Anti-CD24 antibody drug conjugates and uses thereof

Abstract

Provided herein are antibody-drug conjugates containing an anti-CD24 antibody conjugated to a cytotoxic agent via a linker, in which the antibody selectively binds to human CD24 protein expressed on cancer cells but not human CD24 expressed in non-cancerous cells; and uses thereof for treating cancer.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to antibody-drug conjugates containing an anti-CD24 antibody conjugated to a cytotoxic agent via a linker, in which the antibody selectively binds to human CD24 protein expressed on cancer cells but not human CD24 expressed in non-cancerous cells; and uses thereof for treating cancer. BACKGROUND OF THE INVENTION

[0002] CD24 is a small heavily glycosylated mucin-like glycosylphosphatidyl-inositol (GPI) linked cell surface protein. CD24 is expressed at higher level on hematopoietic cell, including B cells, T cells, neutrophils, eosinophils, dendritic cells, and macrophages, as well as non-hematopoietic cells, including neural cells, ganglion cells, epithelia cells, keratinocytes, muscle cells, pancreatic cells, and epithelial stem cells. In general, CD24 tends to be expressed at higher levels in progenitor cells and metabolically active cells and to a lesser extent in terminally differentiated cells. The function of CD24 is unclear in most cell types, but diverse immunological functions of CD24 have been reported.

[0003] Although CD24 is found in many normal tissues and cell types, CD24 is overexpressed in nearly 70% of human cancers. High levels of CD24 expression detected by immunohistochemistry have been found 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 role in tumor progression and metastasis. Overexpression of CD24 in cancer has also been identified as a marker indicative of poor prognosis and a more aggressive course of the disease for cancer patients. In breast cancer, expression of CD24 is significantly higher in invasive carcinoma than benign or precancerous lesions. In non-small cell lung cancer, CD24 expression has been identified as an independent marker for the overall survival of the patient. Furthermore, in esophageal squamous cell carcinoma, CD24 overexpression is suggestive of tumor lymph node metastasis, poor tumor grade as well as survival time. Similar observations were found in many other cancers including colon cancer, hepatocellular carcinoma, glioma, ovarian cancer, and prostate cancer. While CD24 has been heavily used as a prognosis marker for cancer, it has not been utilized as a neoantigen that can be a potential target for cancer therapy.

[0004] Mature CD24 is a small highly glycosylated sialoglycoprotein of 31 amino acids with 16 potential O-glycosylation sites and 2 predicted N-glycosylation sites. Glycosylation is one of the most complex post-translational modifications of proteins. A shift from the normal glycosylation pathway is known to occur in many cancer cells, leading to altered glycan expression and resulting in hyper-glycosylation or hypo-glycosylation of many cellular proteins. The altered glycosylation patterns found in cancer cells are the result of many contributory factors including dysregulation at the transcriptional level, dysregulation of chaperone proteins during glycosylation, and altered glycosidase and glycotransferase activities. Tumor-associated glycan changes include longer or shorter branching of N-glycans, higher or lower density of O-glycans, generation of truncated version of normal counterparts (Tn, sTn, and T antigens), and generation of unusual forms of terminal structures with sialic acid and fucose (sLea and sLex epitopes).

[0005] Antibody-drug conjugates (“ADC”) combine the cell binding specificity advantages monoclonal antibody targeting with the high-efficiency cytotoxicity of cytotoxic drugs and has become one of the current strategies for targeted cancer therapy. Each monoclonal antibody must be characterized separately, a suitable linker designed, and an appropriate cytotoxic agent identified that retains its potency upon delivery to tumor cells. Other considerations include whether the entire ADC is internalized when binding with a target; whether a cytostatic or cytotoxic drug is appropriate when considering potential normal tissue exposure; the type and / or stage of the cancer being treated; and, whether the linker connecting the antibody to the drug payload is a cleavable or a non-cleavable linkage. Furthermore, the antibody to drug moiety conjugation ratio (“DAR”) must be such that the binding activity of the antibody and / or the potency of the drug are unaffected.

[0006] Accordingly, there is a need in the art for improved ways of identifying and treating cancer, in particular for methods and compositions capable of differentiating cancerous from non-cancerous cells. SUMMARY OF THE INVENTION

[0007] Provided herein is an antibody-drug conjugate (ADC) comprising an anti-CD24 antibody, a therapeutic agent, which may be a cytotoxic agent, and a linker. The anti-CD24 antibody may be conjugated to the therapeutic agent with the linker. The anti-CD24 antibody may bind to a glycan-shielded epitope that is exposed on cancer cells but not on non-cancerous cells. Also provided is a pharmaceutical composition comprising the ADC and a pharmaceutically acceptable excipient.

[0008] The anti-CD24 antibody may bind to a peptide comprising SEQ ID NO: 26. The anti-CD24 antibody may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO. 1 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 2. The anti-CD24 antibody may comprise a heavy chain variable region comprising the sequence set forth in one of SEQ ID NOs: 3-10 and a light chain variable region comprising the sequence set forth in one of SEQ ID NOs: 11-16. The anti-CD24 antibody may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 6 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 16. The anti-CD24 antibody may comprise a heavy chain variable region comprising the sequence set forth in one of SEQ ID NOs: 17-20 and a light chain variable region comprising the sequence set forth in one of SEQ ID NOs: 21-24. The anti-CD24 antibody may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 17 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 21. The anti-CD24 antibody may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 19 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 23.

[0009] The cytotoxic agent may comprise a substance that inhibits or prevents the expression activity or function of cells or may cause the destruction of cells. The cytotoxic agent may comprise a radioactive isotope, a chemotherapeutic agent, or a toxin. The toxin may be a small molecule toxin or a protein toxin of bacterial, fungal, plant or animal origin. The cytotoxic agent may comprise a bioactive fragment or variant of the protein toxin. The cytotoxic agent may comprise an 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, dihydroxy 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, or a glucocorticoid. The cytotoxin may comprise a radioisotope, which may be At211, Ac225,1131, 1125, Y90, Rei 86, Rei 88, Sml53, Bi212 or 213, Ra223, Pb212, Tbl49, P32, or a radioactive isotope of Lu. The radioactive isotope of Lu may be Lul77.

[0010] The cytotoxic agent may comprise auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), or a synthetic analog of dolastatin. In one example, the cytotoxic agent comprises monomethylauristatin E (MMAE). The cytotoxic agent may comprise a maytansinoid comprising DM1 or DM4. The cytotoxic agent may comprise a topoisomerase I inhibitor. The topoisomerase I inhibitor may comprise SN-38 or Dxd.

[0011] The linker may be cleavable or non-cleavable. The cleavable linker may comprise a hydrazone, disulfide, or peptide linker. The disulfide linker may further comprise one or more disulfide groups. The peptide linker may comprise a dipeptide linker. The dipeptide linker may comprise mc-Val-Cit-PAB (N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbo nyl)-N-[4-(hydroxymethyl)phenyl]-). The disulfide linker may comprise a reducible or glutathione-sensitive disulfide linker. The reducible or glutathione-sensitive disulfside linker may comprise SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-l-pyrrolidinyl ester). The peptide linker may comprise 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-lH-pyrrol-l-yl)methyl]cyclohexyl]carbonyl]amino]methyl]-lH-l,2,3-tria zol-l-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-). The cleavable linker may comprise mc-GGFG ((S)-6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N-(2-((2-((l-((2-((4-(hydroxymethyl)phenyl)amino)-2 -oxoethyl )amino)-l-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethy l)hexanami de).

[0012] The cytotoxic agent and linker together may comprise a structure selected from the group consisting of O

[0001] The linker may form a covalent linkage to the cytotoxic agent at a first location and a covalent linkage to the anti-CD24 antibody at a second location, and the first and second location may be different.

[0002] Provided herein is a method of treating cancer in a subject in need thereof. The method may comprise administering the ADC or pharmaceutical composition to the subject. Also provided are the ADC or pharmaceutical composition for use in treating cancer, and use of the ADC or pharmaceutical composition in the manufacture of a medicament for treating cancer. The cancer may be a lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer, neuroblastoma, or a cancerthat binds to the anti-CD24 antibody. DESCRIPTION OF THE DRAWINGS

[0003] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0004] FIG. 1 shows binding comparisons of binding affinities of IgG, anti-CD24 (H3L3) alone and various anti-CD24 ADCs (ONC-784 ADCs) to HT29Luc cells. The binding affinity of select variants shows comparable binding to parent antibody H3L3. Kd is shown in table below graph in nM. Detection of bound antibodies was done using fluorescently labeled secondary antibody against human IgG

[0005] FIG. 2 shows comparisons of binding affinities of IgG, anti-CD24 antibody alone (H3L3), and various anti-CD24 ADCs (ONC-784 ADCs) to HT29Luc CD24KO cells. H3L3 and ONC-784 ADC variants did not bind HT-29 cells that do not express CD24. HT-29 cells that underwent CRISPR gene engineering to remove CD24 were subjected to a binding experiment with H3L3 and ADC variants. Detection of bound antibodies was done using fluorescently labeled secondary antibody against human IgG.

[0006] FIG. 3 shows in vitro cytotoxicity studies comparing anti-CD24 antibody alone (H3L3) with anti-CD24 ADC variants (ONC-784 ADCs) in MCL cell line Jeko-1 cells expressing luciferase at 24 and 48 hours. Jeko-1 cells expressing luciferase were incubated with increasing concentrations of ONC-784 variants or a commercially available irrelevant IgGl antibody as control. Cell viability was evaluated by evaluating luciferase activity in live cells and comparison to the luciferase activity in cells incubated with the control antibody. Left - 24h post addition of ADC, Y-axis in linear scale; Right - 48h post addition of ADC, Y-axis in log scale.

[0007] FIG. 4 shows a summary of the therapeutic efficacy comparing IgG, anti-CD24 antibody alone (H3L3) and anti-CD24 ADC variants (ONC-784 ADCs) in a hematological cancer model (JeKo-1 cells). Mice injected with 106 Jeko-1 cells were treated with the different ADC variants at the doses and frequencies indicated in the legend, one week post tumor cell injection.

[0008] FIG. 5 shows a summary of therapeutic efficacy comparing IgG, anti-CD24 antibody alone (H3L3) and anti-CD24 ADC variants (ONC-784 ADCs) in a solid tumor model of colon cancer (HT-29 cells). Mice injected with 106 HT-29 cells were treated when tumors reached -100 cubic mm volume with the different ONC-784 ADC variants at a 5 mg / Kg dose biweekly.

[0009] FIG. 6 shows a summary of therapeutic efficacy comparing IgG, anti-CD24 antibody alone (H3L3) and anti-CD24 ADC variants (ONC-784 ADCs) in a solid tumor model of breast cancer (MDA-mb-468 cells). Mice with an established subcutaneous tumors (-100 cubic mm) were treated with ADC bi-weekly, as indicated in the yellow arrows under the X-axis. Dose of ADC is shown in the legend.

[0010] FIG. 7 shows a summary of therapeutic efficacy comparing IgG, anti-CD24 antibody alone (H3L3) and a 5 mg / kg dose of anti-CD24 ADC variants (ONC-784 ADCs) in a solid tumor model of ovarian cancer (SKOV-3 cells). Mice with an established subcutaneous tumors (-100 cubic mm) were treated with ADC bi-weekly, as indicated in the yellow arrows under the X-axis.

[0011] FIG. 8 shows a dosing scheme used to compare the effects of a vehicle, H3L3, and ONC-784 ADCs in aNude-MDA-MB-468 mouse model.

[0012] FIG. 9 shows a summary of therapeutic efficacy comparing a PBS vehicle (negative control), anti-CD24 antibody alone (H3L3), and ONC-784 ADCs at various concentrations in a model of breast cancer (MDA-MB-468 cells).

[0013] FIG. 10A-C show results from testing the ONC-784-B7 in an MDA-MB-468 breast cancer model. FIG. 10A shows a dosing scheme. FIG. 10B shows specific killing of MDA-MB-468 WT cells that express CD24 by ONC-784-B7. FIG. 10C shows no effect of ONC-784-B7 on MDA-MB-468 CD24 knock-out cells that lack CD24 expression. DETAILED DESCRIPTION

[0014] Targeting of cancer expressed epitopes is a widely adopted approach for the treatment of cancer. However, many such epitopes do not make good drug targets because they are also expressed on normal tissues, which can lead to toxicity issues. An ideal Tumor-Specific Antigen (“TSA”) will have broad expression in cancer but minimal or no expression in essential host organs. Attributes of less ideal but equally workable TSAs are those expressed but differentially modified in normal vs cancer tissues, so-called Tumor-Associated Antigens (“TAA”). Examples of well characterized tumor antigens are MAGE-A3, MUC-1 and NY-ESO 1.

[0015] Identification of novel TSAs and TAAs is a limiting factor in the development of new or more effective cancer therapies, particularly for those cancers where tumor antigens do not currently exist. CD24 is a good cancer target for the following reasons: i) it is broadly overexpressed in over 70% of all human cancers and is differentially glycosylated in cancer, ii) it appears to be oncogenic and is associated with poor prognoses in various cancers and significantly shorter patient survival, and iii) it is a marker for cancer stem cells which can cause relapse and metastasis by giving rise to new tumors. The inventors have discovered anti-CD24 antibodies whose binding to CD24 is blocked by glycosylation that occurs in normal cells but not cancer cells. As a result, the antibodies bind to cancer cell lines and cancer tissues, but with minimal reactivity to a variety of normal tissues and hematopoietic cells.

[0016] In detail, the invention provides a molecule comprising an antigen-binding fragment of an antibody that immunospecifically binds to CD24, particularly human CD24, expressed on the surface of a live cell at an endogenous or transfected concentration together with a cytotoxic agent conjugated to the antibody via a linker. 1. Definitions

[0017] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. The word “about” in association with a numeric value denotes a reasonable approximation of that value. In certain cases, “about” may be construed as being within as much as 10% of the specific value with which it is associated. For example, the phrase “about 100” would encompass any value between 90 and 110.

[0018] For recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 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 contemplated. When reciting a list of numbers, ranges thereof are also contemplated (e.g., 1, 2, 3, 4, or 5 includes ranges of 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, etc.).

[0019] As used herein, the term “antibody” is intended to denote an immunoglobulin molecule that possesses a “variable region” antigen recognition site. The term “variable region” is intended to distinguish such domain of the immunoglobulin from domains that are broadly shared by antibodies (such as an antibody Fc domain). The variable region comprises a “hypervariable region” whose residues are responsible for antigen binding. The hypervariable region comprises amino acid residues from a “Complementarity Determining Region” or “CDR” (i.e., typically at approximately residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and at approximately residues 27-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain) and / or those residues from a “hypervariable loop” (i.e., residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and 26-32 (Hl), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain). “Framework Region” or “FR” residues are those variable domain residues other than the hypervariable region residues as herein defined. The term antibody includes monoclonal antibodies, multi-specific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelid antibodies, single chain antibodies, disulfide-linked Fvs (sdFv), intrabodies, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id and anti-anti-Id antibodies to antibodies of the invention). In particular, such antibodies include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG2, IgGa, IgG4, IgAi and IgA2) or subclass.

[0020] As used herein, the term “antigen binding fragment” of an antibody refers to one or more portions of an antibody that contain the antibody’s CDR and optionally the framework residues that comprise the antibody’s “variable region” antigen recognition site, and exhibit an ability to immunospecifically bind antigen. Such fragments include Fab’, F(ab’)2, Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins comprising the antibody’s “variable region” antigen recognition site and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or receptor ligand, etc.). As used herein, the term “fragment” refers to a peptide or polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.

[0021] Human, chimeric or humanized antibodies are particularly preferred for in vivo use in humans, however, murine antibodies or antibodies of other species may be advantageously employed for many uses (for example, in vitro or in situ detection assays, acute in vivo use, etc.).

[0022] A “chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such as antibodies having a variable region derived from a non-human antibody and a human immunoglobulin constant region. Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, the contents of each of which are incorporated herein in their entirety), veneering or resurfacing (EP 592,106; EP 519,596, the contents of each of which are incorporated herein by reference), and chain shuffling (U.S. Pat. No. 5,565,332, the contents of which are incorporated herein by reference).

[0023] The invention particularly concerns “humanized antibodies.” As used herein, the term “humanized antibody” refers to an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (usually a mouse or rat) immunoglobulin. The non-human immunoglobulin providing the CDRs is called the “donor” and the human immunoglobulin providing the framework is called the “acceptor.” Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, preferably about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of natural human immunoglobulin sequences. A humanized antibody is an antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. For example, a humanized antibody would not encompass a typical chimeric antibody, because, e.g., the entire variable region of a chimeric antibody is non-human. The donor antibody may be referred to as having been “humanized,” by the process of “humanization,” because the resultant humanized antibody is expected to bind to the same antigen as the donor antibody that provides the CDRs. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or a non-human primate having the desired specificity, affinity, and capacity. In some instances, Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin that immunospecifically binds to an FcyRIIB polypeptide, that has been altered by the introduction of amino acid residue substitutions, deletions or additions (i.e., mutations).

[0024] The term “drug-to-antibody ratio” or “DAR” refers to the number of drug molecules, e.g., auristatin, attached to the antibody of the ADC. The DAR of an ADC can range from 1 to 8, although higher loads, e.g., 10, are also possible depending on the number of linkage sites on an antibody. The term DAR may be used in reference to the number of drugs loaded onto an individual antibody, or, alternatively, may be used in reference to the average or mean DAR of a group of ADCs. An ADC typically has anywhere from 1 to 8 drugs moieties conjugated to the antibody, including drug loaded species of 2, 4, 6, or 8.

[0025] “Treatment” or “treating,” when referring to protection of an animal from a disease, means preventing, suppressing, repressing, or completely eliminating the disease. Preventing the disease involves administering a composition of the disclosure to an animal prior to onset of the disease. Suppressing the disease involves administering a composition of the disclosure to an animal after induction of the disease but before its clinical appearance. Repressing the disease involves administering a composition of the disclosure to an animal after clinical appearance of the disease. In one example, “treatment” or “treating” means reducing one or more symptoms of the disease. 2. Antibody-Drug Conjugates

[0026] Provided herein is an antibody-drug conjugate. The antibody-drug-conjugate (ADC) may comprise a binding protein, which may comprise an antibody or antigen binding fragment thereof. The binding protein may be chemically linked by a linker to one or more therapeutic agents, which may comprise one or more cytotoxic agents (for example, cytotoxic chemical drugs) and cytostatic agents as a payload. In one example, the ADC comprises a monoclonal antibody, the therapeutic agent, and a linker that enables attachment or conjugation of the therapeutic agent to the antibody. The therapeutic agent should have one or more of: i) sufficiently high cytotoxicity; ii) sufficiently low immunogenicity; iii) high stability; iv) functional groups that can be modified without significantly affecting potency; v) bystander killing effects; vi) proper water solubility; and vii) an intracellular target.

[0027] The antibody may comprise an anti-CD24 antibody described herein. The anti-CD24 antibody or binding fragment thereof as described herein may be linked to one or more therapeutic agents by way of one or more linkers. The ADC may comprise the following structural formula (I): [n(D-L-XY)]-Ab (I)

[0028] or a salt thereof, where each “D” represents, independently of the others, the therapeutic agent (“drug”); each “L” represents, independently of the others, a linker; “Ab” represents an anti-CD24 antigen binding moiety, such as an anti-CD24 antibody or binding fragment described herein; each “XY” represents a linkage formed between a functional group on the linker and a “complementary” functional group on the antibody, and n represents the number of therapeutic agents linked to, or drug-to-antibody ratio (DAR), of the ADC. a. Therapeutic agent

[0029] The cytotoxic agent may comprise a substance that inhibits or prevents the expression activity of cells, function of cells and / or causes destruction of cells. The cytotoxic agent may comprise one or more radioactive isotopes, chemotherapeutic agents, immune modulators, and toxins such as small molecule toxins or protein-based toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to, auristatins, auromycins, maytansinoids, topoisomerase I or II inhibitors, ricin, ricin A-chain, combrestatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, ccl065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy 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, and glucocorticoid and other chemotherapeutic agents, immune modulators, such as cytokines and Toll like receptor agonists, radioisotopes such as At211, Ac225,1131,1125, Y90, Rei 86, Rei 88, Sml 53, Bi212 or 213, Ra223, Pb212, Tbl49, P32 and radioactive isotopes of Lu including Lul77. The antibody may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form.

[0030] The term “auristatin” as used herein refers to a family of antimitotic agents. Auristatin derivatives are also included within 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.

[0031] The term “maytansinoids” as used herein refers to a family of antimitotic agents, with examples of maytansinoids include, but are not limited to, maytansine and the maytansine thiomethyl analogs S-methyl DM1 (DM1) and S-methyl DM4 (DM4).

[0032] The term “topoisomerase I inhibitors” as used herein, refers to a family of antimitotic agents, including camptothecin derivatives, SN-38, and exatecan derivatives, such as Dxd. b. Linker

[0033] The linker linking the therapeutic agent to the antibody of an ADC may be short, long, hydrophobic, hydrophilic, flexible or rigid, or may be composed of segments that each independently have one or more of the above-mentioned properties such that the linker may include segments having different properties. The linker may be polyvalent and may covalently link more than one therapeutic agent to a single site on the antibody. The linker may be monovalent and may link a single therapeutic agent to a single site on the antibody.

[0034] The linker may link the one or more therapeutic agents to the antibody by forming a covalent linkage to the therapeutic agent at one location and a covalent linkage to antibody at another. The covalent linkage may be formed by reactions between functional groups on the linker and functional groups on one or more cytotoxic agents and the antibody. The linker may be unconjugated, and may comprise a functional group capable of covalently linking the linker to one or more therapeutic agents and a functional group capable of covalently linking the linker to the antibody. The linker may be partially conjugated, and may comprise a functional group that covalently links the linker to the antibody and that is covalently linked to the one or more therapeutic agents, or vice versa. The linker may be covalently linked to both the one or more therapeutic agents and the antibody. The linker may comprise one or more moieties comprising the functional groups on the linker and covalent linkages formed between the linker and the antibody. The linker may be chemically stable to conditions outside the cell, and may be one or more of cleaved, immolated, and otherwise specifically degraded inside the cell.

[0035] The linker may not be specifically cleaved, immolated, or degraded inside a cell. The choice of stable versus unstable linker may depend upon the toxicity of the therapeutic agent. For cytotoxic agents that are toxic to normal cells, the ADC may comprise a stable linker. For cytotoxic agents that are selective or targeted and have lower toxicity to normal cells, chemical stability of the linker to the extracellular environment may be less important. A wide variety of linkers useful for linking therapeutic agents to antibodies in the context of ADCs is known in the art. Any of these linkers, as well as other linkers, may be used to link the therapeutic agents to the antibody of the ADCs described herein,

[0036] The linker may be polyvalent. Exemplary polyvalent linkers that may be used to link a plurality of therapeutic agents to a single antibody molecule are described, for example, in WO 2009 / 07345; WO 2010 / 068795; WO 2010 / 138719; WO 201 1 / 120053; WO 2011 / 171020; WO 2013 / 096901; WO 2014 / 008375; WO 2014 / 093379: WO 2014 / 093394; WO 2014 / 093640, the content of which are incorporated herein by reference in their entireties. The linker may be a dendritic-type linker. Additional examples of dendritic-type linkers can be found in US 2006 / 116422; US 2005 / 271615; de Groot et al (2003) Angew. Chern. Int. Ed. 42:4490-4494; Amir et al (2003) Angew. Chera. Int. Ed. 42:4494-4499; Shamis et al (2004) J. Am. Chern. 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.

[0037] The linker may be monovalent. Exemplary monovalent linkers that may be used are described, for example, in Molting, 2013, Antibody-Drug Conjugates, Methods in Molecular Biology 1045:71-100; Kitson et al., 2013.

[0038] The linker may be cleavable in vivo. The cleavable linker may include a chemically or enzymatically unstable or degradable linkage. The cleavable linker may rely on processes inside the cell to liberate the therapeutic agent, such as reduction in the cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by specific proteases or other enzymes within the cell. The cleavable linker may incorporate one or more chemical bonds that are either chemically or enzymatically cleavable while the remainder of the linker is non-cleavable. The cleavable linker may be a hydrozone, disulfide, or peptide linker. In one example, the peptide linker is a dipeptide linker. The dipeptide linker may comprise Valine-Cit (VC) and may comprise mc-Val-Cit-PAB (N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbonyl)-N-[4-(hydroxymethyl)phenyl]-). The dipeptide linker may comprise Vai-Ala.

[0039] The linker may comprise a chemically labile group such as hydrazone and / or disulfide groups. Linkers comprising chemically labile groups exploit differential properties between the plasma and some cytoplasmic compartments. The intracellular conditions to facilitate therapeutic agent release for hydrazone containing linkers are the acidic environment of endosomes and lysosomes, while the disulfide containing linkers are reduced in the cytosol, which contains high thiol concentrations, e.g., glutathione, in certain embodiments, the plasma stability of a linker comprising a chemically labile group may be increased by introducing steric hindrance using substituents near the chemically labile group. In one example, 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-lH-pyrrol-l- yl)methyl]cyclohexyl]carbonyl]amino]methyl]-lH-l,2,3-triazol-l-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-). In another example, the cleavable linker comprises mc-GGFG ((S)-6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N-(2-((2-((l-((2-((4-(hydroxymethyl)phenyl)amino)-2-oxoethyl)amino)-l -oxo-3 -phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide).

[0040] Acid-labile groups, such as hydrazone, remain intact during systemic circulation in the blood’s neutral pH environment (pH 7.3-7.5) and undergo hydrolysis and release the cytotoxic agent once the ADC is internalized into mildly acidic endosomal (pH 5.0-6.5) and lysosomal (pH 4.5-5.0) compartments of the cell. This pH dependent release mechanism has been associated with nonspecific release of the therapeutic agent. To increase the stability of the hydrazone group of the linker, the linker may be varied by chemical modification, e.g., substitution, allowing tuning to achieve more efficient release of the cytotoxic agent in the lysosome with a minimized loss in circulation. Hydrazone-containing linkers may contain additional cleavage sites, such as additional acid- liable cleavage sites and / or enzymatically labile cleavage sites.

[0041] The cleavable linker may comprise a disulfide group. Disulfides are thermodynamically stable at physiological pH and are designed to release the drug upon internalization inside cells, wherein the cytosol provides a significantly more reducing environment compared to the extracellular environment. Scission of disulfide bonds generally requires the presence of a cytoplasmic thiol cefaclor, such as (reduced) glutathione (GSH), such that disulfide-containing linkers are reasonably stable in circulation, selectively releasing the therapeutic agent in the cytosol. The intracellular enzyme protein disulfide isomerase, or similar enzymes capable of cleaving disulfide bonds, may also contribute to the preferential cleavage of disulfide bonds inside cells. GSH is reported to be present in cells in the concentration range of 0.5-10 mM compared with a significantly lower concentration of GSH or cysteine, the most abundant low-molecular weight thiol, in circulation at approximately 5 pM. Tumor cells, where irregular blood flow leads to a hypoxic state, result in enhanced activity of reductive enzymes and therefore even higher glutathione concentrations, in certain embodiments, the in vivo stability of a disulfidecontaining linker may be enhanced by chemical modification of the linker, for example, the use of steric hinderance adjacent to the disulfide bond. In one example, the glutathione-sensitive disulfide linker comprises glutathione-sensitive disulfide linker is SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-l-pyrrolidinyl ester). The disulfide linker may comprise SPP (N-succinimidyl-4-(2-pyridyldithio)pentanoate).

[0042] The non-cleavable linker may comprise succinimidyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate (SMCC). c. Cytotoxic agent-linker combinations

[0043] The cytotoxic agent may comprise tesirine and the linker may comprise Val-Ala. The cytotoxic agent may comprise Dxd and the linker may comprise GGFG.

[0044] The cytotoxic agent and the linker may comprise GGFG-Dxd. The cytotoxic agent and the linker may comprise the following structure.

[0045] The cytotoxic agent and the linker may comprise VC-MMAE. The cytotoxic agent and the linker may comprise the following structure.

[0046] The cytotoxic agent and the linker may comprise CL2A-SN38. The cytotoxic agent and linker may comprise the following structure.

[0047] The cytotoxic agent and the linker may comprise SPDB-DM4. The cytotoxic agent and the linker may comprise the following structure.

[0048] The cytotoxic agent and the linker may comprise SPP-DM1. The cytotoxic agent and the linker may comprise the following structure.

[0049] The cytotoxic agent and the linker may comprise SMCC-DM1. The cytotoxic agent and the linker may comprise the following structure. d. Anti-CD24 antibody

[0050] The anti-CD24 antibody may specifically target a cancer-specific glycoform of CD24. Specifically, the anti-CD24 antibody or antigen binding fragment thereof may bind to a glycan-shielded epitope that is exposed on cancer cells but not on non-cancerous cells. The anti-CD24 antibody or antigen binding fragment thereof may bind to a CD24 peptide comprising the amino acid sequence SNSGLAPN (SEQ ID NO: 27). The anti-CD24 antibody may be as described in WO2019222082, the contents of which are incorporated herein by reference.

[0051] The anti-CD24 antibody may comprise a heavy chain variable region and a light chain variable region of 3B6. The heavy chain variable region may comprise the following sequence.

[0052] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD KTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQIDNO: 1)

[0053] The light chain variable region may comprise the following sequence.

[0054] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCLQGTSYPWTFGGGTKLEIKR (SEQ ID NO: 2)

[0055] The anti-CD24 antibody or antigen binding fragment thereof may comprise a heavy chain variable region and a light chain variable region of an affinity-matured version of 3B6. The anti-CD24 antibody may comprise a heavy chain variable region comprising one of the following sequences.

[0056] EVKFEESGGGLVQPGGSIKLSCAASGVAFSGAWMDWVRQSPEKGLEWVAEIRD KTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGVYYCTGAMDYWGQGTS VTVSS (SEQIDNO: 3)

[0057] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD KSTNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQIDNO: 4)

[0058] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD NTTNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQ ID NO: 5)

[0059] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD KPNSYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSV TVSS (SEQ ID NO: 6)

[0060] EVKFEESGGGLVQPGGSIKLSCAASGVPFSGAWMDWVRQSPEKGLEWVAEIRD KTKNYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQ ID NO: 7)

[0061] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD KTKNYVTYYAESVKGRFTISRDDSKGRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQ ID NO: 8)

[0062] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQTPEKGLEWVAEIRDR ETKYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTSVT VSS (SEQ ID NO: 9)

[0063] EVKFEESGGGLVQPGGSIKLSCAASGVTFSEAWMDWVRQSPEKGLEWVAEIRD KQNEYVTYYAESVKGRFTISRDDSKSRVYLQMNNLRTEDTGIYYCTGAMDYWGQGTS VTVSS (SEQ ID NO: 10)

[0064] The anti-CD24 antibody may comprise a light chain variable region comprising one of the following sequences.

[0065] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGTPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTSTPWTFGGGTKLEIKR (SEQ ID NO: 11)

[0066] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGASLPWTFGGGTKLEIKR (SEQ ID NO: 12)

[0067] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGTPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGASVPWTFGGGTKLEIKR (SEQ ID NO: 13)

[0068] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTYLPWTFGGGTKLEIKR(SEQ ID NO: 14)

[0069] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGTSLPWTFGGGTKLEIKR (SEQ ID NO: 15)

[0070] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLQQRPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDLGIYYCMQGSSLPWTFGGGTKLEIKR (SEQ ID NO: 16)

[0071] In one example, the anti-CD24 antibody or antigen binding fragment thereof comprises the heavy and light chain variable regions of PP6373, which may comprise the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 6 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 16.

[0072] The anti-CD24 antibody or antigen binding fragment thereof may be a humanized version of PP6373 and may comprise a heavy chain variable region comprising one of the following sequences.

[0073] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVGEIRD KPNSYVTYYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTGAMDYWGQGTL VTVSS (SEQ ID NO: 17) (Hl)

[0074] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRD KPNSYVTYYAESVKGRFTISRDDSKSTAYLQMNSLKTEDTAVYYCTGAMDYWGQGTL VTVSS (SEQ ID NO: 18) (H2)

[0075] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRD KPNSYVTYYAESVKGRFTISRDDSKSTAYLQMNSLKTEDTAIYYCTGAMDYWGQGTLV TVSS (SEQ ID NO: 19) (H3)

[0076] EVQFVESGGGLVQPGGSLKLSCAASGVTFSEAWMDWVRQASGKGLEWVAEIRD KPNSYVTYYAESVKGRFTISRDDSKSTVYLQMNSLKTEDTAIYYCTGAMDYWGQGTLV TVSS (SEQ ID NO: 20) (H4)

[0077] The humanized anti-CD24 antibody may comprise a light chain variable region comprising one the following sequences.

[0078] DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPQRLIYQV SKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO: 21) (LI)

[0079] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPQRLIYQV SKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO: 22) (L2)

[0080] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPKRLIYQV SKLDPGIPDRFSGSGSETDFTLKISRVEAEDVGIYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO: 23) (L3)

[0081] DIVMTQTPLSLSVTIGQPASISCKSSQSLLYSNGKTYLNWLLQKPGQSPKRLIYQV SKLDPGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGSSLPWTFGGGTKVEIKR (SEQ ID NO: 24) (L4)

[0082] In one example, the humanized the anti-CD24 antibody or antigen binding fragment thereof is related to HILI and comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 17 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 21.

[0083] In another example, the humanized anti-CD24 antibody is related to H3L3 and comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 19 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 23. The ADC comprising this anti-CD24 antibody or antigen binding fragment thereof may be referred to as ONC-784.

[0084] In a further example, the humanized anti-CD24 antibody is related to H2L3 and comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 18 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 23.

[0085] The heavy chain of the anti-CD24 antibody may comprise a heavy chain constant region. The heavy chain constant region may comprise a constant region from an immunoglobulin (Ig), which may be one of IgG, IgM, or IgA. The IgG may be one of IgGl, IgG2, IgG3, or IgG4. In one example, the constant region comprises an Fc region. The Fc region may be of IgGl. The Ig may be human. In one example, the heavy chain constant region comprises the following sequence.

[0086] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQS SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP AP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALP APIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 25)

[0087] The light chain of the anti-CD24 antibody may comprise a light chain constant region. The light chain constant region may comprise the following sequence.

[0088] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26) e. Antibody-Drug Conjugate Compositions

[0089] Some tumor targeting antibodies can be used to prevent or limit the growth of tumors directly by affecting the biology of the tumor. For example, the humanized anti-VEGF monoclonal antibody (bevacizumab; Avastin) blocks the growth of tumors by preventing VEGF- induced tumor vascularization. Other tumor targeting antibodies are used to inhibit tumor cell growth or kill cancer cells through modification of the antibody itself. For example, tumor-targeted immunoconjugates consist of an antibody and an effector moiety bonded together by either covalent cross-links or genetic fusion. The effector moiety can be a cytotoxic drug (an antibody-drug conjugate), a protein toxin (an immunotoxin), or a radionuclide (a radioimmunoconjugate). An example of an antibody-drug conjugate is brentuximab vedotin (ADCETRIS®, Seattle Genetics), which consists of the chimeric monoclonal antibody brentuximab (cAClO, which targets the cell-membrane protein CD30) linked to three to five units of the antimitotic agent monomethyl auristatin E (MMAE, reflected by the ‘vedotin’ in the drug’s name).

[0090] The anti-CD24 antibody or antigen binding fragment thereof may comprise one or more of an ADC, immunotoxin, and radioimmunoconjugate. The anti-CD24 antibody component may allow specific delivery of the ADC to cancer cells and tissues, while limiting exposure of normal cells and tissues and thus preventing off-target toxicity. f. Antibody-Radiolabel Compositions

[0091] Also provided herein are an anti-CD24 antibody or antigen binding fragment thereof as described herein conjugated to a detectable label. The detectable label may be a dye, a fluorescent label, or a radiolabel. In one example, the anti-CD24 antibody conjugate may be an antibody-radiolabel conjugate. The radiolabel may be attached to the antibody or antigen binding fragment thereof directly or via a chelator, a bifunctional coupling agent, or an N-hydroxysuccinimide (NHS) ester. The chelator may be a bifunctional chelator. The chelator may be one or more of desferrioxamine (DFO), a squaramide derivative thereof (DFO-Sq), DFO*, DFO-cyclo*, 3,4,3-(LI-l,2-HOPO), 1,4,7,10-tetraazacyclododecane-1,4,7,1 O-tetraacetic acid (DOTA), a gallium (III) complex of (l,4,7-triazanonane-l,4,7-triyl) triacetic acid (NOTA), 1,4,7-tri azacyclononane, 1-glutaric acid-4,7-acetic acid (NODAGA), 1,1,4,7,7-diethylene triaminepentaacetic acid (DTPA), 2-hydrazinonicotinic acid (HYNIC), 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,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 another chelator known in the art. The chelator may be attached to the antibody or antigen binding fragment thereof via a linker, which may be -NCS, -NHS, maleimide, tetrafluorophenyl (TFP) ester, -N-suc-TFP ester, or another linker known in the art. The radiolabel may be 18F, 76Br, nC, 131I,211At, 225Ac, 123T, 89Zr, 64Cu, 68Ga, 52Mn, 86Y, 66Ga, 44Sc, 90Y, mIn, "mTc, 177Lu, 67Cu, [18F]A1F, or another radiolabel known in the art. In one example, the chelator is DFO and the radiolabel is 89Zr.

[0092] Provided herein are methods of detecting CD24 expressing cancer cells by contacting the cells with the anti-CD24 antibody or antibody binding fragment thereof conjugated to the radiolabel. The antibody-radiolabel conjugate may be detected via positron emission tomography. 3. Production

[0093] The anti-CD24 antibodies described herein may be prepared using a eukaryotic expression system. The expression system may entail expression from a vector in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The system may also be a viral vector, such as a replication-defective retroviral vector that may be used to infect eukaryotic cells. The antibodies may also be produced from a stable cell line that expresses the antibody from a vector or a portion of a vector that has been integrated into the cellular genome. The stable cell line may express the antibody from an integrated replication-defective retroviral vector. The expression system may be GPExTM.

[0094] The anti-CD24 antibody described herein or antigen binding fragment thereof can be purified using, for example, chromatographic methods such as affinity chromatography, ion exhange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxylapatite chromatography. In some embodiments, fusion proteins can be engineered to contain an additional domain containing amino acid sequence that allows the polypeptides to be captured onto an affinity matrix. For example, the antibodies described herein comprising the Fc region of an immunoglobulin domain can be isolated from cell culture supernatant or a cytoplasmic extract using a protein A column. In addition, a tag such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus. Other fusions that can be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography also can be used to purify polypeptides. 4. Pharmaceutical Compositions

[0095] Provided herein is a pharmaceutical composition comprising the ADC. The pharmaceutical composition may comprise a prophylactically or therapeutically effective amount of the ADC and a pharmaceutically acceptable carrier excipient. The term “pharmaceutically acceptable” may mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

[0096] Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, ethanol and the like. The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0097] Generally, the ingredients of pharmaceutical composition may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients may be mixed prior to administration.

[0098] The pharmaceutical composition may be formulated as neutral or a salt form. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. 5. Methods of Treatment

[0099] The ADC or pharmaceutical composition may be used to treat cancer. Provided herein is a method of treating cancer, which may comprise administering the ADC or pharmaceutical composition to a subject in need thereof. Further provided herein are the ADC or pharmaceutical composition for treating cancer, and use of the ADC or pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0100] As used herein, the term “cancer” refers to a neoplasm or tumor resulting from abnormal uncontrolled growth of cells. As used herein, cancer explicitly includes leukemia and lymphomas. The term refers to a disease involving cells that have the potential to metastasize to distal sites.

[0101] The cancer may be one or more of the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Berketts lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyelocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyosarcoma, and osteosarcoma; and other tumors, including melanoma, xenodenna pegmentosum, keratoactanthoma, seminoma, thyroid follicular cancer, and teratocarcinoma.

[0102] The cancer may be caused by aberrations in apoptosis. The cancers may be, but are not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. The cancer may comprise a malignancy or dysproliferative changes (such as metaplasias and dysplasias), or hyperproliferative disorders, which may be of one or more of the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. The cancer may comprise a sarcoma, melanoma, or leukemia.

[0103] The ADC or pharmaceutical composition may be administered in combination with a second anti-tumor therapy, The combination may be administered simultaneously or sequentially. The combination may be administered separately or as a single composition. The second anti-tumor therapy may include, but is not limited to, current standard and experimental chemotherapies, hormonal therapies, biological therapies, immunotherapies, radiation therapies, 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 disease, infectious disease or intoxication. Such agents include, for example, any of the above-discussed biological response modifiers, cytotoxins, antimetabolites, alkylating agents, antibiotics, or anti-mitotic agents, as well as immunotherapeutics.

[0104] In one example, the second anti-turn or therapy is an anti-tumor immunotherapy. The immunotherapy may comprise one or more molecules that disrupt or enhance alternative immunomodulatory pathways (such as TIM3, TIM4, 0X40, CD40, GITR, 4-1-BB, B7-H1, PD-1, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27 or LAG3) or modulate the activity of effecter molecules such as cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-beta, IFNg, Flt3, BLys) and chemokines (e.g., CCL21) in order to enhance the immunomodulatory effects. Specific embodiments include a bi-specific antibody comprising the ADC described herein and anti-PD-1 (pembrolizumab (Keytruda®) or nivolumab (Opdivo®)), anti-B7-Hl (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-lBB. The immunotherapy may comprise one or more molecules that activate different stages or aspects of the immune response to achieve a broader immune response. In one example, the immunotherapy comprises one or more anti-PD-1 or anti-4-lBB antibodies, which may be administered without exacerbating autoimmune side effects. 6. Methods of Administration

[0105] The compositions disclosed herein may be administered by a method including, but not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In one example, the composition disclosed herein is administered intramuscularly, intravenously, or subcutaneously. The composition may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. EXAMPLES

[0106] The disclosure has multiple aspects, illustrated by the following non-limiting examples. The present inventors have now generated various variants of an anti-CD24 antibody conjugated to a cytotoxic agent via a linker to form the ADCs as shown in Table 1. The variants differ between the linker chemistry, including both cleavable or non-cleavable linkers, and various cytotoxic agents. Example 1 ADC Production

[0107] This example demonstrates the production of certain ADCs disclosed and validated herein. Various H3L3 ADC variants were generated utilizing different cytotoxic agents and linkers for testing in vitro and in vivo (Table 1) and compared against a H3L3 antibody alone and IgGl. Table 1 ONC-784 ADC ID Payload DAR Linker Linker: Cleavable or Non-Cleavable ONC-784-B-1 Tesirine 2.6 Val-Ala Cleavable ONC-784-B-3 Deruxtecan (Dxd) 8 GGFG Cleavable ONC-784-B-4 SN38 8 CL2A Cleavable ONC-784-B-5 DM4 3.86 SPDB Cleavable ONC-784-B-6 Dxd 3.87 GGFG Cleavable ONC-784-B-7 MMAE 4.04 VC Cleavable ONC-784-B-8 DM1 3.8 SMCC Non-cleavable ONC-784-B-10 DM1 3.6 SPP Cleavable Conjugation of the H3L3 Antibody to the Combined Linker and Cytotoxic Agent

[0108] For VC-MMAE (ONC-784-B-7) and GGFG-Dxd (ONC-784-B-3) bulk conjugations, H3L3 antibody (10 mg) in original buffer (PBS, pH 7.0) was pipetted into a 50 mL tube and then was reduced by the confirmed optimum TCEP / mAb ratio. A reaction buffer (50 mM PB, pH 7.0) was added to the tubes to make the mAb concentration at 5 mg / mL. The reaction vial was placed in an incubator-shaker at 37 °C with rotate speed at 60 rpm. After reduction for 2 hours, 10 mM linker-payload in DMA was added to the sample to make the drug to mAh ratio at 7.0. DMA solvent was added into the sample to make the organic solvent at 10%. The reaction was incubated at 4°C for another 1 hour. After 1 hour, the sample was purified via spin desalting column (40 K, 10 mL).

[0109] For CL2A-SN38 (ONC-784-B-4) bulk conjugations, H3L3 antibody (10 mg) in original buffer (PBS, pH 7.0) was pipetted into a 50 mL tube. And then was reduced by 10 eq. of TCEP. Then a reaction buffer (50 mM PB, pH 7.0) was added to the tube to make the mAb concentration at 8 mg / mL. The reaction vial was placed in an incubator-shaker at 37 °C with rotate speed at 60 rpm. After reduction for 2 hours, the reduced solution was purified via spin desalting column (40 K, 10 mL). After purification, 10 mM linker-payload in DMA was added to the purified sample to make the drug to mAb ratio at 12.0. DMA solvent was added into the sample to make the organic solvent at 10%. Finally, conjugation buffer (50 mM PB, pH 7.0) was added to the tube to make the mAb concentration in the reaction at 5 mg / mL. The reaction was incubated at 22°C for another 1 hour. After 1 hour, the samples were purified via spin desalting column again (40 K, 10 mL).

[0110] For SPBD-DM4 (ONC-784-B-5) bulk conjugations, H3L3 antibody (10 mg) in original buffer (PBS, pH 7.0) was pipetted into 50 mL tubes. And then 10 mM linker-payload in DMA was added to the sample to make the drug to mAb ratio at the confirmed optimum ratio. DMA solvent was added into the sample to make the organic solvent at 15%. Then conjugation buffer (50 mM PB, pH 7.0) was added the tube to make the mAb concentration at 5 mg / mL. The reaction vial was placed in an incubator-shaker at 22 °C with rotate speed 60 rpm. After reaction for 2.5 hours, the sample was quenched by 200 mM succinic acid, pH 2.38 and purified via spin desalting column (40 K, 10 mL). Example 2 CD24 Binding Activity of ADCs [0U1] This example demonstrates the cell binding activities of various ADCs disclosed herein. Binding experiments for selected ONC-784 ADC variants were done to evaluate the affinity of the ONC-784 ADC to hypoglycosylated CD24 using a colon cancer cell line, HT29, expressing luciferase (HT29Luc). Saturation analysis of selected variants is shown in Figure 1, and Kd values in nM for the tested ADC and parent antibody are shown in a table below the graph. Binding affinity of the ADC variants were similar to the parent antibody, indicating that adding the linker and cytotoxic agent did not affect the binding to hypoglycosylated CD24 expressed by the cancer cells. Example 3 Specificity of CD24 Binding Activity for ADCs

[0112] This example demonstrates that ADCs disclosed herein bind to hypoglycosylated CD24. To validate that the binding observed with either H3L3 or ONC-784 ADCs is specific to hypoglycosylated CD24, a negative subclone of HT-29 was made using CRISPR technology, in which CD24 was removed. Comparison of binding of H3L3 or ONC-784 ADC variants to the parent cell line (Figure 1) and to CD24KO cells (Figure 2) show specific binding only to the parent cell line suggesting that the binding of both the H3L3 antibody and the ONC-784 ADC is specific to cells that express hypoglycosylated CD24 (Figure 1). Example 4 Anti-Cancer Activity of ADCs

[0113] This example demonstrates that ADCs disclosed herein exhibit anti-cancer activity. In vitro efficacy of the ONC-784 ADC variants of Table 1 were tested using a mantle cell lymphoma (MCL) cell line Jeko-1 and a colon cancer cell line, HT-29, both validated to express hypoglycosylated CD24; as well as the HT29 CD24KO cells made with CRISPR technology.

[0114] In vitro efficacy was evaluated using mantle cell lymphoma (MCL) cell line Jeko-1. Cells were incubated with the different ONC-784 ADC variants of Table 1 in increasing concentrations and viability was evaluated 24 and 48 hours post the addition of each ONC-784 ADC. An example of these results can be seen in Figure 3, in which the different ONC-784 ADCs were compared to cells incubated with a commercially available IgGl antibody.

[0115] ONC-784 ADC variant in vitro efficacy varied between the variants. In the experiments shown, for example, variants ONC-784-B-10 and ONC-784-B-4 were the most potent followed by ONC-784-B-8 and ONC-784-B-5 (Figure 3). These in vitro experiments were carried out for all ONC-784 ADCs at least twice using other cells as well (HT-291uc, MCF-71uc and mouse cells MC38 transfected with human CD24) and in mouse cells MC38 that do not express CD24. A summary of the experiments is shown in Table 2. Table 2 ONC-784 ADC ID JeKo-1 cells HT-29 cells MCF7 cells ONC-784-B-4 +++ ++ ++ ONC-784-B-5 ++ + + ONC-784-B-8 + ++ + ONC-784-B-9 ++ ++ + ONC-784-B-10 +++ ++ ++

[0116] The ONC-784 variants of Table 1 were also tested in vivo using four human tumor xenografts models in NSG or nude mice. Three models of solid tumors: HT-29 colon cancer cells, ovarian cancer cell line, SKOV3, and breast cancer cell line, MDA-MB-468. Jeko-1 Mantle cell lymphoma blood cancer cells were used as a model for hematological cancer that traffic to hematopoietic organs such as bone marrow and spleen; HT-29 and Jeko-1 cancer cells used here express luciferase to allow non-invasive monitoring of tumor progression, therefore most of the ONC-784 ADC variants were tested using these cell lines.

[0117] To directly compare the activity of the different ONC-784ADC variants, immune-compromised NSG mice were injected with the MCL cell line Jeko-1 intravenously and treated at the indicated dose for each ONC-784 ADC; most ONC-784 ADC variants were administered at 5 mg / Kg bi-weekly for a total of 4 doses on days 7, 10 14 and 17 post tumor cells injection. The mice were followed for 3 weeks using bioluminescence, and data is shown in a log scale compared to pre-dose bioluminescence (Figure 4). At 15 mg / Kg, ONC-784-B-4 was superior to ONC-784-B-10. At a 5 mg / Kg dose, ONC-784-B-7 and ONC-784-B-5 were superior to the other ONC-784 ADCs. Example 5 Anti-Cancer Activity of ADCs in a Solid Tumor Model

[0118] To validate the results in a solid tumor model, HT-29 tumor model was established by subcutaneous injection of the cells, followed by bi-weekly administration of ONC-784 ADC variants on days 7, 10, 14, and 17 post-tumor-cell injection (Figure 5). Similar to the results observed in the hematological tumor model, ONC-784-B-5, and ONC-784-B-7 showed high efficacy and tumors were eliminated in most of the mice. Surprisingly, in this model, ONC-784-B-10 also showed good efficacy (Figure 5). Example 6 Anti-Cancer Activity of ADCs in Breast and Ovarian Cancer Models

[0119] Additional studies were conducted with two more mouse models, breast cancer and ovarian cancer models, using MDA-MB-468 and SKOV-3 cells, respectively. ONC-784 ADC variants B4 and BIO significantly reduced tumor size in both models at doses as low as 3 mg / kg (Figures 6 and 7), and variants B5 and B7 significantly reduced tumor size in the breast cancer model at doses as low as 3 mg / kg (Figure 9). In the experiments on B5 and B7 in the breast cancer model, the groups tested were as shown in the following table, using the dosing scheme shown in Figure 8. Table 3 Group (# of mice) Test substance Dose regimen 7 Vehicle (PBS) NA 7 H3L3 1 mg / kg, twice a week 7 H3L3 3 mg / kg, twice a week 7 H3L3 5 mg / kg, twice a week 7 ONC-784-B5 1 mg / kg, twice a week 7 ONC-784-B5 3 mg / kg, twice a week 7 ONC-784-B5 5 mg / kg, twice a week 7 ONC-784-B7 1 mg / kg, twice a week 7 ONC-784-B7 3 mg / kg, twice a week 7 ONC-784-B7 5 mg / kg, twice a week Example 7 Anti-Cancer Activity of ONC-784-B7 in a Breast Cancer Model

[0120] This Example demonstrates that ONC-784-B7 shows anti-cancer activity in a MDA-MB-468 breast cancer model. Additional studies were conducted to evaluate the specificity of ONC-784-B7 to CD24 expressing cells with breast cancer models, using MDA-MB-468 WT and MDA-MB-468 CD24 knockout cells. Mice were injected with MDA-MB-468 WT (that express CD24) or MDA-MB-468 CD24KO (that lack CD24 expression). Once the tumors reached ~100 cubic mm, mice were treated with ONC784-B7 at 1 mg / kg or isotype control IgGl (Ig) intravenously bi-weekly (days 21, 25, 28, 31, 34, 38, and 41) post-tumor-cell injection as indicated in the black arrows under the x-axis (FIG. 10A).

[0121] FIG. 10B and C show tumor kinetics of MDA-MB-468 WT tumors (FIG. 10B) and of MDA-MB-468 CD24 knockout tumors (FIG. 10C). The results demonstrate specific killing of MDA-MB-468 WT cells by ONC-784-B7, while there was no effect on tumor growth of MDA-MB-468 CD24 knockout cells.

Claims

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 with the linker; and wherein the anti-CD24 antibody binds to a glycan-shielded epitope that is exposed on cancer cells but not on non-cancerous cells.

2. The ADC of claim 1, wherein the anti-CD24 antibody binds to a peptide comprising the sequence set forth in SEQ ID NO: 26.

3. The ADC of claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising the sequence set forth in SEQ ID NO: 2.

4. The ADC of claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence set forth in one of SEQ ID NOs: 3-10 and a light chain variable region comprising the sequence set forth in one of SEQ ID NOs: 11-16.

5. The ADC of claim 4, wherein the anti-CD24 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 6 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 16.

6. The ADC of claim 1, wherein the anti-CD24 antibody comprises a heavy chain variable region comprising the sequence set forth in one of SEQ ID NOs: 17-20 and a light chain variable region comprising the sequence set forth in one of SEQ ID NOs: 21-24.

7. The ADC of claim 6, wherein the anti-CD24 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 17 and the light chain variable region ofSEQIDNO: 21.

8. The ADC of claim 6, wherein the anti-CD24 antibody comprises the heavy chain variable region comprising the sequence set forth in SEQ ID NO: 19 and the light chain variable region comprising the sequence set forth in SEQ ID NO: 23.

9. The ADC of claim 1, wherein the cytotoxic agent comprises a substance that inhibits or prevents the expression activity or function of cells, or causes destruction of cells.

10. The ADC of claim 9, wherein the cytotoxic agent comprises a radioactive isotope, a chemotherapeutic agent, or a toxin.

11. The ADC of claim 10, the toxin comprises a small molecule toxin or a protein toxin of bacterial, fungal, plant or animal origin, or a bioactive fragment or variant of the protein toxin.

12. The ADC of claim 10, wherein the chemotherapeutic agent comprises an 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, dihydroxy 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, or a glucocorticoid.

13. The ADC of claim 10, wherein the radioisotope is At211, Ac225,1131,1125, Y90, Rel86, Rel88, Sml53, Bi212 or 213, Ra223, Pb212, Tbl49, P32, or a radioactive isotope of Lu, optionally Lui 77.

14. The ADC of claim 12, wherein the chemotherapeutic agent comprises auristatin E (AE), monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), or a synthetic analog of dolastatin.

15. The ADC of claim 14, wherein the chemotherapeutic agent comprises monomethylauristatin E (MMAE).

16. The ADC of claim 12, wherein the chemotherapeutic agent comprises a maytansinoid comprising DM1 or DM4.

17. The ADC of claim 12, wherein the chemotherapeutic agent comprises a topoisomerase I inhibitor.

18. The ADC of claim 17, wherein the topoisomerase I inhibitor comprises SN-38 or Dxd.

19. The ADC of claim 1, wherein the linker is cleavable or non-cleavable.

20. The ADC of claim 19, wherein the linker is cleavable.

21. The ADC of claim 20, wherein the cleavable linker comprises a hydrazone, disulfide, or peptide linker.

22. The ADC of claim 21, wherein the disulfide linker further comprises one or more disulfide groups.

23. The ADC of claim 21, wherein the peptide linker comprises mc-Val-Cit-PAB (N-[6-(2,5-dihydro-2,5-dioxo-lH-pyrrol-l-yl)-l-oxohexyl]-L-valyl-N5-(aminocarbo nyl)-N-[4-(hydroxymethyl)phenyl]-) or Vai-Ala.

24. The ADC of claim 21, wherein the disulfide linker comprises a reducible or glutathione-sensitive disulfide linker.

25. The ADC of claim 24, wherein the reducible or disulfide linker comprises SPDB (butanoic acid, 4-(2-pyridinyldithio)-, 2,5-dioxo-l-pyrrolidinyl ester).

26. The ADC of claim 21, where in 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-lH-pyrrol-l-yl)methyl]cyclohexyl]carbonyl]amino]methyl]-lH-1,2,3-tria zol-l-yl]-N-[4-(hydroxymethyl)phenyl]-4,8-dioxo-, (2S)-).

27. The ADC of claim 21, where in the peptide linker comprises mc-GGFG ((S)-6-(2,5-Dioxo-2,5-dihydro-lH-pyrrol-l-yl)-N-(2-((2-((l-((2-((4-(hydroxymethyl)phenyl)amino)-2 -oxoethyl)amino)-l-oxo-3 -phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethy l)hexanami de).

28. The ADC of claim 1, wherein the cytotoxic agent and linker together comprise a structure selected from the group consisting of:

29. The ADC of claim 1, where in the linker forms a covalent linkage to the cytotoxic agent at a first location and a covalent linkage to anti-CD24 antibody at a second location, wherein the first location and second location are different.

30. A method of treating a cancer in a subject in need thereof, comprising administering to the subject the ADC of claim 1.

31. The method of claim 31, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renalcancer, testicular cancer, prostate cancer, a neuroblastoma, and a cancer comprising cells that bind to the anti-CD24 antibody.

32. The ADC of claim 1 for treating a cancer.

33. The ADC of claim 32, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer, a neuroblastoma, and a cancer comprising cells that bind to the anti-CD24 antibody.

34. Use of the ADC of claim 1 in the manufacture of a medicament for treating a cancer.

35. The use of claim 3432, wherein the cancer is selected from the group consisting of lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer, a neuroblastoma, and a cancer comprising cells that bind to the anti-CD24 antibody.

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