT12-GL-SG3932, and the 5F12G3 clone was identified as specifically binding to HT12-GL and HT12-GL-SG3932 with high affinity. Following manufacturing recommendations, 5F12G3 was biotinylated (ThermoFisher, CA) and combined with Dynabeads. ™ MyOne ™ Streptavidin T1 beads (ThermoFisher, CA) were conjugated. Prior to use, 5F12G3 magnetic Dynabeads were washed three times with PBS pH 7.2, once with 0.1 M glycine pH 3.5, and then twice more with PBS pH 7.2. The ADC-serum sample was then combined with 5F12G3 magnetic Dynabeads (100 μL ADC-serum mixture, 50 μL slurry) and mixed for 60 min at room temperature. The 5F12G3 magnetic Dynabeads were settled using a magnetic rack and then washed three times with PBS pH 7.2. The washed Dynabeads were resuspended in 100 μL of 0.1 M glycine pH 3.5 and incubated for an additional 5 min at room temperature. The Dynabeads were settled using a magnetic rack, and the eluent was removed and added to 10 μL of 1 M Tris-HCl (ThermoFisher, Waltham, MA USA). The recovered human antibody solution (45 μL) was then reduced with DTT (Pierce, Rockford, IL USA) (5 μL) and analyzed by LC / MS.

[0597] rLC / MS analysis was performed on an Agilent 1290 series HPLC coupled to an Agilent 6520 Accurate-Mass TOF LC / MS (Santa Clara, CA USA) with an electrospray ionization source. Approximately 2 μg (45 μL volume) of serum-destabilizing eluent was loaded onto a Poroshell 300SB-C3 column (2.1 × 75 mm, 255) (Agilent, Santa Clara, CA USA) and eluted at a flow rate of 0.4 mL / min after 6 min using a stepwise gradient from solvent A to 60% solvent B (solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile) (JTBaker, Radnor, PA USA). Total mass spectra were obtained by integrating the entire peak from the chromatogram, which contained both light and heavy chain antibody species. The total mass spectra were then deconvolved using Agilent MassHunter data acquisition and chromatogram processing software (Agilent, Santa Clara, CA USA).

[0598] The DAR for each sample was calculated using the peak height intensities of conjugated and unconjugated species in the unconjugated mass spectra. Hydrolyzed thiosuccinimide bonds (+18 amu) were included in the analysis as “conjugated species”. The following equation was used:

[0599] Equation 1. Calculation of DAR in light chains

[0600] DAR LC = [(LC+1) + (LC+1 hydrolyzed)] / [LC+(LC+1) + (LC+1 hydrolyzed)]

[0601] LC = Peak height of the light chain

[0602] LC+1 = Light chain + Peak height of AZ14170133

[0603] LC+1 hydrolyzed = light chain + AZ14170133 + peak height of hydrolyzed thiosuccinimide species

[0604] Equation 2. Calculation of DAR for heavy chains

[0605] DAR HC= [(HC+1) + (HC+1 hydrolyzed) + 2×((HC+2) + 2×(HC+2 hydrolyzed) + 3×(HC+3) + 3×((HC+3 hydrolyzed)] / [HC+(HC+1) + (HC+1 hydrolyzed) + 2×(HC+2) + 2×(HC+2 hydrolyzed) + 3×(HC+3) + 3×(HC+3 hydrolyzed)]

[0606] HC = Peak height of heavy chain

[0607] HC+1 = Heavy chain + Peak height of AZ14170133

[0608] HC+1 hydrolysis = heavy chain + AZ14170133 + peak height of hydrolyzed thiosuccinimide species

[0609] HC+2 = Heavy chain + 2 Peak height of AZ14170133

[0610] HC+2 hydrolyzed = heavy chain + 2 AZ14170133 + peak height of hydrolyzed thiosuccinimide species

[0611] HC+3=heavy chain+3 Peak height of AZ14170133

[0612] HC+3 hydrolyzed = heavy chain + 3 AZ14170133 + peak height of hydrolyzed thiosuccinimide species

[0613] Equation 3. Calculation of Total DAR

[0614] DAR 总 = 2×(DAR LC + DAR HC )

[0615] Compared to the time-zero sample, HT12-GL-SG3932 showed drug loss over time after incubation in humans at 37°C, as evidenced by the appearance of unconjugated antibody light and heavy chain peaks in simplified MS analysis. No drug loss via linker cleavage was observed, which would be attributed to enzymatic activity. However, unmodified antibody peaks (i.e., regeneration of unmodified cysteine ​​residues via loss of the entire AZ14170133 drug-linker) increased over time. These two observations confirm that drug loss in human serum occurs via the reverse Michael reaction.

[0616] After 15 days of incubation in human serum, HT12-GL-SG3932 showed less than 18.5% drug loss. The drug release mechanism was deconjugation via the reverse Michael reaction rather than linker cleavage. This observation is consistent with other ADCs prepared by maleimide conjugation to cysteine ​​amino acids involving interchain disulfides.

[0617] Example 19 - HT12-GL-SG3932 with standard of care (HMA and Venetotok) or with cytarabine in AML cells Combination therapy in the system

[0618] In vitro studies in AML cell lines investigated the effects of combination therapy of HT12-GL-SG3932 ADC (AZD9829) with standard system therapy (SoC) of DNA hypomethylating agent (HMA) and BCL2 inhibitor venetoc (VEN) and combination therapy of HT12-GL-SG3932 ADC with cytarabine (cytosine arabinoside).

[0619] AML cell lines were cultured in vitro for 6 days with one of the following treatment groups: (1) HT12-GL-SG3932, (2) VEN, (3) HMA (decitabine), (4) cytarabine, (5) a combination of HT12-GL-SG3932 and VEN, (6) a combination of HT12-GL-SG3932 and HMA, (7) a combination of HT12-GL- and cytarabine, or (8) a triple combination of HT12-GL-SG3932 with VEN and HMA. Seven different CD123-expressing AML cell lines with various molecular alterations (i.e., MOLM-13, EOL-1, KASUMI-1, KG-1, THP-1, NOMO-1, OCI-AML3, and OCI-AML5 cells) were tested, and untreated cells from each cell line served as controls. Prefill each well of a 384-well plate with 20 μL of culture medium (RPMI + 10% FBS, IMDM + 10% FBS, or αMEM + 20% FBS) using a MultiDrop Combi dispenser. Then add the test reagents to each plate using the plate reformatting program on an ECHO655 or ECHO550 acoustic dispenser. (See section 12) A 6-dose matrix was prepared, with HT12-GL-SG3932 (40–250 nL / well in aqueous buffer), venetoclax (40 nL / well in DMSO), and / or decitabine (40 nL / well in DMSO) and cytarabine (40 nL / well in DMSO) added in triplicate. Drug concentrations were selected individually for each cell line to focus on doses exhibiting moderate potency, allowing for evaluation of the combined beneficial effects. Cell suspensions (20 μL / well) were then added to the treated plates using multichannel pipettes. The plates were then incubated for 6 days. At the end of the assay, CellTiter-Glo2.0 (Promega G9242, 40 μL / well) was added to each well of the plate using a multichannel pipette or a MultiDrop Combi dispenser. The plates were incubated in the dark at room temperature for 10–15 minutes, and then the luminescence was read using an Envision plate reader to assess cell viability. Data were analyzed in Rstudio, and the beneficial effects of the combination were evaluated using the Synergyfinder package. A Bliss score ≥5 and <50% cell viability were interpreted as indicating a meaningful synergistic effect.

[0620] After 6 days of culture, cell viability data showed a synergistic cytotoxic effect between HT12-GL-SG3932 and VEN (group 5), with Bliss scores >5 in 5 of the 7 cell lines tested at dose ranges of 10 nM to 1 µM VEN and 0.1 nM to 0.3 µM HT12-GL-SG3932 in THP-1 cells. Figure 15A and Figure 17 HT12-GL-SG3932 and HMA (Group 6) showed synergistic cytotoxicity, with Bliss scores >5 in one of the seven cell lines tested in THP-1 cells at dose ranges of 100 nM to 1 µM HMA and 0.1 nM to 0.3 µM HT12-GL-SG3932. Figure 15B and Figure 17 The triple combination of HT12-GL-SG3932 with SoC (VEN and HMA) (Group 8) showed synergistic effects, with Bliss scores >5 in 5 out of 7 cell lines in THP-1 cells at dose ranges of 10 nM to 1 µM VEN, 10 nM to 0.5 µM HMA, and 0.1 nM to 0.3 µM HT12-GL-SG3932. Figure 15C and Figure 17HT12-GL-SG3932 and cytarabine (Group 7) showed synergistic cytotoxicity, with Bliss scores >5 in 5 of the 7 cell lines tested in THP-1 cells at dose ranges of 50 nM to 0.5 µM cytarabine and 0.1 nM to 0.3 µM HT12-GL-SG3932. Figure 16 and Figure 17 Interestingly, all treatment groups showed synergistic effects in THP-1 cells, but none of them showed synergistic effects in KASUMI-1 cells. Figure 17 ).

[0621] These results demonstrate that treatment with HT12-GL-SG3932 is synergistic with treatment with VEN, HMA, cytarabine, or SoC (VEN and HMA) in AML cell lines with different molecular alterations. Therefore, these preclinical findings suggest the potential for improving long-term outcomes in AML patients through combination therapy with HT12-GL-SG3932 and SoC (VEN and AZA) or cytarabine in clinical settings.

[0622] Example 20 - Combination of HT12-GL-SG3932 with Standards of Care (HMA and Venetotok) in an AML patient sample therapy

[0623] In primary AML patient samples, the efficacy of combination therapy of HT12-GL-SG3932 ADC (AZD9829) with standard of care (SoC) of DNA hypomethylating agent (HMA) and BCL2 inhibitor venetoclax (VEN) was tested in vitro.

[0624] Primary AML patient sample cells from 11 patients were thawed and cultured at 0.5 × 10⁻⁶ cells / mL. 6Cells were seeded at 10% heat-inactivated FCS in RPMI 1640 medium with a mixture of cytokines consisting of FLT3-L (50 ng / ml), IL3 (10 ng / ml), IL6 (10 ng / ml), and SCF (50 ng / ml) to support cell viability. Cells were incubated at 37°C in a 5% CO2 atmosphere for 4 days (D4) and 7 days (D7). Twelve hours after thawing, “on day 0 (D0)”, cells were treated with HT12-GL-SG3932 (125–500 nM) as a single agent or in combination with VEN (0.1 μM) or SoC (VEN and HMA (azacitidine, 0.9 μM)). Cells were also treated with DMSO or NIP228-ADC (NIP228-SG3932) (125–500 nM) as controls. Treatment-induced cytotoxicity was assessed by flow cytometry on days 4 and 7 post-treatment. Cytotoxicity of all treatments was measured based on changes in absolute number of blasts reported as mean blast reduction (BR) %.

[0625] For flow cytometry, cells were washed and incubated with viability markers at 4°C for 15 minutes. Following the washing step, cells were incubated with Fc blocking agent (BD) for 10 minutes as a blocking step. Cell surface staining was then performed according to the supplier's instructions, and cells were incubated at 4°C for 20 minutes. Cells were then fixed and permeabilized using a fixation / permeabilization solution kit from BD. All washes were performed using Perm / Wash solution from this step onwards. The nuclear membrane of the cells was permeabilized using Permeabilization Buffer Plus from BD. To complete the process, intracellular staining was performed according to the supplier's instructions, and cells were incubated at 4°C for 20 minutes. Data were analyzed using a Cytoflex cytometer (Beckman Coulter, Brea, CA, USA). The gating strategy was as follows: First, contamination events and dead cells were removed from the histogram using live / dead viability markers. Single cells were plotted on FSC-A versus FSC-H to remove bimodal peaks. Identification of blasts and lymphocytes using CD45 / SSC gating: CD45 expression was moderate / dim for blasts and bright for lymphocytes. Blasts were characterized using cell surface markers CD33, CD34, CD38, CD117, and CD123.

[0626] To assess the cytotoxic effects of the molecules on the absolute number of blast cells after 4 and 7 days of treatment, the relative number and viability of cells were measured using a trypan blue exclusion assay for each condition. Flow cytometry was then used to analyze the percentage of viable blast cells for each condition. Based on these percentages and the trypan blue exclusion assay, the absolute cell number for each individual subpopulation was calculated using the following formula: Relative number of cells of interest = Total viable cell count % of cells of interest.

[0627] Compared to SoC (69% BR), eight out of eleven AML patient samples (i.e., Top1i sensitive samples) Figure 18A (4 days) and Figure 18C (7 days) demonstrated that adding 125 nM HT12-GL-SG3932 to the SoC improved cell killing relative to the SoC (91% vs. 69% BR, respectively) with significant blast reduction (BR) with 500 nM HT12-GL-SG3932 (89% BR) or with 1 nM Top1i payload alone (89% BR). Although not statistically significant, a larger percentage of blast reduction was observed in the triple combination therapy case compared to 125 nM HT12-GL-SG3932 as a single agent (91% vs. 80% BR, respectively). Figure 18A Three patient samples did not respond to either HT12-GL-SG3932 or the Top1i payload (i.e., Top1i insensitive samples). Figure 18B (4 days) and Figure 18D (7 days)

[0628] These in vitro results using primary AML patient samples demonstrate that, in AML patient samples with different molecular alterations, treatment with HT12-GL-SG3932 as a single agent in combination with SoC is more effective than SoC alone. Therefore, these preclinical findings support further clinical investigations of combination therapy with HT12-GL-SG3932 and SoC (VEN and AZA) to improve long-term AML patient outcomes.

[0629] Example 21 - Combination of HT12-GL-SG3932 with Standards of Care (HMA and Venetotok) in an AML PDX Model therapy

[0630] In a primary AML patient-derived xenograft model (AML PDX model), the efficacy of combination therapy of HT12-GL-SG3932 ADC (AZD9829) with standard of care (SoC) of DNA hypomethylating agent (HMA) and BCL2 inhibitor venetoclax (VEN) was tested in vivo.

[0631] Two AML PDX models (DFAM-68555 and DFAM-49600) were used to test the potential beneficial effects of the combination of HT12-GL-SG3932 and SoC (VEN+AZA). AML DFAM-68555 is characterized by the Flt3 mutation, and AML DFAM-49600 is characterized by the Tp53 mutation. AML PDX cells were thawed and counted, and one million (10^10) cells were counted. 6Peripheral blood graft implantation (huCD45%) was sporadically examined in the entire mouse cohort or subgroups by flow cytometry to determine the dosing phase. Mice were randomized by body weight using a matched distribution method built into the StudyLog software. Venetoclax was administered orally at 50 mg / kg or 100 mg / kg daily for 14 days. Azacitidine was administered intraperitoneally at 1.25 mg / kg or 2.5 mg / kg daily for 5 days. HT12-GL-SG3932 was administered intravenously at 2 mg / kg or 4 mg / kg weekly for 2 weeks. Human IgG was administered intraperitoneally at 100 mg / kg 24 hours prior to HT12-GL-SG3932 treatment.

[0632] Closely monitor mice for clinical signs of AML, weight loss, and hind limb paralysis. Promptly euthanize dying mice and record survival days.

[0633] In the SoC-insensitive AML PDX (DFAM-68555) model, HT12-GL-SG3932 as a single agent significantly prolonged median survival (48 days) compared to untreated mice (28 days) and SoC-treated mice (32.5 days). Figure 19 The triple combination of HT12-GL-SG3932 and SoCs (Ven and AZA) further extends median survival to 53 days. Figure 19 The lower-dose combination groups (groups 6-8) showed a worse or comparable survival benefit compared to HT12-GL-SG3932 monotherapy. Figure 19 ).

[0634] Peripheral blood was collected from all remaining AML DFAM-68555 PDX mice on day 36 post-implantation. Blood samples were processed and stained for flow cytometry analysis to measure the percentage of human CD45% / live-dead. Both the HT12-GL-SG3932 monotherapy group and all combination groups significantly reduced the AML disease burden (human CD45%) in the blood. Figure 20 ).

[0635] In the SoC-responsive AML PX (DFAL-49600) model, SoC prolonged median survival to 125 days (62 days) compared to untreated mice. Figure 21 HT12-GL-SG3932, as a single agent, also significantly prolonged median survival (83 days). Figure 21 The triple combination therapy was significantly superior (>200 days) to both HT12-GL-SG3932 monotherapy and SoC. Figure 21Compared with SoC or HT12-GL-SG3932 monotherapy, all lower-dose combination groups (groups 6-8) showed significantly prolonged median survival. Figure 21 ).

[0636] Peripheral blood was collected from all remaining AML DFAL-49600PDX mice at days 42, 57, 70, 85, 99, 113, 126, and 172 post-implantation. Blood samples were processed and stained for flow cytometry analysis to measure the percentage of human CD45% / live-dead. HT12-GL-SG3932 monotherapy, SoC, and all combination therapies significantly reduced the AML disease burden (human CD45%) in the blood at early time points. Figure 22 ).

[0637] These in vivo AML PDX model results demonstrate that treatment with HT12-GL-SG3932 as a single agent is effective in AML PDX models established using primary AML patient samples with multiple molecular alterations. However, combining HT12-GL-SG3932 with the standard of care (SoC) has a beneficial effect on prolonging survival. Therefore, these preclinical findings provide evidence for exploring combination therapy of HT12-GL-SG3932 and SoC (VEN and AZA) in clinical settings to improve long-term AML patient outcomes.

[0638] It should be understood that the detailed description section (rather than the summary and abstract section) is intended to interpret the claims. The summary and abstract section may set forth one or more, but not all, exemplary aspects of this disclosure as conceived by the inventors, and is therefore not intended to limit this disclosure and the appended claims in any way.

[0639] The present disclosure has been described above using functional building blocks that illustrate implementations of specified functions and their relationships. For ease of description, the boundaries of these functional building blocks are arbitrarily defined herein. Alternative boundaries can be defined as long as the specified functions and their relationships are appropriately implemented.

[0640] The foregoing description of specific aspects so fully reveals the general nature of this disclosure that others can readily modify and / or adapt such specific aspects for various applications by applying knowledge in the art, without excessive experimentation and without departing from the general conception of this disclosure. Therefore, based on the teachings and guidance presented herein, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed aspects. It should be understood that the wording or terminology used herein is for descriptive rather than limiting purposes, and that the terminology or terminology of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0641] The breadth and scope of this disclosure should not be limited by any of the foregoing exemplary aspects, but should be defined solely by the following claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof, comprising an antigen-binding domain, the antigen-binding domain comprising: (i) a variable heavy chain region (VH) comprising a VH-complementarity determining region (CDR)1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and 9; a VH-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and 10; and a VH-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 and 11; and (ii) a variable light chain region (VL) comprising a VL-CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and 12; a VL-CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and 13; and a VL-CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 6 and 14.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding domain specifically binds CD123.

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antigen-binding domain comprises a Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nanobody (single-domain antibody), or any combination thereof.

4. The antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the antigen-binding domain comprises a scFv.

5. The antibody or antigen-binding fragment thereof of any one of claims 1 to 4, wherein the antigen-binding domain comprises: (a) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; or (b) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 9, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 10, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 12, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 13, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

14.

6. The antibody or antigen-binding fragment thereof of any one of claims 1 to 5, wherein the antigen-binding domain comprises a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 15.

7. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the antigen-binding domain comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 7 and 15.

8. The antibody or antigen-binding fragment thereof of any one of claims 1 to 7, wherein the antigen-binding domain comprises a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 16.

9. The antibody or antigen-binding fragment thereof of any one of claims 1 to 8, wherein the antigen-binding domain comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and 16.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antigen-binding domain comprises: (a) a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8; or (b) a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:

16.

11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 10, wherein the antigen-binding domain comprises: (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 8; or (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

16.

12. An antibody or antigen-binding fragment thereof, comprising an antigen-binding domain, the antigen-binding domain comprising: (a) a VH comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 64, 68, 70, 72, 74, 76, 80, 82, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, and 116; and (b) a VL comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, and 117.

13. The antibody or antigen-binding fragment thereof of claim 12, wherein the antigen-binding domain comprises: (a) a VH comprising the amino acid sequence set forth in SEQ ID NO: 64 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 65; (b) a VH comprising the amino acid sequence set forth in SEQ ID NO: 68 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 69; (c) a VH comprising the amino acid sequence set forth in SEQ ID NO: 70 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 71; (d) a VH comprising the amino acid sequence set forth in SEQ ID NO: 72 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 73; (e) a VH comprising the amino acid sequence set forth in SEQ ID NO: 74 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 75; (f) a VH comprising an amino acid sequence set forth in SEQ ID NO: 76 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 77; (g) a VH comprising an amino acid sequence set forth in SEQ ID NO: 80 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 81 ; (h) a VH comprising an amino acid sequence set forth in SEQ ID NO: 82 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 83; (i) a VH comprising an amino acid sequence set forth in SEQ ID NO: 88 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 89; (j) a VH comprising an amino acid sequence set forth in SEQ ID NO: 90 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 91 ; (k) a VH comprising an amino acid sequence set forth in SEQ ID NO: 92 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 93; (l) a VH comprising an amino acid sequence set forth in SEQ ID NO: 94 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 95; (m) a VH comprising an amino acid sequence set forth in SEQ ID NO: 96 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 97; (n) a VH comprising an amino acid sequence set forth in SEQ ID NO: 98 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 99; (o) a VH comprising an amino acid sequence set forth in SEQ ID NO: 100 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 101 ; (p) a VH comprising an amino acid sequence set forth in SEQ ID NO: 102 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 103; (q) a VH comprising an amino acid sequence set forth in SEQ ID NO: 104 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 105; (r) a VH comprising an amino acid sequence set forth in SEQ ID NO: 106 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 107; (s) a VH comprising an amino acid sequence set forth in SEQ ID NO: 108 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 109; (t) a VH comprising an amino acid sequence set forth in SEQ ID NO: 110 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 111 ; (u) a VH comprising an amino acid sequence set forth in SEQ ID NO: 112 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 113; (v) a VH comprising an amino acid sequence set forth in SEQ ID NO: 114 and a VL comprising an amino acid sequence set forth in SEQ ID NO: 115; or (w) a VH comprising an amino acid sequence set forth in SEQ ID NO: 116 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

117.

14. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 64 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

65.

15. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 68 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

69.

16. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 70 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

71.

17. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 72 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

73.

18. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 74 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

75.

19. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising: a VH comprising an amino acid sequence set forth in SEQ ID NO: 76 and a VL comprising an amino acid sequence set forth in SEQ ID NO:

77.

20. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 80 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

81.

21. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 82 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

83.

22. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 88 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

89.

23. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 90 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

91.

24. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 92 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

93.

25. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 94 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

95.

26. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 96 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

97.

27. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:98 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

99.

28. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 100 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

101.

29. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 102 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

103.

30. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 104 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

105.

31. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 106 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

107.

32. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 108 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

109.

33. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 110 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

111.

34. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 112 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

113.

35. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 114 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

115.

36. An antibody or antigen-binding fragment thereof that specifically binds CD123, the antibody or antigen-binding fragment thereof comprising an antigen-binding domain comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 116 and a VL comprising the amino acid sequence set forth in SEQ ID NO:

117.

37. A polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 36.

38. A vector comprising the polynucleotide of claim 37 operably linked to a promoter.

39. A cell comprising the polynucleotide of claim 37.

40. A conjugate of Formula I: wherein Q is: X is: wherein a = 0 to 5, b1= 0 to 16, b2= 0 to 16, c1= 0 or 1, d = 0 to 5; and GL is a linker for attachment to the antibody or antigen-binding fragment thereof; and wherein p is an integer from 1 to 20. A - (D L ) p (I) or a pharmaceutically acceptable salt or solvate thereof, wherein A is an antibody or antigen-binding fragment thereof according to any one of claims 1 to 36, D L is a drug linker unit of Formula II: ， wherein R L of Formula IIa: ， 41. The conjugate of claim 42, wherein Q is: (a) an amino acid residue selected from the group consisting of Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or (b) a dipeptide residue selected from the group consisting of: NH-Phe-Lys-C=0, NH-Val-Ala-C=0, NH-Val-Lys-C=0, NH-Ala-Lys-C=0, NH-Val-Cit-C=0, NH-Phe-Cit-C=0, NH-Leu-Cit-C=0, NH-Ile-Cit-C=0, NH-Phe-Arg-C=0, NH-Trp-Cit-C=0, and NH-Gly-Val-C=0; or (c) a tripeptide residue selected from the group consisting of: NH-Glu-Val-Ala-C=0, NH-Glu-Val-Cit-C=0, NH-aGlu-Val-Ala-C=0, and NH-aGlu-Val-Cit-C=0; or (d) a tetrapeptide residue selected from the group consisting of: NH-Gly-Gly-Phe-Gly-C=0; and 42. The conjugate of claim 41, wherein Q is: (a) an amino acid residue selected from the group consisting of Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or (b) a dipeptide residue selected from the group consisting of: NH-Phe-Lys-C=0, NH-Val-Ala-C=0, NH-Val-Lys-C=0, NH-Ala-Lys-C=0, NH-Val-Cit-C=0, NH-Phe-Cit-C=0, NH-Leu-Cit-C=0, NH-Ile-Cit-C=0, NH-Phe-Arg-C=0, NH-Trp-Cit-C=0, and NH-Gly-Val-C=0; or (c) a tripeptide residue selected from the group consisting of: NH-Glu-Val-Ala-C=0, NH-Glu-Val-Cit-C=0, NH-aGlu-Val-Ala-C=0, and NH-aGlu-Val-Cit-C=0; or (d) a tetrapeptide residue selected from the group consisting of: NH-Gly-Gly-Phe-Gly-C=0; and wherein Q X is as follows: Q is an amino acid residue, a dipeptide residue, a tripeptide residue, or a tetrapeptide residue, wherein the superscript labels C(=O) and NH indicate the groups to which the atom is bonded; ​ ， ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ NH-Gly-Phe-Gly-Gly-C=0, and wherein NH represents the N-terminus and C=0 represents the C-terminus of the residue.

42. The conjugate of claim 40 or 41, wherein a is: (a) 0 to 3; or (b) 0 or 1; or (c)0。 43. The conjugate of any one of claims 40 to 42, wherein b1 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g)8。 44. The conjugate of any one of claims 40 to 43, wherein b2 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g)8。 45. The conjugate of any one of claims 40 to 44, wherein c1 is: (a) 0; or (b) 1; or (c)2。 46. The conjugate of any one of claims 40 to 45, wherein d is: (a) 0 to 3; or (b) 1 or 2; or (c) 2; or (d)0。 47. The conjugate of any one of claims 40 to 46, wherein: (a) a is 0, b1 is 0, c1 is 1, d is 2, and b2 is 0, 2, 3, 4, 5, or 8; or (b) a is 1, b2 is 0, c1 is 0, d is 0, and b1 is 0, 2, 3, 4, 5, or 8; or (c) a is 0, b1 is 0, c1 is 0, d is 1, and b2 is 0, 2, 3, 4, 5, or 8; or (d) b1 is 0, b2 is 0, c1 is 0, one of a and d is 0, and the other of a and d is 1 or 5; or (e) a is 1, b2 is 0, c1 is 0, d is 2, and b1 is 0, 2, 3, 4, 5, or 8; or (f) a is 0, b1 is 0, b2 is 8, c1 is 1, and d is 0.

48. The conjugate of any one of claims 40 to 47, wherein GL is 。 49. The conjugate of any one of claims 40 to 48, wherein Q is NH-Val-Ala-C=0.

50. The conjugate of any one of claims 40 to 49, wherein a is 1 or 0.

51. The conjugate of any one of claims 40 to 50, wherein b1 is 0.

52. The conjugate of any one of claims 40 to 51, wherein b2 is 8.

53. The conjugate of any one of claims 40 to 52, wherein c1 is 1.

54. The conjugate of any one of claims 40 to 53, wherein d is 1 or 0.

55. The conjugate of any one of claims 40 to 54, wherein R L is: 。 56. The conjugate of any one of claims 40 to 55, wherein the antibody is an IgG antibody.

57. The conjugate of claim 55, wherein at least one D L unit is conjugated to the hinge region of the IgG antibody.

58. The conjugate of claim 56 or 57, wherein 1 to 3 D L units are bound to the heavy chain of the IgG antibody.

59. The conjugate of any one of claims 56-68, wherein one D L the unit binds to the light chain of the IgG antibody.

60. The conjugate of any one of claims 56-59, wherein 1 to 3 D L units are bound to each heavy chain of the IgG antibody, and one D L unit is bound to each light chain of the IgG antibody.

61. The conjugate of any one of claims 56 to 60, wherein Formula II is a single enantiomer or is in enantiomerically enriched form.

62. The conjugate of any one of claims 56 to 61, wherein p is an integer from 1 to about 10.

63. The conjugate of any one of claims 56 to 62, wherein p is 8.

64. A conjugate comprising the antibody or antigen binding fragment thereof of any one of claims 1 to 37, wherein the antibody or antigen binding fragment thereof is conjugated to a drug linker unit of: (SG3932).

65. A mixture of conjugates comprising the conjugate of any one of claims 40 to 64, wherein the average p in the mixture is about 1 to about 10.

66. A pharmaceutical composition comprising the conjugate of any one of claims 40 to 64 or the mixture of conjugates of claim 65, and a pharmaceutically acceptable diluent, carrier or excipient.

67. A method of producing an antibody or antigen binding fragment thereof that specifically binds CD123, the method comprising culturing the cell of claim 39 under suitable conditions.

68. A method of producing the conjugate of any one of claims 40 to 64 or the mixture of conjugates of claim 65, the method comprising conjugating the antibody or antigen binding fragment thereof to the drug linker unit.

69. A method of treating a proliferative disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen binding fragment thereof of any one of claims 1 to 36.

70. A method of treating a proliferative disease, the method comprising administering to a subject in need thereof a therapeutically effective amount of the conjugate of any one of claims 40 to 64, the mixture of conjugates of claim 65 or the pharmaceutical composition of claim 66.

71. The method of claim 70, wherein the proliferative disease is a cancer.

72. The method of claim 71, wherein the cancer is a hematological cancer.

73. The method of claim 72, wherein the hematological cancer is a leukemia or lymphoma.

74. The method of any one of claims 70 to 73, wherein the cancer is acute myeloid leukemia, acute lymphoid leukemia, myelodysplastic syndrome, refractory anemia with excess blasts, DLBCL non-Hodgkin lymphoma, marginal zone non-Hodgkin lymphoma, mantle zone non-Hodgkin lymphoma, follicular non-Hodgkin lymphoma, Hodgkin lymphoma or minimal residual disease.

75. The conjugate of any one of claims 40 to 64, the mixture of conjugates of claim 65 or the pharmaceutical composition of claim 66 for use in medical therapy.

76. The conjugate of any one of claims 40 to 64, the mixture of conjugates of claim 65 or the pharmaceutical composition of claim 66 for use in the treatment of a proliferative disease.

77. The conjugate, mixture or pharmaceutical composition of claim 76, wherein the proliferative disease is a cancer.

78. Use of the conjugate of any one of claims 40-64, the mixture of conjugates of claim 65, or the pharmaceutical composition of claim 66 in the manufacture of a medicament for the treatment of a proliferative disease.

79. An antibody-drug conjugate (ADC) comprising: (i) an antibody or antigen-binding fragment thereof that binds CD123, the antibody or antigen-binding fragment thereof comprising: a HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; a HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; a HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; and a LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) wherein one or more cysteine residues of the antibody or antigen-binding fragment are conjugated to: (SG3932), and wherein the ADC has a drug-to-antibody ratio (DAR) of about 8.

80. The ADC of claim 79, wherein the antibody or antigen-binding fragment thereof comprises a variable heavy (VH) chain comprising the amino acid sequence set forth in SEQ ID NO: 7 and a variable light (VL) chain comprising the amino acid sequence set forth in SEQ ID NO:

8.

81. The ADC of claim 79 or 80, comprising a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 116 and a light chain (LC) comprising the amino acid sequence of SEQ ID NO:

117.

82. A pharmaceutical composition comprising the ADC of any one of claims 79-81.

83. A method of treating cancer, the method comprising administering to a patient the ADC of any one of claims 79-81 or the pharmaceutical composition of claim 82.

84. The method of claim 83, wherein the cancer is acute myeloid leukemia or myelodysplastic syndrome.

85. The ADC of any one of claims 79-81 or the pharmaceutical composition of claim 84 for use in treating cancer.

86. The ADC for use of claim 85, wherein the cancer is acute myeloid leukemia or myelodysplastic syndrome.

87. The method of claims 70-74 or 83, further comprising administering venetoclax and / or a hypomethylating agent, optionally wherein the hypomethylating agent is 5-azacitidine or decitabine.

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