Anti-CD24 antibody drug conjugate and its uses

Abstract

In this specification, an antibody-drug conjugate containing an anti-CD24 antibody that selectively binds to a human CD24 protein expressed in cancer cells but not to human CD24 expressed in non-cancer cells, conjugated to a cytotoxic agent via a linker, and the use thereof for cancer treatment are provided.

Description

Technology Field

[0001] Field of invention

[0002] The present invention relates to an antibody-drug conjugate containing an anti-CD24 antibody that selectively binds to the human CD24 protein expressed in cancer cells but not to the human CD24 expressed in non-cancer cells, which is conjugated to a cytotoxic agent via a linker, and to the use thereof for cancer treatment. Background Technology

[0003] Background of the Invention

[0004] CD24 is a small, highly glycosylated mucin-like glycosylphosphatidyl-inositol (GPI)-binding cell surface protein. CD24 is expressed at high levels in hematopoietic cells, including B cells, T cells, neutrophils, eosinophils, dendritic cells, and macrophages, as well as in non-hematopoietic cells, including neurons, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells, and epithelial stem cells. Generally, CD24 tends to be expressed at high levels in progenitor cells and cells with high metabolic activity, and at relatively low levels in terminally differentiated cells. Although the function of CD24 in most cell types has not yet been clearly elucidated, various immunological functions of CD24 have been reported.

[0005] CD24 is found in many normal tissues and cell types, but it is overexpressed in approximately 70% of human cancers. High levels of CD24 expression detected by immunohistochemistry were observed in epithelial ovarian cancer (83%), breast cancer (85%), non-small cell lung cancer (45%), prostate cancer (48%), and pancreatic cancer (72%). CD24 is one of the most overexpressed proteins in cancer cells. CD24 expression is upregulated during tumorigenesis, suggesting its involvement in tumor progression and metastasis. In cancer, CD24 overexpression has also been identified as a marker indicating a poor prognosis and a more aggressive disease course in cancer patients. In breast cancer, CD24 expression is significantly higher in invasive carcinomas than in benign or precancerous lesions. In non-small cell lung cancer, CD24 expression has been identified as an independent marker for overall patient survival. Furthermore, CD24 overexpression in esophageal squamous cell carcinoma is strongly associated with tumor lymph node metastasis, low tumor grade, and survival. Similar observations have been confirmed in various other cancers, including colon cancer, hepatocellular carcinoma, glioma, ovarian cancer, and prostate cancer. Although CD24 has been widely used as a prognostic marker for cancer, it has not yet been sufficiently utilized as a neoantigen that could serve as a potential target for cancer treatment.

[0006] Mature CD24 is a small, highly glycosylated sialoglycoprotein composed of 31 amino acids, possessing 16 potential O-glycosylation sites and 2 predicted N-glycosylation sites. Glycosylation is one of the most complex post-translational modifications of proteins. It is known that many cancer cells deviate from normal glycosylation pathways, leading to altered glycan expression and consequently hyper-glycosylation or hypo-glycosylation of many cellular proteins. These altered glycosylation patterns found in cancer cells are the result of several causative factors, including dysregulation at the transcriptional level, dysregulation of chaperone proteins during the glycosylation process, and changes in glycosidase and glycotransferase activity. Tumor-related glycan changes include changes in the branching length of N-glycans (becoming longer or shorter), changes in O-glycan density (becoming higher or lower), the production of shortened forms of normal glycans (Tn, sTn, and T antigens), and the production of abnormal terminal structures including sialic acid and fucose (sLea and sLex epitopes).

[0007] Antibody-drug conjugates (ADCs) combine the cell binding specificity resulting from the targeting of monoclonal antibodies with the high-efficiency cytotoxicity of cytotoxic drugs, and have now become a key strategy for targeted cancer therapy. Each monoclonal antibody must be individually characterized, an appropriate linker must be designed, and a suitable cytotoxic drug must be selected to maintain its efficacy even after delivery to tumor cells. Other considerations include whether the entire ADC enters the cell upon binding; whether a proliferation inhibitor or a cytotoxic drug is suitable given the potential exposure to normal tissues; the type and / or stage of the cancer being treated; and whether the linker connecting the antibody and the drug payload is a cleavable or non-cleavable link. Additionally, the antibody-to-drug moiety conjugation ratio (DAR) must be at a level that does not affect the binding activity of the antibody and / or the efficacy of the drug.

[0008] Therefore, there is a need in the technical field for improved methods for identifying and treating cancer, particularly methods and compositions capable of distinguishing between cancer cells and non-cancer cells. means of solving the problem

[0009] Summary of the Invention

[0010] In this specification, an antibody-drug conjugate (ADC) comprising an anti-CD24 antibody, a therapeutic agent (which may be a cytotoxic agent), and a linker is provided. The anti-CD24 antibody may be conjugated to the therapeutic agent via the linker. The anti-CD24 antibody may bind to an epitope shielded by a glycan that is exposed to cancer cells but not to non-cancer cells. Additionally, a pharmaceutical composition comprising the ADC and a pharmaceutically acceptable excipient is provided.

[0011] The anti-CD24 antibody may bind to a peptide containing sequence identification number 26. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in sequence identification number 1 and a light chain variable region containing the sequence described in sequence identification number 2. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in one of sequence identification numbers 3-10 and a light chain variable region containing the sequence described in one of sequence identification numbers 11-16. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in sequence identification number 6 and a light chain variable region containing the sequence described in sequence identification number 16. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in one of sequence identification numbers 17-20 and a light chain variable region containing the sequence described in one of sequence identification numbers 21-24. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in sequence identification number 17 and a light chain variable region containing the sequence described in sequence identification number 21. The anti-CD24 antibody may include a heavy chain variable region containing the sequence described in sequence identification number 19 and a light chain variable region containing the sequence described in sequence identification number 23.

[0012] Cytotoxic agents may include substances that inhibit or prevent the expression activity or function of cells and / or cause cell destruction. Cytotoxic agents may include radioisotopes, chemotherapy agents, or toxins. Toxins may be small molecule toxins or protein toxins derived from bacteria, fungi, plants, or animals. Cytotoxic agents may also include biologically active fragments or variants of protein toxins.Cytotoxic agents include auristatin, auromycin, maytansinoid, topoisomerase I or II inhibitor, ricin, ricin A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, CCL065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxyanthracindione dione), actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin,. Sapaonaria Officinalis inhibitor ( Sapaonaria officinalisIt may include an inhibitor or a glucocorticoid. The cytotoxin may include a radioisotope, which may be At211, Ac225, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, Ra223, Pb212, Tb149, P32, or a radioisotope of Lu. A radioisotope of Lu may be Lu177.

[0013] The cytotoxic agent may include synthetic analogs of auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), or dolastatin. In one embodiment, the cytotoxic agent includes monomethylauristatin E (MMAE). The cytotoxic agent may include a mytancinoid containing DM1 or DM4. The cytotoxic agent may include a topoisomerase I inhibitor. The topoisomerase I inhibitor may include SN-38 or Dxd.

[0014] The linker may be cleavable or non-cleavable. The cleavable linker may include a hydrazone, disulfide, or peptide linker. The disulfide linker may further include one or more disulfide groups. The peptide linker may include a dipeptide linker. The dipeptide linker may include mc-Val-Cit-PAB(N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-N-[4-(hydroxymethyl)phenyl]-). The disulfide linker may include a reducible or glutathione-sensitive disulfide linker. A reducible or glutathione-sensitive disulfide linker may include SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-1-pyrrolidinyl ester). A peptide linker may include CL2A ((6,12,15,18,21,24,27,30,33-Nonaoxa-3,9-diazapentatriacontanamide, 2-(4-aminobutyl)-35-[4-[[[[4 -[(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]cyclohexyl]carbonyl]amino]methyl]-1H-1,2,3-triazol-1-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-).The cleavable linker may include mc-GGFG((S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide).

[0015] The cytotoxic agent and the linker together may comprise a structure selected from the group consisting of the following components:

[0016] ; ;

[0017] ;

[0018] ;

[0019] ; and

[0020] .

[0021] The linker can form a covalent bond with a cytotoxic agent at the first position and a covalent bond with an anti-CD24 antibody at the second position, and the first and second positions may be different from each other.

[0022] The present specification provides a method for treating cancer in a subject requiring cancer treatment. The method may include the step of administering an ADC or a pharmaceutical composition to the subject. Additionally, the use of an ADC or pharmaceutical composition in the manufacture of a medicament for cancer treatment and an ADC or pharmaceutical composition used for cancer treatment is provided. The cancer may be lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, testicular cancer, prostate cancer, neuroblastoma, or cancer that binds to an anti-CD24 antibody. Brief explanation of the drawing

[0023] Drawing description The patent or patent application contains at least one color drawing. A copy of the publication of this patent or patent application containing the color drawing(s) is provided by the Patent Office upon request and payment of a prescribed fee. Fig. 1 is This shows the results of comparing the binding affinities of IgG, anti-CD24 (H3L3) alone, and various anti-CD24 ADCs (ONC-784 ADC) to HT29Luc cells. The binding affinities of the selected variants are similar to those of the parent antibody, H3L3. Kd values ​​are indicated in nM units in the table below the graph. The bound antibodies were detected using a fluorescently labeled secondary antibody against human IgG. Figure 2 is This shows the results comparing the binding affinities of IgG, anti-CD24 antibody alone (H3L3), and various anti-CD24 ADCs (ONC-784 ADC) on HT29Luc CD24KO cells. H3L3 and ONC-784 ADC variants did not bind to HT-29 cells that do not express CD24. Binding experiments with H3L3 and ADC variants were performed using HT-29 cells that had undergone CRISPR genetic engineering to remove CD24. The bound antibodies were detected using a fluorescently labeled secondary antibody against human IgG. Fig. 3 The anti-CD24 antibody alone (H3L3) and the anti-CD24 ADC variant (ONC-784 ADC) in Jeko-1 cells, an MCL cell line expressing luciferase in vitroThis shows the results of a cytotoxicity study comparing the results after 24 and 48 hours. Luciferase-expressing Jeko-1 cells were cultured with various concentrations of the ONC-784 variant or a commercially available irrelevant IgG1 antibody as a control. Cell viability was evaluated by measuring luciferase activity in live cells and comparing it to the luciferase activity of cells cultured with the control antibody. The left side shows the results after 24 hours of ADC addition, with the Y-axis on a linear scale; the right side shows the results after 48 hours of ADC addition, with the Y-axis on a logarithmic scale. Fig. 4 is This shows a summary comparing the therapeutic efficacy of IgG, anti-CD24 antibody alone (H3L3), and anti-CD24 ADC variant (ONC-784 ADC) in a blood cancer model (JeKo-1 cells). 10 6 Mice injected with Jeko-1 cells were treated with various ADC variants at the doses and frequencies indicated in the legend one week after tumor cell injection. Fig. 5 is This shows a summary comparing the therapeutic efficacy of IgG, anti-CD24 antibody alone (H3L3), and anti-CD24 ADC variant (ONC-784 ADC) in a solid colon cancer tumor model (HT-29 cells). 10 6 Mice injected with HT-29 cells were treated with various ONC-784 ADC variants at a dose of 5 mg / kg at twice-weekly intervals when the tumor volume reached approximately 100 mm³. Fig. 6 is This shows a summary comparing the therapeutic efficacy of IgG, anti-CD24 antibody alone (H3L3), and anti-CD24 ADC variant (ONC-784 ADC) in a breast cancer solid tumor model (MDA-mb-468 cells). Mice with subcutaneous tumors (approx. 100 mm³) were treated with ADCs at twice-weekly intervals as indicated by the yellow arrows under the X-axis. ADC dosages are indicated in the legend. Fig. 7 isThis shows a summary comparing the therapeutic efficacy of IgG, anti-CD24 antibody alone (H3L3), and an anti-CD24 ADC variant (ONC-784 ADC) at a dose of 5 mg / kg in an ovarian cancer solid tumor model (SKOV-3 cells). Mice with subcutaneous tumors (approx. 100 mm³) were treated with the ADC at twice-weekly intervals as indicated by the yellow arrows below the X-axis. Fig. 8 is This shows the administration plan used to compare the effects of vehicle, H3L3, and ONC-784 ADC in the Nude-MDA-MB-468 mouse model. Fig. 9 is This shows a summary comparing the therapeutic efficacy of PBS vehicle (negative control), anti-CD24 antibody alone (H3L3), and various concentrations of ONC-784 ADC in a breast cancer model (MDA-MB-468 cells). Figures 10A-C are This shows the results of testing ONC-784-B7 in an MDA-MB-468 breast cancer model. Figure 10A shows the administration plan. Figure 10B shows that ONC-784-B7 specifically kills MDA-MB-468 WT cells expressing CD24. Figure 10C shows that ONC-784-B7 has no effect on MDA-MB-468 CD24 knockout cells lacking CD24 expression. Specific details for implementing the invention

[0024] Detailed description

[0025] Targeting epitopes expressed in cancer is a widely adopted approach for cancer treatment. However, since many of these epitopes are also expressed in normal tissues, they can cause toxicity issues and are therefore not suitable as effective drug targets. An ideal tumor-specific antigen ("TSA") is one that is widely expressed in cancer but minimally or not at all in the host's essential organs. Less ideal but still useful TSAs are tumor-associated antigens ("TAAs"), which are expressed in both normal and cancer tissues but are modified differently. Examples of well-characterized tumor antigens include MAGE-A3, MUC-1, and NY-ESO-1.

[0026] The discovery of novel tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) is a limiting factor in the development of new or more effective cancer therapies, particularly in the case of cancers where tumor antigens are currently unknown. CD24 is a good cancer target for the following reasons: i) it is broadly overexpressed in over 70% of all human cancers and differentially glycosylated in cancer; ii) CD24 appears to be oncogenic and is associated with poor prognosis and significantly shortened patient survival in various cancers; and iii) CD24 is a marker for cancer stem cells that can generate new tumors and cause recurrence and metastasis. The inventors have discovered an anti-CD24 antibody in which CD24 binding is blocked by glycosylation that occurs in normal cells but not in cancer cells. Consequently, this antibody binds to cancer cell lines and cancer tissues but exhibits minimal reactivity to various normal tissues and hematopoietic cells.

[0027] Specifically, the present invention provides a molecule comprising an antigen-binding fragment of an antibody that is immune-specifically bound to CD24, particularly human CD24, wherein the antibody is expressed on the surface of living cells at an endogenous or transformed concentration, and a cytotoxic agent is conjugated to the antibody via a linker.

[0028] 1. Definition

[0029] The terms used herein are for the purpose of describing specific embodiments only and are not intended to be restrictive. As used herein and in the appended claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise indicated in the context. The word "about" used with numeric values ​​indicates a reasonable approximation of the value. In some cases, "about" may be interpreted as being within 10% of the specific value associated. For example, the expression "about 100" includes all values ​​between 90 and 110.

[0030] When referring to a numerical range in this specification, each number in between is explicitly considered with the same precision. For example, in the case of the 6-9 range, 7 and 8 are included in addition to 6 and 9, and in the case of the 6.0-7.0 range, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered. When describing a list of numbers, the range of the corresponding numbers is also considered (e.g., 1, 2, 3, 4, or 5 includes the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, etc.).

[0031] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule having an antigen recognition site called a "variable region." The term "variable region" is intended to be distinguished from a domain widely shared by antibodies (e.g., the antibody Fc domain). The variable region includes a "hypervariable region" having residues responsible for antigen binding. The hypervariable region comprises amino acid residues of the "Complementarity Determining Region" or "CDR" (i.e., generally about 24-34 (L1), 50-56 (L2), and 89-97 (L3) residues of the light chain variable domain and about 27-35 (H1), 50-65 (H2), and 95-102 (H3) residues of the heavy chain variable domain) and / or amino acid residues of the "hypervariable loop" (i.e., about 26-32 (L1), 50-52 (L2), and 91-96 (L3) residues of the light chain variable domain and about 26-32 (H1), 53-55 (H2), and 96-101 (H3) residues of the heavy chain variable domain). "Framework Region" or "FR" residue is a variable domain residue other than the supervariable region residue defined in this specification.Antibodies include monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single-chain antibodies, disulfide-linked Fv (sdFv), intrabody or anti-idiotypic (anti-Id) antibodies (e.g., including anti-Id and anti-anti-Id antibodies against the antibodies of the present invention). In particular, these antibodies include all types (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) and classes (e.g., IgG1, IgG2). IgG3, It includes immunoglobulin molecules of IgG4, IgA1, and IgA2) or subclasses.

[0032] As used herein, the term “antigen binding fragment” of an antibody refers to one or more parts of an antibody that contain framework residues comprising the antibody’s CDR and optionally the antibody’s “variable domain” antigen recognition site, and exhibit the ability to bind immunospecifically to an antigen. Such fragments include Fab’, F(ab’)2, Fv, single-strand (ScFv) and their mutants, naturally occurring variants, and fusion proteins comprising the antibody’s “variable domain” antigen recognition site and a heterogeneous protein (e.g., toxins, antigen recognition sites for other antigens, enzymes, receptors, or receptor ligands, etc.). The term “fragment” as used in this specification refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0033] Human, chimeric, or humanized antibodies are in humans In vivo (in vivo ) Although particularly desirable for use, murine antibodies or antibodies of other species are also used for various purposes (e.g., in vitro or in situ Detection test, acute In vivo It can be used advantageously for (use, etc.).

[0034] A "chimeric antibody" is a molecule in which different parts of the antibody are derived from different immunoglobulin molecules; for example, it is an antibody having a variable region derived from a non-human antibody and a constant region from a human immunoglobulin. Chimeric antibodies comprising one or more CDRs of non-human species and a framework region of a human immunoglobulin molecule may be produced using various techniques known in the art, including, for example, CDR grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Patent Nos. 5,225,539, 5,530,101 and 5,585,089, the contents of which are incorporated herein in their entirety), veneering or resurfacing (EP 592,106; EP 519,596, the contents of which are incorporated herein by reference), and chain shuffling (U.S. Patent No. 5,565,332, the contents of which are incorporated herein by reference).

[0035] As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human framework region and one or more CDRs of a non-human (typically mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “acceptor.” The constant region is not required to be present, but if present, it must be substantially identical to the constant region of the human immunoglobulin. That is, it must be at least about 85-90% identical, preferably about 95% or more identical. Thus, all parts of the humanized immunoglobulin, possibly excluding the CDR, are substantially identical to the corresponding parts of the natural human immunoglobulin sequence. A humanized antibody is an antibody comprising humanized light chain and humanized heavy chain immunoglobulin. For example, a humanized antibody will not contain a typical chimeric antibody. This is because, for example, the entire variable region of the chimeric antibody is a non-human antibody. Donor antibodies are said to be "humanized" through a "humanization" process because the resulting humanized antibodies are expected to bind to the same antigen as the donor antibody providing the CDR. In most cases, the humanized antibody may be a human immunoglobulin (recipient antibody), and the hypervariable region residues of the recipient antibody are replaced with hypervariable region residues of a non-human species (donor antibody), such as mice, rats, rabbits, or non-human primates, that possess the desired specificity, affinity, and ability. In some cases, the Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in the recipient or donor antibody. These modifications can further enhance antibody performance.Typically, the humanized antibody may include at least one, generally two, variable domains substantially all, wherein all or substantially all hypervariable regions correspond to the hypervariable regions of non-human immunoglobulins, and all or substantially all FRs correspond to the hypervariable regions of human immunoglobulin sequences. Additionally, the humanized antibody may optionally include at least a portion of an immunoglobulin constant region (Fc), which is typically the constant region of human immunoglobulin that binds immunospecifically to the FcγRIIB polypeptide and is modified by the introduction of one or more amino acid residue substitutions, deletions, or additions (i.e., mutations).

[0036] The term "drug-to-antibody ratio" or "DAR" refers to the number of drug molecules (e.g., auristatin) bound to the antibody of an ADC. The DAR of an ADC can range from 1 to 8, but higher drug loadings, such as 10, are also possible depending on the number of binding sites on the antibody. The term DAR may be used to indicate the number of drugs bound to individual antibodies, or to indicate the average or arithmetic mean DAR of multiple ADCs. ADCs generally have 1 to 8 drug moieties conjugated to the antibody, and this includes species with drug loadings of 2, 4, 6, or 8.

[0037] When referring to protecting animals from disease, “treatment” or “treating” means preventing, inhibiting, suppressing, or completely eliminating disease. Preventing disease includes administering a composition of the present disclosure to an animal before the onset of disease. Inhibiting disease includes administering a composition of the present disclosure to an animal after the induction of disease but before its clinical manifestation. Suppressing disease includes administering a composition of the present disclosure to an animal after the clinical manifestation of disease. In one embodiment, “treatment” or “treating” means alleviating one or more symptoms of disease.

[0038] 2. Antibody-drug conjugate

[0039] Antibody-drug conjugates are provided herein. Antibody-drug conjugates (ADCs) may comprise a binding protein, which may comprise an antibody or an antigen-binding fragment thereof. The binding protein may be chemically linked to one or more therapeutic agents via a linker, which may comprise one or more cytotoxic agents (e.g., cytotoxic chemical drugs) and cell proliferation inhibitors as a payload. In one embodiment, the ADC comprises a monoclonal antibody, a therapeutic agent, and a linker that enables the therapeutic agent to be attached or conjugated to the antibody. The therapeutic agent must have one or more of the following: i) sufficiently high cytotoxicity; ii) sufficiently low immunogenicity; iii) high stability; iv) a functional group that can be modified without significantly affecting efficacy; v) bystander killing effect; vi) adequate solubility; and vii) an intracellular target.

[0040] The antibody may include the anti-CD24 antibody described herein. The anti-CD24 antibody or its binding fragment described herein may be linked to one or more therapeutic agents through one or more linkers. The ADC may include the following structural formula (I) or a salt thereof:

[0041] [n(DL-XY)]-Ab

[0042] (I)

[0043] Here, each "D" represents a therapeutic agent ("drug") independently of the others; each "L" represents a linker independently of the others; "Ab" represents an anti-CD24 antigen-binding portion, such as an anti-CD24 antibody or binding fragment described herein; each "XY" represents a connection formed between a functional group of the linker and a "complementary" functional group of the antibody, and n represents the number of therapeutic agents or the drug-to-antibody ratio (DAR) linked to the ADC.

[0044] a. Treatment

[0045] Cytotoxic agents may include substances that inhibit or prevent the expression activity or function of cells and / or cause cell destruction. Cytotoxic agents may include one or more radioisotopes, chemotherapy agents, immunomodulators, and toxins (including fragments and / or variants), such as small molecule toxins or protein-based toxins derived from bacteria, fungi, plants, or animals. Examples of cytotoxic agents include auristatin, auromycin, mytancinoids, topoisomerase I or II inhibitors, lysine, lysine A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, CCL065, ethidium bromide, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, dihydroxyanthracindione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrine, abrine A-chain, modesin A-chain, alpha-sarcine, gelonin, mitozelin, retrictosine, phenomycin, enomycin, curicin, crotin, and caliceamycin. Sapaonaria Officinalis Inhibitors, and glucocorticoids and other chemotherapeutic agents, immunomodulators such as cytokines and Toll-like receptor agonists, radioisotopes such as At211, Ac225, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, Ra223, Pb212, Tb149, P32, and radioisotopes of Lu including Lu177 are included but not limited thereto. Additionally, antibodies may be conjugated to anticancer prodrug activating enzymes capable of converting anticancer prodrugs into an active form.

[0046] As used herein, the term "auristatin" refers to a class of antimitotic agents. Auristatin derivatives are also included in the definition of the term "auristatin." Examples of auristatins include, but are not limited to, auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), and synthetic analogs of dolastatin.

[0047] The term “mytansinoid” as used in this specification refers to a class of antimitotic agents, and examples of mytansinoids include, but are not limited to, maytansine and maytansine thiomethyl analogs S-methyl DM1 (DM1) and S-methyl DM4 (DM4).

[0048] As used herein, the term “topoisomerase I inhibitor” refers to a class of antimitotic agents including camptothecin derivatives and exatecan derivatives such as SN-38 and Dxd.

[0049] b. Linker

[0050] The linker connecting the antibody and the therapeutic agent of the ADC may be short or long, may be hydrophobic, hydrophilic, flexible, or rigid, or may be composed of segments having one or more of the aforementioned characteristics independently, so that the linker may include segments with different characteristics. The linker may be polyvalent and may covalently connect two or more therapeutic agents to a single site of the antibody. The linker may be monovalent and may connect a single therapeutic agent to a single site of the antibody.

[0051] One or more therapeutic agents may be linked to an antibody by forming a covalent bond with a therapeutic agent at one location and a covalent bond with an antibody at another location. Covalent bonds may be formed by a reaction between the functional group of the linker and the functional groups of one or more cytotoxic agents and antibodies. The linker may not be conjugated and may include a functional group capable of forming a covalent bond with one or more therapeutic agents and a functional group capable of forming a covalent bond with an antibody. The linker may be partially conjugated and may include a functional group that forms a covalent bond with an antibody and one or more therapeutic agents, or vice versa. The linker may be covalently bonded to one or more therapeutic agents and antibodies. The linker may consist of one or more parts including the functional group of the linker and the covalent bond formed between the linker and the antibody. The linker may be chemically stable under extracellular conditions and may undergo one or more of cleavage, loss, or other specific degradation within the cell.

[0052] Linkers may not be specifically cleaved, lost, or degraded within the cell. The choice between stable and unstable linkers may depend on the toxicity of the therapeutic agent. In the case of cytotoxic agents that are toxic to normal cells, the ADC may include a stable linker. In the case of selective or targeted cytotoxic agents that are less toxic to normal cells, the chemical stability of the linker against the extracellular environment may be less important. Various linkers useful for attaching therapeutic agents to antibodies in the context of ADCs are known in the art. These linkers, as well as other linkers, may be used to attach therapeutic agents to the antibodies of the ADCs described herein.

[0053] The linker may be multivalent. Exemplary multivalent linkers that can be used to link multiple therapeutic agents to a single antibody molecule are described, for example, in WO 2009 / 07345; WO 2010 / 068795; WO 2010 / 138719; WO 2011 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379; WO 2014 / 093394; WO 2014 / 093640, the contents of which are incorporated herein by reference in their entirety. The linker may be a dendritic-type linker. Additional examples of dendritic linkers are in US 2006 / 116422; US 2005 / 271615; It can be found in de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494; Amir et al (2003) Angew. Chera. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chem. Soc, 126:1726-1731; Sun et al (2002) Bioorganic & Medicinal Chemistry Letters 12:2233-2215; Sun et al (2003) Bioorganic & Medicinal Chemistry 1:1761-1768; King et al (2002) Tetrahedron Letters 43:1987-1990, each of which is incorporated herein by reference.

[0054] The linker may be monovalent. Exemplary available monovalent linkers are described, for example, in Molting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013.

[0055] The linker is In vivo (in vivo )It may be cleavable. The cleavable linker may include chemically or enzymatically unstable or degradable bonds. The cleavable linker may release a therapeutic agent depending on intracellular processes such as reduction within the cytoplasm, exposure to acidic conditions within the lysosome, or cleavage by specific intracellular proteases or other enzymes. The cleavable linker may include one or more chemical bonds that are chemically or enzymatically cleavable, while the remainder of the linker is non-cleavable. The cleavable linker may be a hydrazone, disulfide, or peptide linker. In one embodiment, the peptide linker is a dipeptide linker. The dipeptide linker may include Valine-Cit(VC) and may include mc-Val-Cit-PAB(N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-N-[4-(hydroxymethyl)phenyl]-). The dipeptide linker may include Val-Ala.

[0056] The linker may contain a chemically unstable functional group (labile group), such as a hydrazone and / or a disulfide group. Linkers containing a chemically unstable functional group utilize the differential properties between plasma and some cytoplasmic compartments. In the case of a linker containing a hydrazone, the intracellular conditions that promote therapeutic release are the acidic environments of endosomes and lysosomes, whereas a linker containing a disulfide is reduced in the cytoplasm containing high thiol concentrations (e.g., glutathione), and in certain embodiments, the plasma stability of a linker containing a chemically unstable functional group may be enhanced by introducing a substituent near the corresponding functional group to induce steric hindrance. In one embodiment, the cleavable linker comprises CL2A((6,12,15,18,21,24,27,30,33-Nonaoxa-3,9-diazapentatriacontanamide, 2-(4-aminobutyl)-35-[4-[[[[4 -[(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]cyclohexyl]carbonyl]amino]methyl]-1H-1,2,3-triazol-1-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-). In another embodiment, the cleavable linker comprises mc-GGFG ((S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino )-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide) Includes.

[0057] Acid-unstable functional groups, such as hydrazone, remain intact during systemic circulation in the neutral pH environment of blood (pH 7.3–7.5); however, when the ADC enters the cell's weakly acidic endosome (pH 5.0–6.5) and lysosome (pH 4.5–5.0) compartments, they undergo hydrolysis to release cytotoxic agents. This pH-dependent release mechanism is associated with the non-specific release of therapeutic agents. To enhance the stability of the hydrazone group in the linker, the linker can be chemically modified (e.g., substitution) to induce more efficient release of cytotoxic agents from lysosomes and minimize circulatory losses. Linkers containing hydrazone may include additional cleavage sites, such as additional acid-unstable cleavage sites and / or enzymatically unstable cleavage sites.

[0058] Cleavable linkers may contain disulfide groups. Disulfides are thermodynamically stable at physiological pH and are designed to release drugs upon entering the cell, where the cytoplasm provides a much more reducing environment compared to the extracellular environment. Since the cleavage of disulfide bonds generally requires the presence of cytoplasmic thiols (e.g., (reduced) glutathione (GSH)), disulfide-containing linkers are relatively stable in the circulatory system and selectively release therapeutic agents in the cytoplasm. Intracellular enzymes such as protein disulfide isomerases or similar enzymes capable of cleaving disulfide bonds can also contribute to the preferential cleavage of disulfide bonds within the cell. GSH is reported to be present intracellularly at concentrations of 0.5–10 mM, whereas in the circulatory system, the concentrations of GSH or cysteine, the most abundant low-molecular-weight thiol, are much lower at approximately 5 μM. In tumor cells where hypoxic conditions occur due to irregular blood flow, the activity of the reductase increases, leading to a higher glutathione concentration, and in certain embodiments, of a linker containing disulfide In vivoStability can be improved by chemically modifying the linker, for example, by utilizing steric hindrance adjacent to the disulfide bond. In one embodiment, the glutathione-sensitive disulfide linker comprises SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-1-pyrrolidinyl ester). The disulfide linker may comprise SPP (N-succinimidyl-4-(2-pyridyldithio)pentanoate).

[0059] The non-cleavable linker may include succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC).

[0060] c. cytotoxic agents - Linker combination

[0061] The cytotoxic agent may include tesirine, and the linker may include Val-Ala. The cytotoxic agent may include Dxd, and the linker may include GGFG.

[0062] The cytotoxic agent and linker may include GGFG-Dxd. The cytotoxic agent and linker may include the following structure.

[0063]

[0064] The cytotoxic agent and linker may include VC-MMAE. The cytotoxic agent and linker may include the following structure.

[0065]

[0066] The cytotoxic agent and linker may include CL2A-SN38. The cytotoxic agent and linker may include the following structure.

[0067]

[0068] The cytotoxic agent and linker may include SPDB-DM4. The cytotoxic agent and linker may include the following structure.

[0069]

[0070] The cytotoxic agent and the linker may include SPP-DM1. The cytotoxic agent and the linker may include the following structures.

[0071]

[0072] The cytotoxic agent and linker may include SMCC-DM1. The cytotoxic agent and linker may include the following structures.

[0073]

[0074] d. Anti-CD24 antibody

[0075] Anti-CD24 antibodies can specifically target cancer-specific glycoforms of CD24. Specifically, anti-CD24 antibodies or their antigen-binding fragments can bind to glycan-shielded epitopes that are exposed to cancer cells but not to non-cancer cells. Anti-CD24 antibodies or their antigen-binding fragments can bind to a CD24 peptide containing the amino acid sequence SNSGLAPN (sequence identification number 27). Anti-CD24 antibodies may be as described in WO2019222082, the contents of which are incorporated herein by reference.

[0076] The anti-CD24 antibody may include a heavy chain variable region and a light chain variable region of 3B6. The heavy chain variable region may include the following sequence.

[0077] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDKTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 1)

[0078] The light chain variable region may include the following sequence.

[0079] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCLQGTSYPWTFGGGTKLEIKR (SEQ ID NO: 2)

[0080] An anti-CD24 antibody or its antigen-binding fragment may include a heavy chain variable region and a light chain variable region of an affinity-matured version of 3B6. The anti-CD24 antibody may include a heavy chain variable region comprising one of the following sequences.

[0081] EVKFEESGGGLVQPGGSIKLSCAASGVAFSGAWMDWVRQSPEKGLEWVAEIRDKTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGVYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 3)

[0082] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDKSTNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 4)

[0083] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDNTTNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 5)

[0084] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDKPNSYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 6)

[0085] EVKFEESGGGLVQPGGSIKLSCAASGVPFSGAWMDWVRQSPEKGLEWVAEIRDKTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 7)

[0086] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDKTKNYVTYYAESVKGRFTISRDDSKGRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 8)

[0087] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQTPEKGLEWVAEIRDRETKYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 9)

[0088] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRDKQNEYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVTVSS (SEQ ID NO: 10)

[0089] The anti-CD24 antibody may include a light chain variable region containing one of the following sequences.

[0090] DDIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGTPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTSTPWTFGGGTKLEIKR (SEQ ID NO: 11)

[0091] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGASLPWTFGGGTKLEIKR (SEQ ID NO: 12)

[0092] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGTPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGASVPWTFGGGTKLEIKR (SEQ ID NO: 13)

[0093] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTYLPWTFGGGTKLEIKR (SEQ ID NO: 14)

[0094] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTSLPWTFGGGTKLEIKR (SEQ ID NO: 15)

[0095] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGSSLPWTFGGGTKLEIKR (SEQ ID NO: 16)

[0096] In one embodiment, the anti-CD24 antibody or its antigen-binding fragment comprises a heavy chain and a light chain variable region of PP6373, which may include a heavy chain variable region comprising the sequence described in sequence identification number 6 and a light chain variable region comprising the sequence described in sequence identification number 16.

[0097] The anti-CD24 antibody or its antigen-binding fragment may be a humanized version of PP6373 and may include a heavy chain variable region containing one of the following sequences.

[0098] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVGEIRDKPNSYVTYYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTGAMDYWGQGTLVTVSS (SEQ ID NO. 17) (H1)

[0099] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRDKPNSYVTYYAESVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCTGAMDYWGQGTLVTVSS (SEQ ID NO. 18) (H2)

[0100] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRDKPNSYVTYYAESVKGRFTISRDDSKSTAYLQMNSLKTEDTAIYYCTGAMDYWGQGTLVTVSS(Sequence Identification Number: 19)(H3)

[0101] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRDKPNSYVTYYAESVKGRFTISRDDSKSTVYLQMNSLKTEDTAIYYCTGAMDYWGQGTLVTVSS(Sequence Identification Number 20)(H4)

[0102] The humanized anti-CD24 antibody may include a light chain variable region containing one of the following sequences.

[0103] DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPQRLIYQVSKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO: 21) (L1)

[0104] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPQRLIYQVSKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR(Sequence identification number 22)(L2)

[0105] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPKRLIYQVSKLDPGIPDRFSGSGSETDFTLKISRVEAEDVGIYYCMQGSSLPWTFGGGTKVEIKR(Sequence identification number 23)(L3)

[0106] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPKRLIYQVSKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO. 24) (L4)

[0107] In one embodiment, the humanized anti-CD24 antibody or its antigen-binding fragment is associated with H1L1 and comprises a heavy chain variable region comprising the sequence described in sequence identification number 17 and a light chain variable region comprising the sequence described in sequence identification number 21.

[0108] In another embodiment, the humanized anti-CD24 antibody is associated with H3L3 and comprises a heavy chain variable region comprising the sequence described in sequence identification number 19 and a light chain variable region comprising the sequence described in sequence identification number 23. An ADC comprising this anti-CD24 antibody or its antigen-binding fragment may be referred to as ONC-784.

[0109] In another embodiment, the humanized anti-CD24 antibody is associated with H2L3 and includes a heavy chain variable region comprising the sequence described in sequence identification number 18 and a light chain variable region comprising the sequence described in sequence identification number 23.

[0110] The heavy chain of the anti-CD24 antibody may include a heavy chain constant region. The heavy chain constant region may include a constant region of immunoglobulin (Ig), and this Ig may be one of IgG, IgM, or IgA. The IgG may be one of IgG1, IgG2, IgG3, or IgG4. In one embodiment, the constant region includes an Fc region. The Fc region may be of IgG1. The Ig may be human. In one embodiment, the heavy chain constant region includes the following sequence.

[0111] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG identification number 25)

[0112] The light chain of the anti-CD24 antibody may include a light chain constant region. The light chain constant region may include the following sequence.

[0113] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26)

[0114] e. Antibody-drug conjugate composition

[0115] Some tumor-targeted antibodies can be used to directly prevent or limit tumor growth by affecting the biological activities (biology) of the tumor. For example, humanized anti-VEGF monoclonal antibodies (bevacizumab; Avastin) block tumor growth by preventing tumor angiogenesis induced by VEGF. Other tumor-targeted antibodies are used to inhibit tumor cell growth or kill cancer cells through modifications of the antibody itself. For example, tumor-targeted immunoconjugates bind together through covalent cross-linking or gene fusion It consists of an antibody and an effector portion. The effector portion may be a cytotoxic drug (antibody-drug conjugate), a protein toxin (immunotoxin), or a radionuclide (radioimmunoconjugate). An example of an antibody-drug conjugate is brentuximab vedotin (ADCETRIS®, Seattle Genetics), which consists of a chimeric monoclonal antibody brentuximab (cAC10, targeting the cell membrane protein CD30) to which 3-5 monomethyl auristatin E (MMAE, reflected in the 'vedotin' in the drug name), an anti-mitotic agent, are bound.

[0116] The anti-CD24 antibody or its antigen-binding fragment may comprise one or more of an ADC, an immunotoxin, and a radioimmunoconjugate. The anti-CD24 antibody component can enable the ADC to be selectively delivered to cancer cells and cancer tissues, while simultaneously limiting exposure to normal cells and tissues, thereby consequently preventing off-target toxicity.

[0117] f. Antibody-radiolabeled composition

[0118] The present specification also provides an anti-CD24 antibody or an antigen-binding fragment thereof conjugated to a detectable label. The detectable label may be a dye, a fluorescent label, or a radiolabel. In one embodiment, the anti-CD24 antibody conjugate may be an antibody-radiolabel conjugate. The radiolabel may be attached to the antibody or its antigen-binding fragment directly or via a chelator, a difunctional coupling agent, or an N-hydroxysuccinimide (NHS) ester. The chelator may be a difunctional chelator. Chelating agents include desferrioxamine (DFO), its squaramide derivative (DFO-Sq), DFO*, DFO-cyclo*, 3,4,3-(LI-1,2-HOPO), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), gallium(III) complex of (1,4,7-triazanonane-1,4,7-triyl)triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), 1,1,4,7,7-diethylene triaminepentaacetic acid (DTPA), and 2-hydrazinonicotinic acid (HYNIC). It may be one or more of 3,6,9,15-tetraazabicyclo)[9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA), N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED), tris(hydroxypyridinone) (THP) or other chelating agents known in the art.The chelating agent may be attached to the antibody or its antigen-binding fragment via -NCS, -NHS, maleimide, tetrafluorophenyl (TFP) ester, -N-suc-TFP ester, or other linkers known in the art. The radiolabel. 18 F, 76 Br, 11 C, 131 I, 211 At, 225 Ac, 123 I, 89 Zr, 64 Cu, 68 Ga, 52 Mn, 86 Y, 66 Ga, 44 Sc, 90 Y, 111 In, 99m Tc, 177 Lu, 67 Cu, [ 18 It may be [F]AlF or other radioactive markers known in the art. In one embodiment, the chelating agent is DFO and the radioactive marker is 89 It is Zr.

[0119] The present specification provides a method for detecting cancer cells expressing CD24 by contacting cancer cells expressing CD24 with an anti-CD24 antibody conjugated with a radiolabel or an antibody-binding fragment thereof. The antibody-radiolabel conjugate can be detected via positron emission tomography.

[0120] 3. Production

[0121] The anti-CD24 antibody described herein may be produced using a eukaryotic cell expression system. The expression system may involve expression via a vector in mammalian cells such as Chinese Hamster Ovary (CHO) cells. Additionally, it may be a viral vector, such as a replication-defective retroviral vector, that can be used to infect eukaryotic cells. Furthermore, the antibody may be produced from a stable cell line that expresses the antibody from a vector or a portion of a vector integrated into the cell genome. This stable cell line may express the antibody from an integrated replication-defective retroviral vector. The expression system may be GPExTM.

[0122] The anti-CD24 antibody or its antigen-binding fragment described herein may be purified using chromatographic methods such as, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, the fusion protein may be engineered to contain an additional domain containing an amino acid sequence that allows the polypeptide to be captured in an affinity matrix. For example, the antibody described herein containing the Fc region of an immunoglobulin domain may be isolated from cell culture supernatant or cytoplasmic extract using a protein A column. Additionally, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid in polypeptide purification. These tags can be inserted anywhere within the polypeptide, including at carboxyl or amino terminuses. Other useful fusion agents include enzymes that aid in polypeptide detection, such as alkaline phosphatases. Immunoassay chromatography can also be used to purify polypeptides.

[0123] 4. Pharmaceutical composition

[0124] The present specification provides a pharmaceutical composition comprising an ADC. This pharmaceutical composition may comprise a prophylactic or therapeutically effective amount of the ADC and a pharmaceutically acceptable carrier excipient. The term “pharmaceutically acceptable” means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeias for use in animals, particularly humans. The term “carrier” means a diluent, adjuvant (e.g., Freund’s adjuvant (full and incomplete forms)), excipient, or vehicle used when administering the therapeutic agent. Such pharmaceutical carriers may be sterile liquids such as water and oil (oils of petroleum, animal, vegetable, or synthetic origin (e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.)). Water is preferred as the carrier when the pharmaceutical composition is administered intravenously. Physiological saline, aqueous glucose solution (aqueous dextrose), and aqueous glycerol solution can also be used as liquid carriers and are particularly suitable for injection solutions.

[0125] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. Additionally, if necessary, the pharmaceutical composition may contain a small amount of a wetting agent or emulsifier, or a pH buffer. Such compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc.

[0126] Generally, the components of a pharmaceutical composition may be supplied individually or mixed in the form of a unit dosage regimen, for example, in the form of a dry lyophilized powder or a water-free concentrate, contained in a sealed container such as an ampoule or sachet marked with the amount of active agent. When the pharmaceutical composition is administered by infusion, it may be provided with an infusion bottle containing sterile pharmaceutical-grade water or physiological saline. When the pharmaceutical composition is administered by injection, an ampoule containing sterile water for injection or physiological saline may be provided so that the components can be mixed before administration.

[0127] Pharmaceutical compositions may be formulated in a neutral form or in a salt form. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations derived from sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0128] 5. Treatment methods

[0129] An ADC or pharmaceutical composition may be used for cancer treatment. Additionally, the present specification provides a method for cancer treatment, which may include administering an ADC or pharmaceutical composition to a patient requiring cancer treatment. Furthermore, the present specification provides an ADC or pharmaceutical composition for cancer treatment and a use of the ADC or pharmaceutical composition in the manufacture of a medicament for cancer treatment.

[0130] As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal and uncontrolled growth of cells. As used herein, "cancer" explicitly includes leukemia and lymphoma. The term refers to a disease involving cells that have the potential to metastasize to distant sites.

[0131] Cancer may be one or more of the following: carcinomas including bladder cancer, breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, stomach cancer, cervical cancer, thyroid cancer, and skin cancer; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, and Burkitt's lymphoma; hematopoietic tumors of myeloid lineage including acute and chronic myelogenous leukemia and promyelocytic leukemia; Tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and Schwannoma; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; and other tumors, including melanoma, xenoderma pigmentosum, keratoactanthoma, seminoma, thyroid follicular cancer and tetratocarcinoma.

[0132] Cancer can arise from aberrations in apoptosis. These cancers may include, but are not limited to, follicular lymphomas, p53 mutation carcinomas, hormone-dependent tumors of the breast, prostate and ovarian cancers, and precancerous lesions such as familial adenomatous polyposis and myelodysplastic syndrome. Cancer may include malignant tumors or dysproliferative changes (e.g., metaplasias and dysplasias), or hyperproliferative disorders, which may occur in one or more of the ovaries, bladder, breasts, colon, lungs, skin, pancreas, or uterus. Cancer may include sarcoma, melanoma, or leukemia.

[0133] The ADC or pharmaceutical composition may be administered in combination with a second anti-tumor therapy, and the combination may be administered simultaneously or sequentially. The combination may be administered alone or as a single composition. The second anti-tumor therapy may include, but is not limited to, current standard and experimental chemotherapy, hormone therapy, biological therapy, immunotherapy, radiation therapy, or surgery. The second anti-tumor therapy may comprise a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies, or other agents known to those skilled in the art for the treatment and / or prevention of cancer, autoimmune diseases, infectious diseases, or poisoning. Such agents include, for example, the biological response modifiers, cytotoxins, antimetabolites, alkylating agents, antibiotics, or antimitotic agents discussed above, as well as immunotherapies.

[0134] In one embodiment, the second anti-tumor therapy is an anti-tumor immunotherapy. The anti-tumor immunotherapy may include a molecule that inhibits or enhances an alternative immunomodulatory pathway (e.g., CTLA-4, TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, PD-1, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27, or LAG3), or a molecule that enhances the immunomodulatory effect by regulating the activity of effector molecules such as cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-beta, IFNγ, Flt3, BLys) and chemokines (e.g., CCL21). Specific embodiments of the present invention include the ADCs described herein and bispecific antibodies including anti-PD-1 (pembrolizumab (Keytruda®) or nivolumab (Opdivo®)), anti-B7-H1 (atezolizumab (Tecentriq®) or durvalumab), anti-B7-H3, anti-B7-H4, anti-LIGHT, anti-LAG3, anti-TIM3, anti-TIM4, anti-CD40, anti-OX40, anti-GITR, anti-BTLA, anti-CD27, anti-ICOS, or anti-4-1BB. Anti-tumor immunotherapy may include one or more molecules that activate various stages or aspects of the immune response to achieve a broader immune response. In one embodiment, anti-tumor immunotherapy includes one or more anti-PD-1 or anti-4-1BB antibodies It includes, and can be administered without exacerbating autoimmune side effects.

[0135] 6. Method of Administration

[0136] The composition disclosed herein may be administered by methods including, but not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural administration, and mucosal administration (e.g., intranasal and oral routes). In one embodiment, the composition disclosed herein is administered intramuscularly, intravenously, or subcutaneously. The composition may be administered by a convenient route, such as infusion or bolus injection, or absorption through the epithelial or mucosal walls (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biological active agents. Administration may be systemic or local.

[0137] Examples

[0138] The present disclosure includes various aspects described through the following non-limiting examples. The inventors have produced various variants of an anti-CD24 antibody conjugated to a cytotoxic agent via a linker, as shown in Table 1. These variants vary depending on the chemical properties of the linker, including both cleavable and non-cleavable linkers, and the various cytotoxic agents.

[0139] Examples 1

[0140] ADC production

[0141] This example demonstrates the production of a specific ADC disclosed and verified herein. Various H3L3 ADC variants In a test tube and In vivo For testing, different cytotoxic agents and linkers were used (Table 1), and H3L3 antibody alone and IgG1 were compared.

[0142] Table 1

[0143]

[0144] H3L3 Conjugation of antibody binding linker and cytotoxic agent

[0145] For bulk conjugation of VC-MMAE (ONC-784-B-7) and GGFG-Dxd (ONC-784-B-3), 10 mg of H3L3 antibody contained in the original buffer (PBS, pH 7.0) was pipetted into a 50 mL tube and reduced to the identified optimal TCEP / mAb ratio. Reaction buffer (50 mM PBS, pH 7.0) was added to the tube to adjust the mAb concentration to 5 mg / mL. The reaction vessel was placed in a shaking incubator at 37°C and a rotation speed of 60 rpm. After reduction for 2 hours, 10 mM linker payload dissolved in DMA was added to the sample to adjust the drug-to-mAb ratio to 7.0. DMA solvent was added to the sample to adjust the organic solvent concentration to 10%. The reaction mixture was incubated for another 1 hour at 4°C. After 1 hour, the sample was purified using a spin desalination column (40K, 10mL).

[0146] For bulk conjugation of CL2A-SN38 (ONC-784-B-4), 10 mg of H3L3 antibody immersed in the original buffer (PBS, pH 7.0) was pipetted into a 50 mL tube. Then, 10 eq. of TCEP was added to reduce it. Subsequently, reaction buffer (50 mM PBS, pH 7.0) was added to the tube to adjust the mAb concentration to 8 mg / mL. The reaction vessel was placed in a shaking incubator at 37°C and a rotation speed of 60 rpm. After reduction for 2 hours, the reduced solution was purified using a spin desalting column (40K, 10 mL). After purification, 10 mM linker-payload dissolved in DMA was added to the purified sample to adjust the drug-to-mAb ratio to 12.0. DMA solvent was added to the sample to adjust the organic solvent concentration to 10%. Finally, conjugation buffer (50 mM PB, pH 7.0) was added to the tube to adjust the mAb concentration in the reaction mixture to 5 mg / mL. The reaction mixture was incubated at 22°C for another hour. After 1 hour, the sample was purified again using a spin desalting column (40K, 10 mL).

[0147] For bulk conjugation of SPBD-DM4 (ONC-784-B-5), 10 mg of H3L3 antibody contained in the original buffer (PBS, pH 7.0) was pipetted into a 50 mL tube. Then, 10 mM linker-payload dissolved in DMA was added to the sample to adjust the drug-to-mAb ratio to the identified optimal ratio. DMA solvent was added to the sample to adjust the organic solvent concentration to 15%. Subsequently, conjugation buffer (50 mM PBS, pH 7.0) was added to the tube to adjust the mAb concentration to 5 mg / mL. The reaction vessel was placed in a shaking incubator at 22°C and a rotation speed of 60 rpm. After 2.5 hours of reaction, the reaction was stopped with 200 mM succinic acid (pH 2.38) and purified using a spin desalting column (40K, 10 mL).

[0148] Examples 2

[0149] ADC's CD24 binding activity

[0150] This example demonstrates the cell binding activity of various ADCs disclosed herein. The affinity of ONC-784 ADC for low-glycosylated CD24 was evaluated using the luciferase-expressing colon cancer cell line HT29 (HT29Luc) through binding experiments with selected ONC-784 ADC variants. The results of the saturation analysis for the selected variants are shown in Figure 1, and the Kd values ​​(nM) of the tested ADC and parent antibody are presented in the table below the graph in nM units. The binding affinity of the ADC variants was similar to that of the parent antibody, indicating that the addition of a linker and a cytotoxic agent did not affect binding to low-glycosylated CD24 expressed in cancer cells.

[0151] Examples 3

[0152] to the ADC Specificity of CD24 binding activity for

[0153] This example demonstrates that the ADC disclosed herein binds to hypoglycosylated CD24. To verify whether the binding observed in H3L3 or ONC-784 ADCs is specific to hypoglycosylated CD24, a negative subclone of HT-29 with CD24 removed was produced using CRISPR technology. A comparison of the binding of H3L3 or ONC-784 ADC variants to parent cell lines (Fig. 1) and CD24KO cells (Fig. 2) confirmed that they bind specifically only to parent cell lines, suggesting that both the H3L3 antibody and the ONC-784 ADC specifically bind to cells expressing hypoglycosylated CD24 (Fig. 1).

[0154] Examples 4

[0155] ADC's Anticancer activity

[0156] This example demonstrates that the ADC disclosed herein exhibits anticancer activity. The ONC-784 ADC variant of Table 1 In a test tube Efficacy was evaluated using Jeko-1, a mantle cell lymphoma (MCL) cell line verified to express low-glycosylated CD24, HT-29, a colon cancer cell line, and HT29 CD24KO cells produced by CRISPR technology.

[0157] In a test tube Efficacy was evaluated using the mantle cell lymphoma (MCL) cell line Jeko-1. Cells were cultured with various ONC-784 ADC variants from Table 1 at gradually increasing concentrations, and cell viability was evaluated 24 and 48 hours after the addition of each ONC-784 ADC. An example of these results can be seen in Figure 3, where various ONC-784 ADCs were compared with cells cultured with commercially available IgG1 antibodies.

[0158] ONC-784 ADC variant In a test tubeEfficacy varied among the variants. For example, in the presented experiment, the ONC-784-B-10 and ONC-784-B-4 variants showed the strongest efficacy, followed by ONC-784-B-8 and ONC-784-B-5 (Fig. 3). These In a test tube Experiments were performed at least twice for all ONC-784 ADCs using different cells (HT-29luc, MCF-7luc, and mouse cells MC38 transfected with human CD24) and mouse cells MC38 that do not express CD24. A summary of the experiments is presented in Table 2.

[0159] Table 2

[0160]

[0161] The ONC-784 variants in Table 1 were used in four human tumor xenograft models using NSG mice or nude mice In vivo It was also tested in [another region]. Three solid tumor models were used: HT-29 colon cancer cells, the ovarian cancer cell line SKOV3, and the breast cancer cell line MDA-MB-468. Jeko-1 mantle cell lymphoma cells were used as a model of blood cancer that migrates to hematopoietic organs such as the bone marrow and spleen; since the HT-29 and Jeko-1 cancer cells used here express luciferase, allowing for non-invasive monitoring of tumor progression, most ONC-784 ADC variants were tested using these cell lines.

[0162] To directly compare the activities of various ONC-784ADC variants, immune-compromised NSG mice were intravenously injected with the MCL cell line Jeko-1 and treated with the doses specified for each ONC-784 ADC; most ONC-784 ADC variants were administered at a dose of 5 mg / kg at twice-weekly intervals for a total of four times, on days 7, 10, 14, and 17 following tumor cell injection. Mice were tracked by bioluminescence for 3 weeks, and data were presented on a logarithmic scale compared to pre-administration bioluminescence values ​​(Fig. 4). At a dose of 15 mg / kg, ONC-784-B-4 showed superior efficacy compared to ONC-784-B-10. At a dose of 5 mg / kg, ONC-784-B-7 and ONC-784-B-5 showed superior efficacy compared to other ONC-784 ADCs.

[0163] Examples 5

[0164] Anticancer activity of ADCs in solid tumor models

[0165] To validate the results in a solid tumor model, an HT-29 tumor model was established by subcutaneously injecting HT-29 tumor cells, and ONC-784 ADC variants were administered at twice-weekly intervals on days 7, 10, 14, and 17 following the tumor cell injection (Fig. 5). Similar to the results observed in the hematological cancer model, ONC-784-B-5 and ONC-784-B-7 showed high efficacy, and tumors were eliminated in most mice. Surprisingly, ONC-784-B-10 also demonstrated excellent efficacy in this model (Fig. 5).

[0166] Examples 6

[0167] Anticancer activity of ADCs in breast cancer and ovarian cancer models

[0168] Further studies were conducted using two additional mouse models of breast and ovarian cancer, utilizing MDA-MB-468 and SKOV-3 cells, respectively. ONC-784 ADC variants B4 and B10 significantly reduced tumor size in both models even at a low dose of 3 mg / kg (Figs. 6 and 7), while variants B5 and B7 significantly reduced tumor size in the breast cancer model even at a low dose of 3 mg / kg (Fig. 9). For the experiments on B5 and B7 in the breast cancer model, the tested groups are as shown in the following table, and the administration schedule shown in Fig. 8 was used.

[0169] Table 3

[0170]

[0171] Examples 7

[0172] In a breast cancer model ONC Anticancer activity of -784-B7

[0173] This example demonstrates that ONC-784-B7 exhibits anticancer activity in an MDA-MB-468 breast cancer model. To evaluate the specificity of ONC-784-B7 for CD24-expressing cells, breast cancer models using MDA-MB-468 WT and MDA-MB-468 CD24 knockout cells were further studied. Mice were injected with MDA-MB-468 WT (CD24-expressing) or MDA-MB-468 CD24KO (CD24-deficient) cells. When the tumor size reached approximately 100 mm³, mice were treated by intravenous injection of ONC784-B7 1 mg / kg or isotype control IgG1 (Ig) at twice-weekly intervals (days 21, 25, 28, 31, 34, 38, and 41) following the injection of tumor cells; the timing of administration is indicated by the black arrow below the x-axis (Fig. 10A).

[0174] Figures 10B and 10C show the tumor kinetics of MDA-MB-468 WT tumors (Figure 10B) and MDA-MB-468 CD24 knockout tumors (Figure 10C). The results showed that ONC-784-B7 specifically killed MDA-MB-468 WT cells, while it did not affect the tumor growth of MDA-MB-468 CD24 knockout cells.

Claims

Claim 1 An antibody-drug conjugate (ADC) comprising an anti-CD24 antibody, a cytotoxic agent, and a linker, wherein the anti-CD24 antibody is conjugated to the cytotoxic agent through the linker; and the anti-CD24 antibody binds to a glycan-shielded epitope that is exposed to cancer cells but not to non-cancer cells. Claim 2 An antibody-drug conjugate (ADC) according to claim 1, wherein the anti-CD24 antibody binds to a peptide comprising the sequence described in sequence identification number 26. Claim 3 An antibody-drug conjugate (ADC) according to claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence described in sequence identification number 1 and a light chain variable region comprising the sequence described in sequence identification number 2. Claim 4 An antibody-drug conjugate (ADC) according to claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising a sequence described in one of sequence identification numbers 3-10 and a light chain variable region comprising a sequence described in one of sequence identification numbers 11-16. Claim 5 An antibody-drug conjugate (ADC) according to claim 4, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence described in sequence identification number 6 and a light chain variable region comprising the sequence described in sequence identification number 16. Claim 6 An antibody-drug conjugate (ADC) according to claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising a sequence described in one of sequence identification numbers 17-20 and a light chain variable region comprising a sequence described in one of sequence identification numbers 21-24. Claim 7 In claim 6, an antibody-drug conjugate (ADC) comprising an anti-CD24 antibody comprising a heavy chain variable region containing the sequence described in sequence identification number 17 and a light chain variable region of sequence identification number 21. Claim 8 An antibody-drug conjugate (ADC) according to claim 6, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence described in sequence identification number 19 and a light chain variable region comprising the sequence described in sequence identification number 23. Claim 9 An antibody-drug conjugate (ADC) according to claim 1, comprising a cytotoxic agent that inhibits or prevents the expression activity or function of cells, or causes the destruction of cells. Claim 10 In paragraph 9, an antibody-drug conjugate (ADC) in which the cytotoxic agent comprises a radioisotope, a chemotherapy agent, or a toxin. Claim 11 In paragraph 10, an antibody-drug conjugate (ADC) comprising a toxin that is a small molecule toxin or protein toxin of bacterial, fungal, plant or animal origin, or a biologically active fragment or variant of a protein toxin. Claim 12 In paragraph 10, the chemotherapy agents are auristatin, auromycin, maytansinoid, topoisomerase I or II inhibitor, ricin, ricin A-chain, combrestatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, taxol, cisplatin, CCL065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxyanthracindione. anthracin dione), actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Sapaonaria Officinalis inhibitor ( Sapaonaria officinalis An antibody-drug conjugate (ADC) comprising an inhibitor or a glucocorticoid. Claim 13 In paragraph 10, an antibody-drug conjugate (ADC) in which the radioisotope is At211, Ac225, I131, I125, Y90, Re186, Re188, Sm153, Bi212 or 213, Ra223, Pb212, Tb149, P32, or the radioisotope of Lu, optionally Lu177. Claim 14 In paragraph 12, an antibody-drug conjugate (ADC) comprising a synthetic analog of auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), or dolastatin. Claim 15 In paragraph 14, an antibody-drug conjugate (ADC) comprising a chemotherapy agent monomethylauristatin E (MMAE). Claim 16 In paragraph 12, an antibody-drug conjugate (ADC) comprising a mytancinoid comprising DM1 or DM4. Claim 17 In paragraph 12, an antibody-drug conjugate (ADC) comprising a topoisomerase I inhibitor as a chemotherapy agent. Claim 18 In paragraph 17, an antibody-drug conjugate (ADC) in which the topoisomerase I inhibitor comprises SN-38 or Dxd. Claim 19 An antibody-drug conjugate (ADC) of claim 1, wherein the linker is cleavable or non-cleavable. Claim 20 In paragraph 19, an antibody-drug conjugate (ADC) in which the linker is cleavable. Claim 21 An antibody-drug conjugate (ADC) according to claim 20, wherein the cleavable linker comprises a hydrazone, disulfide, or peptide linker. Claim 22 An antibody-drug conjugate (ADC) according to claim 21, wherein the disulfide linker further comprises one or more disulfide groups. Claim 23 An antibody-drug conjugate (ADC) according to claim 21, wherein the peptide linker comprises mc-Val-Cit-PAB (N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]-L-valyl-N5-(aminocarbonyl)-N-[4-(hydroxymethyl)phenyl]-) or Val-Ala. Claim 24 An antibody-drug conjugate (ADC) according to claim 21, wherein the disulfide linker comprises a reducible or glutathione-sensitive disulfide linker. Claim 25 In paragraph 24, an antibody-drug conjugate (ADC) in which the reducible or disulfide linker comprises SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-1-pyrrolidinyl ester). Claim 26 An antibody-drug conjugate (ADC) according to claim 21, wherein the peptide linker comprises CL2A ((6,12,15,18,21,24,27,30,33-Nonaoxa-3,9-diazapentatriacontanamide, 2-(4-aminobutyl)-35-[4-[[[[4 -[(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]cyclohexyl]carbonyl]amino]methyl]-1H-1,2,3-tria zol-1-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-). Claim 27 An antibody-drug conjugate (ADC) according to claim 21, wherein the peptide linker comprises mc-GGFG((S)-6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-((1-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide). Claim 28 An antibody-drug conjugate (ADC) according to claim 1, wherein the cytotoxic agent and the linker together comprise a structure selected from the group consisting of the following components. ; ; ; ; ; and . Claim 29 An antibody-drug conjugate (ADC) according to claim 1, wherein the linker forms a covalent bond with a cytotoxic agent at a first position and forms a covalent bond with an anti-CD24 antibody at a second position, and said first position and second position are different. Claim 30 A method for treating cancer in a subject requiring cancer treatment, comprising administering the ADC of claim 1 to the subject. Claim 31 A method according to claim 31, wherein the cancer is selected from the group comprising lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, testicular cancer, prostate cancer, neuroblastoma, and cancers including cells that bind to an anti-CD24 antibody. Claim 32 In paragraph 1, an antibody-drug conjugate (ADC) for cancer treatment. Claim 33 An antibody-drug conjugate (ADC) in paragraph 32, wherein the cancer is selected from the group comprising lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, testicular cancer, prostate cancer, neuroblastoma, and cells that bind to an anti-CD24 antibody. Claim 34 A use of the antibody-drug conjugate (ADC) of claim 1 for the manufacture of a medicament for cancer treatment. Claim 35 In paragraph 34, the use is selected from the group consisting of cancers including lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, testicular cancer, prostate cancer, neuroblastoma, and cells that bind to anti-CD24 antibodies.

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