Anti-CD24 Compositions and Uses Thereof

Abstract

To provide improved ways of identifying and treating cancer, in particular for methods and compositions capable of differentiating cancerous cells from non-cancerous cells.SOLUTION: Provided herein are anti-CD24 antibodies that selectively bind human CD24 expressed in cancer cells, but not human CD24 expressed in non-cancerous cells, and the use of such antibodies in cancer therapy.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to anti-CD24 antibodies that selectively bind to human CD24 expressed on cancer cells but not to human CD24 expressed on non-cancerous cells. The present disclosure also relates to the use of such antibodies in cancer therapy. [Background technology]

[0002] CD24 is a small, highly glycosylated, mucin-like glycosylphosphatidyl-inositol (GPI)-linked cell surface protein. CD24 is expressed at high levels on hematopoietic cells, such as B cells, T cells, neutrophils, eosinophils, dendritic cells, and macrophages, as well as non-hematopoietic cells, such as neurons, ganglion cells, epithelial cells, keratinocytes, muscle cells, pancreatic cells, and epithelial stem cells. In general, CD24 tends to be expressed at higher levels on progenitor cells and metabolically active cells, and at lower levels on terminally differentiated cells. Although the function of CD24 is unknown in most cell types, 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 suggestive of poor prognosis in cancer patients and a more aggressive course of the disease. In breast cancer, CD24 expression is significantly higher in invasive cancers than in benign or precancerous lesions. In non-small cell lung cancer, CD24 expression has been identified as an independent marker of overall patient survival. Moreover, in esophageal squamous cell carcinoma, overexpression of CD24 is associated with tumor lymph node metastasis, worse tumor grade, and shorter survival. Similar observations have been found in many other cancers, including colon cancer, hepatocellular carcinoma, glioma, ovarian cancer, and prostate cancer. Although CD24 has been widely used as a prognostic marker in cancer, it has not been exploited as a neoantigen that could be a potential target for cancer therapy due to its expression in normal cell types and potential toxicity.

[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, which leads to altered glycan expression, resulting in hyper- or hypo-glycosylation of many cellular proteins. The altered glycosylation patterns seen in cancer cells are the result of many factors, including dysregulation at the transcriptional level, dysregulation of chaperone proteins during glycosylation, and altered activity of glycosidases and glycotransferases. Tumor-associated glycan changes include longer or shorter N-glycan branches, higher or lower density O-glycans, the production of truncated versions of their normal counterparts (Tn antigen, sTn antigen, and T antigen), and the production of aberrant terminal structures bearing sialic acid and fucose (sLea and sLex epitopes). Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need in the art for improved methods of identifying and treating cancer, particularly methods and compositions that can distinguish cancerous cells from non-cancerous cells. [Means for solving the problem]

[0006] Provided herein are monoclonal anti-CD24 antibodies whose binding to CD24 is blocked by glycosylation that occurs on normal cells but not on cancer cells. The antibodies can bind to a glycan-shielded epitope that is exposed on cancer cells but not on non-cancerous cells. The antibodies can bind to a peptide comprising the sequence set forth in SEQ ID NO:48.

[0007] In another embodiment, the monoclonal antibody can bind to cancerous cells with minimal or no reactivity to non-cancerous cells.

[0008] In another embodiment, the monoclonal antibody is capable of binding to tumor cells with minimal or no reactivity to non-tumor cells.

[0009] In another embodiment, the monoclonal antibody has minimal or no reactivity to hematopoietic cells and can bind to circulating cancer cells.

[0010] In another embodiment, the monoclonal antibody may not bind to CD24 on cells lacking a cancer-specific glycosylation pattern, but may bind to CD24 on cells with a cancer-specific glycosylation pattern.

[0011] In another embodiment, the composition, which may be a pharmaceutical composition, comprises a monoclonal antibody, or one or more antigen-binding fragments thereof.

[0012] In another embodiment, the composition is used to kill cancer cells via antibody mediated cellular cytotoxicity (ADCC).

[0013] In another embodiment, the composition is used to kill cancer cells via antibody-mediated cellular phagocytosis (ADCP).

[0014] In another embodiment, the composition is used to kill cancer cells via a combination of ADCC and ADCP.

[0015] In another embodiment, the composition comprises chimeric antigen receptor T cells that can be used to confer cancer cell specificity to T cells.

[0016] In another embodiment, the composition comprises the monoclonal antibody 3B6.

[0017] In another embodiment, the composition comprises a monoclonal antibody comprising the sequence shown in SEQ ID NO:1 and the sequence shown in SEQ ID NO:2.

[0018] In another embodiment, the composition comprises a monoclonal antibody generated by affinity maturation of monoclonal antibody 3B6.

[0019] In another embodiment, the composition comprises a monoclonal antibody comprising a heavy chain selected from any one of the sequences shown in SEQ ID NO:3 to SEQ ID NO:10.

[0020] In another embodiment, the composition comprises a monoclonal antibody comprising a light chain selected from any one of the sequences shown in SEQ ID NO:11 to SEQ ID NO:16.

[0021] In another embodiment, the composition comprises the monoclonal antibody PP6373, produced by affinity maturation of the monoclonal antibody 3B6.

[0022] In another embodiment, the composition comprises a monoclonal antibody comprising the sequence shown in SEQ ID NO:6 and the sequence shown in SEQ ID NO:16.

[0023] In another embodiment, the composition comprises a monoclonal antibody generated by humanizing monoclonal antibody PP6373.

[0024] In another embodiment, the composition comprises a monoclonal antibody comprising a heavy chain selected from any one of the sequences set forth in SEQ ID NO:29 to SEQ ID NO:32.

[0025] In another embodiment, the composition comprises a monoclonal antibody comprising a light chain selected from any one of the sequences set forth in SEQ ID NO:33 to SEQ ID NO:36.

[0026] In another embodiment, the pharmaceutical composition comprises the monoclonal antibody H2L3 generated by humanizing the monoclonal antibody PP6373.

[0027] In another embodiment, the pharmaceutical composition comprises the monoclonal antibody H3L3 generated by humanizing the monoclonal antibody PP6373.

[0028] In another embodiment, the composition comprises a monoclonal antibody comprising a heavy chain variable sequence comprising the sequence set forth in SEQ ID NO:30 and a light chain variable region comprising the sequence set forth in SEQ ID NO:35.

[0029] In another embodiment, the composition comprises a monoclonal antibody comprising a heavy chain variable region comprising the sequence set forth in SEQ ID NO:31 and a light chain variable region comprising the sequence set forth in SEQ ID NO:33.

[0030] In another embodiment, the composition comprises a single chain monoclonal antibody comprising the sequence shown in SEQ ID NO:17.

[0031] In another embodiment, the composition comprises a bispecific antibody comprising a first antibody domain comprising the anti-CD24 antibody or antigen-binding fragment thereof and a second antibody domain comprising a second antibody or antigen-binding fragment thereof, the bispecific antibody may be used to bridge cancer and immune effector T cells in patients in need of cancer treatment or prevention.

[0032] In another embodiment, said second antibody domain has a different binding specificity than said first antibody domain.

[0033] In another embodiment, said second antibody domain attracts immune effector T cells to said cancer cells.

[0034] In another embodiment, the second antibody or antigen-binding fragment thereof binds to CD3.

[0035] In another embodiment, the second antibody or antigen-binding fragment thereof binds to a TCR-α chain, a TCR-β chain, a TCR-γ chain, or a TCR-δ chain.

[0036] In another embodiment, the first antibody domain comprises an antibody comprising the sequence set forth in SEQ ID NO:17 and the second antibody domain comprises the sequence set forth in SEQ ID NO:18.

[0037] In another embodiment, the first antibody domain comprises an antibody comprising any one of the sequences shown in SEQ ID NOs: 23 to 27 and 37 to 41.

[0038] In another embodiment, the compositions comprising bispecific antibodies can be used to treat cancer cells via antibody-mediated cellular cytotoxicity (ADCC).

[0039] In another embodiment, the composition comprises a bispecific antibody with enhanced ADCC activity.

[0040] In another embodiment, the composition comprises a bispecific antibody used to treat cancer cells via antibody-mediated cellular phagocytosis (ADCP).

[0041] In another embodiment, the composition comprising a bispecific antibody has enhanced ADCP activity.

[0042] In another aspect, the composition comprises a chimeric antigen receptor for use in immunotherapy, the receptor comprising a single chain antibody comprising any one of the sequences set forth in SEQ ID NO:1 to SEQ ID NO:36.

[0043] In another embodiment, the chimeric antigen receptor is used in an immunotherapy, wherein the receptor comprises a single chain antibody comprising the sequence shown in SEQ ID NO:28.

[0044] In another embodiment, the pharmaceutical composition is used in combination with a second anti-cancer therapeutic agent.

[0045] Provided herein is a method of treating cancer in a patient in need of cancer treatment comprising administering to the patient any one or more of the antibodies, bispecific antibodies, chimeric antigen receptors or compositions described herein, wherein the cancer is lung cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, renal cancer, testicular cancer, prostate cancer or neuroblastoma, wherein the cancer is capable of binding to an anti-CD24 antibody composition described herein.

[0046] Further provided herein is a method of diagnosing malignant tissue or metastatic lesions by using the anti-CD24 antibody composition, which may bind to malignant tissue or metastatic lesions at a level above a threshold amount and thus be indicative of malignant tissue or metastatic lesions.

[0047] Also provided herein is a method of identifying circulating cancer cells using the anti-CD24 antibody compositions. The anti-CD24 antibody compositions may bind to circulating cancer cells at levels above a threshold amount, and thus may be indicative of circulating cancer cells. Further provided herein is the use of the compositions described herein in the manufacture of a medicament for treating a disease or condition described herein. [Brief description of the drawings]

[0048] [Figure 1]1 is a bar graph of ELISA results showing that binding of anti-CD24 monoclonal antibody 3B6 is hindered by the presence of carbohydrate (glycan), whereas the commercially available anti-CD24 monoclonal antibody ML5 is not. 3B6 binds strongly to CD24 with N-glycan and sialic acid modifications removed (N-SA-CD24) and CD24 with N-glycan, sialic acid, and O-glycan modifications removed (N-SA-O-CD24), but binds very weakly to both CD24 with N-glycan modifications removed (N-CD24) or fully modified (N-glycan + sialic acid + O-glycan) CD24. CD24GST represents the negative control CD24-GST fusion.

[0049] [Figure 2A] Binding assays show that 3B6 binds to neuroblastoma cell lines and medulloblastoma tumors. Figure 2A shows normalized affinity plots of anti-CD24 monoclonal antibodies ML5, 3B6, and SN3 and control antibodies tested against six neuroblastoma cell lines IMR32, SK-N-SH, SH-SY5Y, SK-N-BE(2), SK-N-AS, and SK-N-BE(2)C. Although 3B6 has some affinity for all neuroblastoma cell lines except SK-N-AS, the affinity of 3B6 was significantly lower than that of commercially available anti-CD24 antibodies ML5 (BD Bioscience, Cat. No. 555426) and SN3 (Thermo Fisher, Cat. No. MA5-11833). [Figure 2B] Figure 2B shows radiofluorography images of 3B6 treatment of four medulloblastoma tumors. 3B6 bound to three of the four tumors.

[0050] [Diagram 3] 1 shows plots of a competition ELISA comparing the ability of 3B6 mutants to block 3B6 binding to a CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

[0051] [Figure 4]1 shows plots of a competition ELISA comparing the ability of 3B6 mutants to block 3B6 binding to a CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

[0052] [Diagram 5] 1 shows plots of a competition ELISA comparing the ability of 3B6 mutants to block 3B6 binding to a CD24-GST fusion protein. PP6226 has the same variable region as 3B6.

[0053] [Figure 6] 1 is a bar graph of ELISA results showing the relative affinity of affinity matured chimeric anti-CD24 antibodies for CD24 expressed by CHO cells. Twelve of the clones had increased affinity and different specificity for fully glycosylated CD24, N-CD24, SA-CD24, and N-SA-CD24 compared to 3B6 (PP6226).

[0054] [Figure 7] Figure 1 shows a titration assay of various affinity matured chimeric anti-CD24 antibodies tested against the lung cancer cell line NCI-H727 (left panel) and the neuroblastoma cell line IMR32 (right panel). The maximum antibody concentration tested was 5 μg / mL with a titration factor of 2× for a minimum concentration of 0.01 μg / mL. An unstained (0 μg / mL) negative control is also shown.

[0055] [Figure 8]Quantitative comparison of binding between different CD24 glycoforms and anti-CD24 antibodies (parental PP6229 and affinity matured PP6373). Fc-depleted CD24 was coated onto ELISA plates and treated with either buffer (CD24), NanA (SA-), or NanA + N-glycanase (SA-N-) prior to addition of PP6626 (left panel) or PP6373 (right panel) at the indicated doses. The maximum antibody concentration tested was 7812.50 ng / mL, using a titration factor of 5x relative to a minimum concentration of 0.02 ng / mL.

[0056] [Figure 9] Mapping of the 3B6 binding site by peptide inhibition assays. Of five overlapping CD24 peptides tested, only one (peptide 4) contains the antigenic epitope.

[0057] [Figure 10] Figure 1 shows mapping of the PP6373 binding site by peptide inhibition assays. Of five overlapping CD24 peptides tested, only one (SNSGLAPNT (SEQ ID NO: 46)) contains an antigenic epitope.

[0058] [Figure 11] 1 shows mapping of the PP6373 epitope using truncated peptides from the peptide 4 antigenic epitope sequence. The data indicates that the optimal epitope is contained within the sequence SNSGLAPN (SEQ ID NO: 48).

[0059] [Figure 12] 1 is a plot showing that PP6373 reduces tumor growth in vivo in a mouse model. Nude mice bearing palpable lung cancer xenografts were administered either control human IgG or PP6373 at two time points indicated by the arrows, and tumor growth was measured weekly thereafter.

[0060] [Figure 13]1 is a plot showing that PP6373 induced cytotoxicity (ADCC) against human cancer cell line H727. H727 cells co-cultured with effector cells PBL at 5 μg / mL using PP6373 and human IgGFC induced ADCC.

[0061] [Figure 14] This is a plot showing that PP6373 (d6873) without core fucosylation induces stronger ADCC against human cancer cell line H727 than PP6373. H727 cells co-cultured with effector cells PBL at 5 μg / mL using d6373, PP6373, and human IgGFC induced ADCC.

[0062] [Figure 15] Flow cytometry plots showing that the combination of PP6373-hole and OKT3-knob exhibits higher bispecificity than the combination of PP6373-knob and OKT3-hole. Jurkat cells were stained with tissue culture supernatants of 293T cells transfected with PP6373, OKT3, PP6373-knob & OKT3-hole, or PP6373-hole & OKT3-knob, and then incubated with biotinylated SA-N-CD24 protein. PE-streptavidin signals were measured by flow cytometry. Three independent experiments were performed.

[0063] [Figure 16] Flow cytometry plots showing that PP6373-OKT3 induces higher bispecific activity than OKT3-PP6373. Jurkat cells were stained with tissue culture supernatants of 293T cells transfected with empty plasmid (negative control), PP6373-OKT3, or OKT3-PP6373, and then incubated with biotinylated SA-N-CD24 protein. PE-streptavidin signals were measured by flow cytometry. Three independent experiments were performed.

[0064] [Figure 17]Figure 1 shows flow cytometry plots showing that bispecific antibody PP6373-OKT3 has antitumor activity. Lung cancer cells H727 and activated human T cells were incubated 1:5 with tissue culture supernatants of untreated 293T cells or tissue culture supernatants of 293T cells transfected with empty plasmid (untransfected), PP6373, OKT3 or PP6373-OKT3 for 12 hours. Cytokines (IFNr, TNF, IL10, IL6, IL4, and IL2) in the tissue culture medium were measured by flow cytometry. Three independent experiments were performed.

[0065] [Figure 18] Flow cytometry plots showing that bispecific antibody PP6373-OKT3 induced tumor cell cytotoxicity by T cells. Lung cancer cells H727 and activated human T cells were incubated 1:5 with tissue culture supernatants of untreated 293T cells or tissue culture supernatants of 293T cells transfected with empty plasmid (untransfected), PP6373, OKT3, or PP6373-OKT3 for 12 hours. Lung cancer cells and human T cells were harvested and stained with anti-human CD45 and LIVE / DEAD reagent Aqua. The number of tumor cells that were double negative for anti-CD45 and Aqua was plotted. Three independent experiments were performed.

[0066] [Figure 19] Flow cytometric analysis of high bispecific activity induced by FIT-Ig. Jurkat cells were stained with negative control (untransfected 293T supernatant) or FIT-Ig and then incubated with biotinylated SA-N-CD24 protein. PE-streptavidin signal was measured by flow cytometry. Three independent experiments were performed.

[0067] [Figure 20]Flow cytometry analysis showing that FIT-Ig has higher antitumor activity than PP6373-OKT3 and OKT3-PP6373. Lung cancer cells H727 and activated human T cells were incubated 1:5 with negative control (untransfected 293T supernatant), PP6373-OKT3, OKT3-PP6373, or FIT-Ig for 12 hours. Cytokines (IFNr, TNF, IL10, IL6, IL4, and IL2) in tissue culture medium were measured by flow cytometry. Three independent experiments were performed.

[0068] [Figure 21] Flow cytometry analysis showing that FIT-Ig induces tumor cell cytotoxicity by T cells. Lung cancer cells H727 and activated human T cells were incubated 1:5 with negative control (untransfected 293T supernatant), PP6373-OKT3, OKT3-PP6373, or FIT-Ig for 12 hours. Lung cancer cells and human T cells were harvested and stained with anti-human CD45 and LIVE / DEAD reagent Aqua. The number of tumor cells that were double negative for anti-CD45 and Aqua was plotted. Three independent experiments were performed.

[0069] [Figure 22] Flow cytometry analysis showing that FIT-Ig has higher thermal stability than PP6373-OKT3 and OKT3-PP6373. All bispecific antibodies PP6373-OKT3, OKT3-PP6373, and FIT-Ig were incubated at the indicated temperatures for 20 min, and the supernatants after spinning at 14000 g for 5 min were used to stain Jurkat cells. Biotinylated SA-N-CD24 protein was then incubated with Jurkat cells, and the PE-streptavidin signal was measured by flow cytometry.

[0070] [Figure 23] A schematic diagram of the CarT construct containing anti-CD24-scFv is shown.

[0071] [Figure 24] A plot of induction of cytotoxicity by CD24 CART against the lung cancer cell line A549 is shown.

[0072] [Diagram 25] 1 shows plots of CART activation by tumor cell lines, as indicated by IFNγ production.

[0073] [Figure 26] 1 shows a bar graph of the antitumor activity of CD24 CART against various tumor types. The E / T ratio of the data shown is 5.

[0074] [Figure 27] A ribbon diagram of the 3D structural alignment of chimera PP6373 (FR: white, CDR: light grey) and huVHv1VLv1 (FR: grey, CDR: dark grey).

[0075] [Figure 28] Plots of the expression and relative efficacy of various antibody pairs for binding to CD24-GST are shown.

[0076] [Figure 29] Plots of H2L3 and H3L3 binding to human cancer cell lines NCI-H727 (top) and IMR32 (bottom) are shown. Data shown are mean fluorescence intensity using a broad range of antibodies.

[0077] [Diagram 30] Figure 1 shows cell death plots showing that HL33 is more potent in ADCC than PP6373 at low concentrations. The lung cancer cell line A549 was used as target and human PBLs were used as effectors. The antibody doses used were 3 μg / mL (upper panel) or 9 μg / mL (lower panel).

[0078] [Diagram 31]Figure 1 shows cell death plots showing that H3L3 confers potent ADCC activity to multiple tumor cell lines, including lung cancer cell lines A549 and NCI-H727, and neuroblastoma cell line IMR-32. Human PBMCs were used as effector cells.

[0079] [Diagram 32] Figure 1 shows cell death plots demonstrating that H3L3 confers potent ADCC activity against multiple tumor cell lines, including lung cancer cell lines A549 and NCI-H727, and neuroblastoma cell line IMR-32, using NK cells purified from human PBMC as effector cells.

[0080] [Diagram 33] This is a flow cytometry analysis showing that antibodies that recognize epitopes that are shielded by glycans do not recognize B cells or red blood cells, and have insufficient interactions with neutrophils. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0081] Targeting cancer-expressed epitopes is a widely adopted approach for the treatment of cancer. However, many such epitopes are not good drug targets because they are also expressed in normal tissues and may cause toxicity problems. An ideal tumor-specific antigen (TSA) would show broad expression in cancer but minimal or no expression in essential host organs. A less ideal but equally functional attribute of a TSA is one that is expressed but differentially modified in normal and cancer tissues, the so-called tumor-associated antigen (TAA). Examples of well-characterized tumor antigens are MAGE-A3, MUC-1, and NY-ESO1.

[0082] A limiting factor in the development of new or more effective cancer therapeutics, especially for cancers for which no tumor antigens currently exist, is the identification of novel TSAs and TAAs. CD24 is an excellent cancer target because it is widely overexpressed in over 70% of all human cancers and is differentially glycosylated in cancers, CD24 appears to be oncogenic and is associated with poor prognosis and significantly reduced patient survival in a variety of cancers, and CD24 is a marker for cancer stem cells that may cause recurrence and metastasis by initiating new tumors. The inventors have discovered an anti-CD24 antibody whose binding to CD24 is blocked by glycosylation that occurs in normal cells but not in cancer cells. As a result, the antibody binds to cancer cell lines and cancer tissues, but has minimal reactivity to a variety of normal tissues and hematopoietic cells.

[0083] Provided herein are antibodies and antigen-binding fragments thereof. The antibodies may be monoclonal, human, chimeric, or humanized. The antibodies may be monospecific, bispecific, trispecific, or multispecific. The antigen-binding fragments of the antibodies may immunospecifically bind to CD24, particularly human CD24, preferably expressed on the surface of live cells at endogenous or transfected concentrations. The antigen-binding fragments may bind to CD24. The antibodies may be detectably labeled or may include a conjugated toxin, drug, receptor, enzyme, or receptor ligand.

[0084] In addition to directly targeting tumors, the immune system also has the ability to recognize and eliminate cancer in experimental models and patients. As a result, cancer immunotherapy is emerging as one of the most promising areas of cancer treatment. Active cancer immunotherapy includes agents that amplify the innate immune response (including antibodies against PD-1, PD-L1, or CTLA-4), bispecific molecules such as antibodies that bridge cancer and immune effector T cells, or adoptive cell transfer (ACT) using ex vivo stimulated tumor infiltrating lymphocytes (TIL), activated Natural Killer (NK) cells, or genetically engineered T cells (chimeric antigen receptor (CAR) and T cell receptor (TCR) modified T cells). Many of these techniques require a tumor targeting component for specificity and efficacy.

[0085] 1.Definition The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. The word "about" associated with a numerical value indicates a reasonable approximation of the value. In some cases, "about" may be interpreted as being within 10% of the particular value to which it is attached. For example, the phrase "100 μm" includes any value between 90 and 110.

[0086] With respect to references to numerical ranges herein, each intervening number is expressly contemplated to the same degree of precision, for example, for the range 6 to 9, the numbers 7 and 8 are expressly contemplated in addition to 6 and 9, and for the range 6.0 to 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 expressly contemplated.

[0087] "Treatment" or "treating", when referring to protecting an animal from a disease, means preventing, suppressing, repressing, or completely eliminating the disease. Preventing a disease involves administering a composition of the invention to an animal before the onset of the disease. Suppressing a disease involves administering a composition of the invention to an animal after induction of the disease but before its clinical appearance. Suppressing a disease involves administering a composition of the invention to an animal after the clinical appearance of the disease. Preventing a disease involves administering a composition of the invention to an animal before the onset of the disease. Suppressing a disease involves administering a composition of the invention to an animal after induction of the disease but before its clinical appearance. Suppressing a disease involves administering a composition of the invention to an animal after the clinical appearance of the disease.

[0088] As used herein, the term "antibody" refers to an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" refers to a domain of an immunoglobulin that is distinct from the domains commonly shared by antibodies (such as the antibody Fc domain). The variable region contains the "hypervariable region" whose residues are responsible for antigen binding capacity. The hypervariable region may comprise amino acid residues from the "Complementarity Determining Regions" or "CDRs" (i.e., typically residues at about positions 24-34 (L1), 50-56 (L2), and 89-97 (L3) of the light chain variable domain and residues at about positions 27-35 (H1), 50-65 (H2), and 95-102 (H3) of the heavy chain variable domain) and / or amino acid residues from the "hypervariable loops" (i.e., residues at positions 26-32 (L1), 50-52 (L2), and 91-96 (L3) of the light chain variable domain and residues at positions 26-32 (H1), 53-55 (H2), and 96-101 (H3) of the heavy chain variable domain). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as defined herein. The antibodies disclosed herein may be monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, camelized antibodies, single chain antibodies, disulfide-linked Fv (sdFv), intrabodies, or anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies and anti-anti-Id antibodies to an antibody of the invention). In particular, the antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2) or subclass of immunoglobulin molecule.

[0089] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody, including the antibody's complementarity determining regions (CDRs) and, optionally, the framework residues that comprise the antibody's "variable region" antigen recognition site, that exhibit the ability to immunospecifically bind an antigen. Such fragments include Fab', F(ab')2, Fv, single chain (ScFv), and mutants, naturally occurring variants thereof, as well as fusion proteins that include 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 a receptor ligand, etc.). As used herein, the term "fragment" refers to a peptide or polypeptide that comprises 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 acid 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.

[0090] While human, chimeric, or humanized antibodies are particularly preferred for in vivo use in humans, murine antibodies or antibodies from other species may be advantageously used for many applications (e.g., in vitro or in situ detection assays, emergency in vivo uses, etc.).

[0091] A "chimeric antibody" is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules, such as an antibody 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 a framework region from a human immunoglobulin molecule can be produced using a variety of techniques known in the art, such as, for example, CDR grafting (EP 239,400, WO 91 / 09967, and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, all of which are incorporated herein by reference), veneering or resurfacing (EP 592,106, EP 519,596, all of which are incorporated herein by reference), and chain shuffling (U.S. Pat. No. 5,565,332, all of which are incorporated herein by reference).

[0092] As used herein, the term "humanized antibody" refers to an immunoglobulin that includes a human framework region and one or more CDRs from a non-human (usually mouse or rat) immunoglobulin. The non-human immunoglobulin that provides the CDRs is referred to as the "donor" and the human immunoglobulin that provides the framework is referred to as the "acceptor". Constant regions may be absent, but if present, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85%-90%, preferably about 95% or more identical. Thus, all parts of a humanized immunoglobulin, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human immunoglobulin sequence. A humanized antibody is an antibody that includes a humanized light chain immunoglobulin and a humanized heavy chain immunoglobulin. For example, a typical chimeric antibody would not be encompassed by a humanized antibody, since the entire variable region of the chimeric antibody is non-human, etc. A donor antibody is said to be "humanized" by the process of "humanization" because the resulting humanized antibody is expected to bind to the same antigen as the donor antibody that provided the CDRs. In general, 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 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 contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve antibody performance. In general, a humanized antibody may 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 of human immunoglobulin sequences.A humanized antibody may also optionally comprise at least a portion of an immunoglobulin constant region (Fc), which can be that of a human immunoglobulin that immunospecifically binds to an FcγRIIB polypeptide, altered by the introduction of one or more amino acid residue substitutions, deletions, or additions (i.e., mutations).

[0093] 2. Anti-CD24 antibody composition Described herein are anti-CD24 antibodies that can specifically target cancer-specific glycoforms of CD24. The anti-CD24 antibodies can be used to develop cancer therapeutics, including but not limited to antibody-drug conjugates, ADCC-enhanced therapeutic antibodies, bispecific antibodies, CAR-T therapeutics, and TCR therapeutics. Specifically, the anti-CD24 antibodies or antigen-binding fragments thereof can bind to glycan-masked epitopes that are not present on non-cancerous cells but are exposed on cancer cells. In particular, the anti-CD24 antibodies or antigen-binding fragments thereof can bind to a CD24 peptide that includes the amino acid sequence SNSGLAPN (SEQ ID NO: 48).

[0094] The anti-CD24 antibody may be 3B6, which 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 or antigen-binding fragment thereof may be an affinity matured version of 3B6, which may comprise a heavy chain variable region comprising any one of the sequences set forth in SEQ ID NO:3-10 and a light chain variable region comprising any one of the sequences set forth in SEQ ID NO:11-16. The anti-CD24 antibody or antigen-binding fragment thereof may be PP6373, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO:6 and a light chain variable region comprising the sequence set forth in SEQ ID NO:16. For human therapeutic use, the anti-CD24 antibody or antigen-binding fragment thereof may be a humanized version of PP6373, which may comprise a heavy chain variable region comprising any one of the sequences set forth in SEQ ID NO:29-32 and a light chain variable region comprising any one of the sequences set forth in SEQ ID NO:33-36. In particular, the humanized anti-CD24 antibody or antigen-binding fragment thereof may be H2L3, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO:30 and a light chain variable region comprising the sequence set forth in SEQ ID NO:35, or H3L3, which may comprise a heavy chain variable region comprising the sequence set forth in SEQ ID NO:31 and a light chain variable region comprising the sequence set forth in SEQ ID NO:35.

[0095] 3. Antibody-drug conjugate compositions Tumor-targeted antibodies can be used to directly prevent or limit tumor growth by affecting tumor biology. For example, humanized anti-VEGF monoclonal antibodies (generic name: Bevacizumab; Avastin) block tumor growth by inhibiting VEGF-induced tumor angiogenesis. Other tumor-targeted 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 linked by either covalent crosslinking or genetic fusion. The effector moiety can 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 the chimeric monoclonal antibody brentuximab (cAC10, which targets the cell membrane protein CD30) conjugated to 3-5 units of the mitotic inhibitor monomethyl auristatin E (MMAE, reflected in the "vedotin" in the drug name).

[0096] The anti-CD24 antibody or antigen-binding fragment thereof may be comprised in an antibody-drug conjugate, an immunotoxin, or a radioimmunoconjugate. The anti-CD24 targeting component of such compositions may allow for specific delivery of the conjugate to cancer cells and tissues while limiting exposure of normal cells and tissues, thereby preventing off-target toxicity.

[0097] 4.ADCC antibody composition The anti-CD24 antibody or antigen-binding fragment thereof or an antibody composition comprising one of the foregoing can be used to stimulate cancer cell death via at least one of antibody-mediated cellular cytotoxicity (ADCC) and antibody-mediated cellular phagocytosis (ADCP). ADCC is an immune defense mechanism in which a set of specific immune cells (effector cells) in the body actively engages and lyses target cells (e.g., pathogens). ADCC has been identified as an important cellular innate immune response, which serves as the body's first line of defense against pathogens, acting to limit and contain infection. The ADCC process is designed to kill antibody-coated target cells via a non-phagocytic process, characterized by either the targeted release of cytotoxic granules or the expression of cell death-inducing molecules. ADCC is typically initiated when specific host antibodies (mainly of the IgG class) recognize and bind to membrane surface antigens on target cells and simultaneously engage Fc receptors (FcRs) on the surface of effector cells. The most common effector cells mediating ADCC are natural killer (NK) cells, but monocytes, macrophages, neutrophils, eosinophils, and dendritic cells can also mediate ADCC responses. Although ADCC is a fairly rapid response, efficacy depends on several parameters, including antigen density on the surface of the target cells and the affinity of the antigen-antibody interaction, as well as the properties of the Fc fragment, which determine antibody interactions with various members of the Fc receptor family.

[0098] The binding of the antibody to specific cell surface receptors on target cells, a process called opsonization, is a key event in the ADCC process. The opsonization process attracts phagocytes to target cells and can initiate phagocytosis. Binding of the antibody Fc region to the FcR on phagocytes also promotes the formation of C3b, a cleavage product of complement component 3, a key protein that initiates the engulfment of antibody-opsonized target cells. Antibody-mediated phagocytosis is also called antibody-dependent cell-mediated phagocytosis (ADCP). However, for ADCC, pathogens do not need to be phagocytosed and destroyed. As mentioned above, FcRs on the surface of cytotoxic effector cells are key to triggering ADCC. In humans, the most important FcR classes capable of triggering ADCC are FcγRI (CD64), FcγRIIa, and FcγRIIc (CD32), and FcγRIIIa (CD16). However, FcγRIIb receptors inhibit ADCC responses. Thus, the balance between activating and inhibitory signals from FcγRs is a key determinant of the magnitude of the ADCC response. Upon target recognition, specialized intracellular granules (also called secretory lysosomes) are released by cytotoxic effector cells in a calcium-dependent polarized extracellular release process. The key components released from the granules are perforin, cytolysin, and granzyme B. Perforin inserts and forms pores in the target cell membrane. Calcium is required for this process. Granzyme B causes DNA fragmentation of the target cell. An example of a therapeutic antibody that functions by ADCC is Trastuzumab (Herceptin, Genentech). Trastuzumab targets HER2, which is expressed at abnormally high levels in many breast cancers and is often referred to as HER2-positive breast cancer, and inhibits the growth of HER2-positive breast cancer by inducing ADCC in the host.

[0099] Antibodies used for ADCC-mediated activities usually require some modification to enhance ADCC activity. There are several available techniques that usually involve modifying the antibody so that the oligosaccharides of the Fc region of the antibody do not have fucose sugar units, improving binding to the FcγIIIa receptor. If the antibody is afucosylated, it has the effect of increasing antibody-dependent cellular cytotoxicity (ADCC). For example, BioWa's POTELLIGENT® technology uses a FUT8 gene knockout CHO cell line to produce 100% afucosylated antibodies. FUT8 is the only gene that encodes 1,6-fucosyltransferase, which catalyzes the transfer of fucose from GDP-fucose to GlcNAc in the 1,6 linkage of complex oligosaccharides. ProBioGen has developed a CHO line that is modified to produce low levels of fucosylated glycans on MAbs, but not by FUT knockout. ProBioGen's system introduces a bacterial enzyme that redirects the de novo fucose synthesis pathway to sugar nucleotides that cannot be metabolized by cells. As an alternative approach, Seattle Genetics has a proprietary feed system that produces low levels of fucosylated glycans on MAbs produced in CHO (and possibly other) cell lines. Xencor has developed the XmAb Fc domain technology designed to improve immune system clearance of tumor and other pathological cells. This Fc domain has two amino acid changes that result in a 40-fold increase in affinity for FcγRIIIa. It also increases affinity for FcγRIIa, potentially recruiting other effector cells such as macrophages that play a role in immunity by phagocytosing and digesting foreign material.

[0100] The anti-CD24 antibody or antigen-binding fragment thereof may be incorporated into an antibody that kills cancer via ADCC. The anti-CD24 targeting component of such a composition may allow for specific delivery targeting of cancer cells for ADCC-mediated destruction while sparing normal cells and tissues. The ADCC activity of the anti-CD24 antibody or antigen-binding fragment thereof may be enhanced by one or more modifications described herein.

[0101] 5. Bispecific antibody composition Further provided herein is a bispecific antibody comprising a first antibody domain comprising a first antibody or antigen-binding fragment thereof crosslinked to a second antibody or antigen-binding fragment thereof. The first antibody domain may comprise an anti-CD24 antibody or antigen-binding fragment thereof as described herein, and the second antibody or antigen-binding fragment thereof may bind to other immunostimulatory molecules. In certain embodiments, the second antibody domain comprises an anti-CD3 antibody or antigen-binding fragment thereof. In this case, the bispecific antibody specifically targets tumor cells expressing a cancer-specific glycoform of CD24 and simultaneously binds to CD3 on cytotoxic T cells, thereby attracting T cells to the tumor site, where the T cells infiltrate the tumor and cause tumor cytotoxicity. Other examples of partner antibodies for use in bispecific antibodies to attract cytotoxic T cells or other effector cells to the tumor site are known in the art.

[0102] The second antibody or antigen-binding fragment thereof may target a complementary anti-tumor pathway or mechanism. The second antibody domain may include a cancer immunotherapeutic antibody or antigen-binding fragment thereof that amplifies the natural immune response. Examples of such cancer immunotherapeutic antibodies include anti-PD-1, anti-B7-H1, 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. Such antibodies may be used to treat cancer. The second antibody or antigen-binding fragment thereof may bind to the TCR-α chain, TCR-β chain, TCR-γ chain, or TCR-δ chain.

[0103] The bispecific antibody may comprise any one of the sequences shown in SEQ ID NOs: 17 and 18, or the sequences shown in SEQ ID NOs: 23 to 27, and 37 to 41.

[0104] There are many different bispecific antibody technologies known in the art. Most of these require the two-component antibody to be in a single-chain format so that the two parts can be expressed in a single construct. A preferred method is to express the antibody as a single-chain variable fragment (scFv). Non-limiting examples of bispecific antibody technologies include the Bispecific T-cell Engager (BiTE) method, the Dual-Affinity Re-Targeting (DART) method, the Fabs-in-tandem Immunoglobulin (FIT-Ig) method, and the Knobs-into-Holes method. Such bispecific antibodies comprising the anti-CD24 antibody or antigen-binding fragment thereof are specifically contemplated herein.

[0105] 6. CAR-T Therapeutic Composition Chimeric Antigen Receptor (CAR) T-cell therapy, or CAR-T therapy, is a type of cell treatment in which a cancer patient's T cells are genetically modified ex vivo to express a CAR protein and attack cancer cells. Specifically, T cells are taken from the patient's blood, particularly the patient's own blood (autologous), and transfected with a genetic construct that expresses a recombinant CAR receptor. Large numbers of CAR-T cells can then be grown in the laboratory and infused into the patient to target and destroy the patient's cancer cells. The T cells can also be allogeneic T cells, either from a matched donor, or derived from a universal or "off-the-shelf" T cell line in which one or more TCR genes and HLA class I loci of the allogeneic T cells have been disrupted, such that the resulting T cells cannot recognize allogeneic antigens.

[0106] CAR protein constructs typically have a modular structure that includes the following core components: an extracellular single-chain variable fragment (scFv) derived from an antibody, which is linked to a hinge / spacer peptide and a transmembrane domain, which is further linked to an intracellular T cell signaling domain of a T cell receptor. The scFv is a targeting element that is expressed on the surface of CAR-T cells to confer antigen specificity. The spacer connects the extracellular targeting element to the transmembrane domain, which influences CAR function and the flexibility of the scFv. The transmembrane domain spans the cell membrane, anchoring the CAR to the cell surface and connecting the extracellular domain to the intracellular signaling domain, which influences the expression of the CAR at the cell surface. The costimulatory domain is derived from the intracellular signaling domain of costimulatory proteins such as CD28 and 4-1BB, which enhance cytokine production. The CD3ζ domain is derived from the intracellular signaling portion of the T cell receptor and mediates downstream signaling during T cell activation. Examples of CAR-T therapeutics include those that target the B cell surface antigens CD19 (such as JCAR017 and JCAR014 [Juno Therapeutics]), CTL019 (tisagenlecleucel-T (Kymriah™) [Novartis]), and KTE-C19 (axicabtagene ciloleucel (Yescarta®) [Kite Pharma]), and CD22 (JCAR014 [Juno Therapeutics]). Other examples of CAR-T therapeutics include those that target L1-CAM (JCAR023 [Juno Therapeutics]), ROR-1 (JCAR024 [Juno Therapeutics]), and MUC16 (JCAR020 [Juno Therapeutics]).

[0107] The scFv portion of the CAR is a key component, ensuring specificity for cancer cells while preventing activity against normal cells, associated with off-target toxicity. Thus, typically, the scFv portion is derived from a portion of an antibody that recognizes a target protein that is specifically expressed on cancer cells, but is expressed less frequently or ideally not at all on other cells or tissues. Thus, an scFv fragment derived from any of the anti-CD24 antibodies described herein can be used as the cancer targeting component of a recombinant CAR protein. In particular, the scFv protein can comprise the sequence shown in SEQ ID NO:28.

[0108] CAR-T cells have shown excellent efficacy against hematological tumors such as acute lymphoblastic leukemia (ALL), B-cell acute lymphoblastic leukemia, adult myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL), non-Hodgkin's lymphoma (NHL), chronic lymphocytic leukemia (CLL), primary mediastinal B-cell lymphoma (PMBCL), mantle cell lymphoma (MCL), and multiple myeloma (MM). However, CAR-T therapeutics have only shown limited efficacy against solid tumors. Due to the distinctive expression pattern of CD24 in tumors and normal tissues, the targetable cancer types demonstrated in the data generated using CD24CAR-T include, but are not limited to, brain tumors, head and neck cancer, sarcoma, lung cancer, gastrointestinal cancer, breast cancer, testicular cancer, prostate cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, or hematological malignancies.

[0109] 7. TCR Therapeutic Composition Similar to CAR-T therapeutics, genetically modified T cell receptor therapeutics (TCRs) are a type of cell treatment in which cancer patients' T cells are genetically modified ex vivo to express modified TCRs, improving the ability of the T cell receptor to recognize and attack specific antigenic cellular antigens when the T cells are infused into the patient. However, unlike CAR-T cells, which recognize proteins expressed on the surface, T cell immunotherapeutics using genetically modified TCRs have been more targeted to solid tumors. TCRs can recognize tumor-specific proteins inside the cell. When tumor-specific proteins are broken down into fragments, they appear on the cell surface along with another protein called the Major Histocompatibility Complex, or MHC. TCRs are designed to recognize tumor-specific protein fragment / MHC combinations. Examples of targets for TCR-modified T cells include those targeting MAGE-A3, such as KITE-718 (Kite Pharma), Wilms tumor antigen 1 (WT-1), such as JTCR016 (Juno Therapeutics), and NY-ESO 1.

[0110] TCR is a heterodimer consisting of two subunits, TCRα and TCRβ. Each subunit contains a constant region adjacent to the T cell membrane and anchors the receptor to the cell membrane, and a hypervariable region that functions in antigen recognition. Thus, scFv fragments derived from any of the anti-CD24 antibodies described herein can be used as the cancer targeting component of recombinant TCR proteins. In particular, the scFv protein can comprise the sequence shown in SEQ ID NO:28.

[0111] 8. Peptide Composition The anti-CD24 antibodies or antigen-binding fragments thereof described herein may bind to glycosylated epitopes that are exposed on cancer cells but not on non-cancerous cells. Specifically, the anti-CD24 antibodies or antigen-binding fragments thereof may bind to a CD24 peptide comprising the amino acid sequence SNSGLAPN (SEQ ID NO: 48). Thus, a peptide comprising the sequence set forth in SEQ ID NO: 48 may be used to neutralize anti-CD24 antibodies that bind to an epitope comprising the core sequence of the sequence set forth in SEQ ID NO: 48. This may be used in anti-drug antibody assays to detect neutralizing antibodies. A peptide comprising the sequence set forth in SEQ ID NO: 48 may be used to inhibit potential adverse effects associated with antibodies that bind to an epitope comprising the core sequence of the sequence set forth in SEQ ID NO: 48. The peptides may be modified for better stability for in vivo use using methods known in the art, including, but not limited to, the use of D-amino acids, O to S substitutions in one or more peptide bonds, and the addition of fusion sequences to improve solubility or half-life (e.g., albumin fusion). In yet another embodiment, a molecule comprising the amino acid sequence set forth in SEQ ID NO: 48 may be used as a vaccine for the treatment and prevention of cancer.

[0112] 9. Treatment The anti-CD24 antibody compositions described herein or cell therapy agents comprising such antibody compositions may be used to treat or prevent cancer or another abnormal proliferative disease. Provided herein is a method of such use in a patient in need thereof, which may comprise administering to the patient an anti-CD24 antibody or antigen-binding fragment thereof, or a pharmaceutical composition comprising said antibody or fragment. Such molecules and pharmaceutical compositions may also be used in the manufacture of a medicament for treating or preventing cancer or another abnormal proliferative disease. As used herein, the term "cancer" refers to a neoplasm or tumor resulting from the abnormal, uncontrolled proliferation of cells. As used herein, cancer expressly includes leukemia and lymphoma. The term refers to a disease involving cells that can metastasize to distant sites. The patient may be a human.

[0113] Cancer or other abnormal proliferative disorders may also include one or more of the following (but are not limited to): carcinomas, including cancer of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid, and skin; including squamous cell carcinoma; hematopoietic malignancies of the lymphoid system, including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic malignancies of the myeloid system, including acute and chronic myeloid leukemia and promyelocytic leukemia. Tumors; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; other tumors, including melanoma, seminoma, teratocarcinoma, neuroblastoma, glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, schwannoma; mesenchymal tumors, including fibrosarcoma, rhabdomyosarcoma, osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma. Cancers caused by abnormalities in apoptosis are also contemplated to be treated by the methods and compositions of the present invention. Such cancers include, but are not limited to, follicular lymphoma, carcinomas with p53 mutations, hormone-dependent tumors of the breast, prostate, and ovary, precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes. In certain embodiments, malignant tumors or proliferative changes (such as metaplasia and dysplasia), or hyperproliferative disorders are treated or prevented by the methods and compositions of the invention in the ovary, bladder, breast, colon, lung, skin, pancreas, or uterus. The cancer may be a sarcoma, melanoma, or leukemia.

[0114] The anti-CD24 antibodies and antigen-binding fragments thereof may be used in conjunction with another anti-tumor therapeutic agent, which may be selected from, but is not limited to, current standard and experimental chemotherapeutic agents, hormonal therapeutic agents, biological therapeutic agents, immunotherapeutic agents, radiation therapeutic agents, or surgery. In some embodiments, the anti-CD24 antibodies and antigen-binding fragments thereof may be administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies, or other agents known to those of skill in the art for the treatment or prevention of cancer, autoimmune disease, infection, or poisoning. Such agents include, for example, any of the biological response modifiers, cytotoxins, antimetabolites, alkylating agents, antibiotics, mitotic inhibitors, or immunotherapeutic agents described above.

[0115] The anti-CD24 antibodies and antigen-binding fragments thereof may be used in conjunction with another anti-tumor immunotherapeutic agent, which may include molecules that interfere with or enhance alternative immunoregulatory pathways (such as TIM3, TIM4, OX40, CD40, GITR, 4-1-BB, B7-H1, PD-1, B7-H3, B7-H4, LIGHT, BTLA, ICOS, CD27, or LAG3) or modulate the activity of effector molecules such as cytokines (e.g., IL-4, IL-7, IL-10, IL-12, IL-15, IL-17, GF-β, IFNg, Flt3, BLys) and chemokines (e.g., CCL21) to enhance the immunomodulatory effect. Certain embodiments include bispecific antibodies comprising an anti-CD24 antibody or antigen-binding fragment thereof described herein in combination with anti-PD-1 (pembrolizumab (Keytruda®) or nivolumab (Opdivo®)), anti-B7-H1 (atezolizumab (Tecentriq®) or durvalumab (Imfinzi)), 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. In yet another embodiment, the anti-CD24 antibody or antigen-binding fragment thereof is administered in combination with molecules that activate different stages or aspects of the immune response to achieve a broader immune response. In a more preferred embodiment, the anti-CD24 antibodies and antigen-binding fragments thereof are combined with anti-PD-1 or anti-4-1BB antibodies without exacerbating autoimmune side effects.

[0116] 10. Production The anti-CD24 antibody or antigen-binding fragment thereof may be produced using a eukaryotic expression system. The expression system may involve expression from a vector in mammalian cells, such as Chinese Hamster Ovary (CHO) cells. The expression system may also be a viral vector, such as a replication-defective retroviral vector, which may be used to infect eukaryotic cells. The anti-CD24 antibody or antigen-binding fragment thereof may be produced from a stable cell line that expresses the antibody from a vector or part of a vector integrated into the cell genome. The stable cell line may express the antibody from an integrated replication-defective retroviral vector. The expression system may be GPEx™.

[0117] The anti-CD24 antibodies and antigen-binding fragments thereof described herein can be purified using chromatographic methods, such as, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. In some embodiments, the fusion protein may be designed to include an additional domain that includes an amino acid sequence that allows the polypeptide to be captured on an affinity matrix. For example, the antibodies described herein that include the Fc region of an immunoglobulin domain may be isolated from cell culture supernatants or cytoplasmic extracts using a protein A or protein G column. Additionally, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ (Kodak) can be used to aid in the purification of the polypeptide. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl group or the amino terminus. Other fusions that may be useful include enzymes that aid in the detection of the polypeptide, such as alkaline phosphatase. Immunoaffinity chromatography may be used to purify the polypeptide.

[0118] vaccine Provided herein is a method of treating cancer or providing prophylaxis of cancer as described herein in a patient. The method may vaccinate the patient against cancer. The method may include administering a composition comprising a sequence as set forth in SEQ ID NO: 48 to a patient in need thereof. The composition may also be administered to a patient in need of treatment for adverse effects associated with a therapy involving the use of an anti-CD24 antibody or a cell expressing a receptor that binds to CD24. The composition may also be used in the manufacture of a medicament for treating cancer or providing prophylaxis of cancer.

[0119] 11. Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any of the above-described anti-CD24 antibodies, cellular therapeutics, or peptide compositions, and a physiologically acceptable carrier or excipient. The pharmaceutical composition may comprise a prophylactically or therapeutically effective amount of an anti-CD24 antibody or antigen-binding fragment thereof, and a pharma- ceutical acceptable carrier.

[0120] In certain embodiments, the term "pharmaceutical acceptable" means approved by a regulatory agency of a federal or state government, or by the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more specifically 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 can 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, aqueous dextrose, and glycerol solutions can also be used as liquid carriers, particularly for injectables. 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, water, ethanol, and the like. The compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0121] Generally, the ingredients of the pharmaceutical compositions can be supplied in unit dosage form, either separately or mixed together, for example as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0122] The pharmaceutical compositions may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include, but are not limited to, those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0123] 12. Method of Administration Methods of administering the compositions and pharmaceutical compositions include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and transmucosal administration (e.g., intranasal and oral routes). In certain embodiments, the compositions are administered intramuscularly, intravenously, or subcutaneously. The compositions may be administered by any convenient route, such as, for example, by infusion or bolus injection, 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 may be systemic or local. EXAMPLES

[0124] The present disclosure has multiple aspects, as illustrated by the following non-limiting examples.

[0125] Example 1 Generation of monoclonal antibodies against underglycosylated CD24 Overexpression of NEU1 and CD24 in tumors suggests dysregulation of glycosidases. Dysregulation of glycosidases suggests that CD24, like MUC1, may be hypoglycosylated in tumors. Binding of the antibody 3B6 to CD24 is hindered by sialic acid glycans (Figure 1). As detected by ELISA, 3B6 binds strongly to N-SA-CD24 and N-SA-O-CD24, but weakly to N-CD24 or fully glycosylated CD24, compared with the commercially available anti-CD24 antibody ML5 (BD biosciences). This suggested that the epitope bound by 3B6 is indeed the protein backbone and that 3B6 binding is hindered by glycosylation of the epitope.

[0126] Fluorescence-activated cell sorting (FACS) and immunofluorescence (IFA) staining results show that 3B6 binds to multiple cancer cell lines, including neuroblastoma and medulloblastoma (Figure 2A-B). 3B6 binds to the neuroblastoma cell lines IMR32, SK-N-SH, SH-SY5Y, SK-N-BE(2), and SK-N-BE(2)C, but not to SK-N-AS (Figure 2A). 3B6 also binds to three out of four medulloblastoma tumors from patients evaluated by IFA staining (Figure 2B). These data suggest that 3B6 can bind to cancerous cell lines and tumors.

[0127] Example 2 affinity maturation The binding affinity of 3B6 to CD24 was considerably lower than that of the commercially available antibodies ML5 (BD Bioscience) and SN3 (Thermo Fisher). To increase the binding affinity and specificity of 3B6 to its antigen, affinity maturation of 3B6 was performed. First, the heavy chain (IgH) and light chain (IgL) of the 3B6 antibody were cloned, and the Ig variable region sequences were identified as follows:

[0128] 3B6 IgH (SEQ ID NO: 1, CDRs are underlined and in bold)

[0129] [ka]

[0130] 3B6 IgL (SEQ ID NO:2, CDRs are underlined and in bold)

[0131] [ka]

[0132] The VH and VL fragments from the parent antibody 3B6 were converted into scFv format and cloned into a phage display vector. The scFvs were monovalently displayed on the phage, allowing the selection of phage clones with higher affinity. To verify the display level of the scFvs, the scFvs were fused with a Flag-6xHis detection tag. Phage ELISA was performed to verify the binding of the parent antibody to the antigen in the phage display format. The binding signal from the phage supernatant was significant, so the project proceeded to library construction.

[0133] Three rounds of selection and screening were performed. The concentrations of antigen CD24-GST and biotinylated CD24-GST were reduced in the screening to select for higher binder clones. 48 clones from each CDR mutagenesis library were selected, cultured, assayed for binding, and sequenced. After confirming the sequences of affinity matured scFv clones, the affinity matured clones' scFvs were reformatted into full-length antibody genes and transiently expressed in mammalian cells. All affinity matured antibodies were produced at small scale in 0.01 liters. The parent antibodies were also scaled up for direct comparison. Antibodies were produced by transfecting the indicated heavy and light chain plasmids (Table 1) into suspension HEK293 cells using chemically defined medium in the absence of serum. Five days after transfection, the conditioned medium was collected and clarified. Total antibodies in the conditioned medium were purified using MabSelect SuRe™ Protein A medium (GE Healthcare).

[0134] [Table 1]

[0135] The purified affinity matured and parental antibodies were evaluated for their affinity to the antigen by competitive ELISA. Antibody PP6226 (parental 3B6 variable region) was coated onto plates at 2 μg / mL. The affinity matured antibodies were first incubated with CD24-GST and then with the plates, followed by incubation with a secondary detection antibody. As shown in Figures 3 to 5, 16 antibodies were generated with varying abilities to compete with the parental clone. The amino acid sequences of the heavy and light chains of these antibodies are SEQ ID NOs: 3 to 10 (heavy chain) and SEQ ID NOs: 11 to 16 (light chain).

[0136] To determine whether affinity-matured clones bind more avidly to CD24 and whether the interaction is glycan-regulated, CD24 was treated with N-glycanase (N-CD24), sialidase NanA (SA-CD24), or both (N-SA-CD24). Sixteen clones described in Figures 3-5 were tested using ELISA. As shown in Figure 6, despite significant affinity for CD24-GST, PP6231 and PP6230 failed to bind to CD24 expressed by mammalian cells, regardless of glycosylation. Meanwhile, most other clones maintained preferential binding to sialidase and / or N-glycanase-treated CD24. However, the relative effects of sialidase and N-glycanase on antibody binding varied considerably between clones, so each clone needs to be tested individually to determine its sensitivity to glycan interference.

[0137] Six clones that bind strongly to SA-N-CD24 but minimally to CD24 were selected and tested for binding to two cancer cell lines, the lung cancer cell line H727 and the neuroblastoma cell line IMR32. As shown in Figure 7, despite the six clones showing similar binding to SA-N-CD24, their binding to cancer cells was significantly different. Importantly, PP6373 shows significantly stronger binding to both cancer cell lines tested. Therefore, this clone is selected for further studies. The heavy chain sequence of PP6373 is shown in SEQ ID NO: 6, and the light chain sequence is shown in SEQ ID NO: 16. Compared to the parental sequence, the heavy chain has three mutations in CDR2 and the light chain has one mutation in CDR3 of the light chain. As shown in Figure 8, these mutations not only increase the binding to SA-N-CD24 by almost 100-fold, but also more tightly control the interaction by desialylation. It is also noteworthy that PP6373 obtained binding capacity to CD24 without deglycosylation at a level 1000-fold lower than that to SA-N-CD24, however, since CHO cells are known to have incomplete glycosylation, this binding may reflect a higher sensitivity of the antibody to detect minor glycoforms in recombinant CD24 prepared from CHO cells.

[0138] Example 3 Antigenic epitopes recognized by 3B6 and PP6373 To determine the antigenic epitopes recognized by 3B6 and the affinity matured clone PP6373, overlapping peptides covering the mature CD24 amino acid sequence (SEQ ID NO: 42) were synthesized and preincubated with the 3B6 antibody before adding 3B6 to plates precoated with NO-CD24 protein (CD24Fc pretreated sequentially with N-glycosidase, NanA, and O-glycosidase). As shown in Figure 9, of the five peptides tested (SEQ ID NOs: 43-47), only peptide 4 (SEQ ID NO: 46) showed significant blocking of the 3B6-CD24 interaction, suggesting that the CD24 binding epitope is contained within this sequence. To confirm that PP6373 recognizes the same epitope, the five peptides were titrated over a wide dose range. As shown in Figure 10, only peptide 4 dose-dependently inhibited the binding of PP6373 to SA-N-CD24.

[0139] To define the minimal PP6373 binding site, peptide 4 was truncated one amino acid at a time and the inhibition of PP6373 binding to SA-N-CD24 was compared. As shown in Figure 11, deletion of three amino acids from the C-terminus abolished the inhibition, whereas deletion of one or two amino acids significantly improved the inhibition (left panel). Furthermore, deletion of amino acids from the N-terminus of peptide 4 also abolished the inhibition (right panel). These data identify SNSGLAPN (SEQ ID NO: 48) as the optimal epitope recognized by PP6373.

[0140] Identification of the antigenic epitope allows the generation of additional antibodies with similar properties. In one embodiment, a synthetic peptide containing the sequence SNSGLAPN (SEQ ID NO: 48) could be used by one skilled in the art to generate new antibodies. The peptide could be conjugated to another immunogenic protein carrier or used in combination with an adjuvant. In another embodiment, the peptide could be used by one skilled in the art to identify other anti-CD24 mAbs that recognize the same epitope to generate cancer-specific antibodies for cancer diagnosis and treatment. In yet another embodiment, the antigenic peptide could be used to neutralize or inhibit potential adverse effects associated with antibodies that bind to epitopes that contain the core sequence of SEQ ID NO: 48. The peptide could be modified to be more stable for in vivo use using methods known in the art, including but not limited to the use of D-amino acids, O to S substitutions in one or more peptide bonds, and the addition of fusion sequences that improve solubility or half-life (e.g., albumin fusion). In yet another embodiment, molecules containing the amino acid sequence of SEQ ID NO: 48 could be used as vaccines for the treatment and prevention of cancer vaccines.

[0141] Example 4 Contrasting expression of antigenic epitopes in normal and malignant tissues To determine whether the epitope recognized by PP6373 is preferentially presented in cancer tissues compared to normal tissues, tissue binding was analyzed by immunofluorescence using biotinylated PP6373. Normal tissue data are summarized in Table 2, and cancer tissue data are summarized in Table 3. In addition, antibody binding to normal, benign and malignant brain tumors has been evaluated. Data are summarized in Table 4.

[0142] [Table 2]

[0143] [Table 3]

[0144] [Table 4]

[0145] As shown in Table 2, PP6373 did not stain normal tissues, except for the pancreas and possibly the spleen. It is notable that most of the staining in the pancreas is seen intracellularly. In the spleen, there were rare numbers of cells that showed staining. In contrast, as shown in Table 3, most cancers tested show strong binding to PP6373. As shown in Table 4, normal CNS tissues are completely devoid of CD24, whereas benign meningiomas show intracellular rather than cell surface staining. Importantly, malignant brain tumors such as astrocytomas, glioblastomas, and oligodendrogliomas showed cell surface staining ranging from 8% to 70%, and in addition, some cancer tissues showed intracellular staining.

[0146] In one embodiment, PP6373 can be used to distinguish malignant brain tumors from normal or benign brain tissue, hi another embodiment, PP6367 can be used to identify cancerous tissue in solid organs such as the liver, lung, breast, and ovary.

[0147] Example 5 PP6373 slows lung cancer growth in vivo To test whether PP6373 can slow tumor growth in vivo, nude mice were subcutaneously injected with human lung cancer cell line H727. When tumors became palpable, tumor-bearing mice were injected twice with 5 mg / kg PP6373 (14 and 21 days after H727 inoculation). As shown in Figure 12, the rate of growth was significantly reduced in tumors treated with PP6373 compared to the IgG control. These data indicate that unmodified PP6373 can exhibit antitumor activity in vivo.

[0148] Our in vitro studies show that PP6367 mediates potent antibody-dependent cellular cytotoxicity, as shown in FIG. 13, consistent with tumor growth retardation in vivo.

[0149] Because ADCC is affected by glycosylation, particularly fucosylation, antibody engineering was used to generate PP6373 lacking core FC fucosylation (d6373). As shown in Figure 14, fucosylation increased the ADCC activity of PP6373.

[0150] The present data indicates that PP6373 can be used for the treatment of cancer. In one embodiment, PP6373 WT IgG1 can be used as a cancer therapeutic antibody administered to cancer patients. In another embodiment, the antibody can be chemically glycoengineered or produced in a cell line lacking fucosyltransferase.

[0151] Example 6 Bispecific antibodies based on PP6373 and OKT3 sequences To weaponize the anti-CD24 antibody, a bispecific antibody that binds both CD24 and CD3 was created. In one embodiment, the anti-CD24 antibody and the anti-CD3 (OKT3) antibody are converted into single chain antibodies with reactivity against CD24 and CD3, respectively, and linked by a flexible linker sequence GGGGSGGGSGGGGS (SEQ ID NO: 49). The sequence of the PP6373 single chain antibody is shown in SEQ ID NO: 17, and the OKT3 single chain sequence is shown in SEQ ID NO: 18.

[0152] In one embodiment, bispecific antibodies are generated by the knob-and-hole technique, where the two partners of the bispecific molecule have complementary mutations in the Fc region, creating knobs and holes to facilitate the formation of bispecific heterodimers. The sequences of the knob-and-hole variants of PP6373 and OKT3 are shown in SEQ ID NOs: 19-22. To evaluate the bispecificity of the various knob-and-hole configurations, an assay was developed that consisted of staining Jurkat cells with the products of co-transfection of the various knob-and-hole products. Briefly, CD3+ Jurkat cells were first stained with tissue culture supernatant of transfected 293T cells. After washing away unbound antibodies, the cells were incubated with biotinylated SA-N-CD24. The amount of SA-N-CD24 on Jurkat cells was detected by PE-streptavidin. As shown in Figure 15, the combination of PP6373 hole and OKT3 knob showed the highest CD24 binding to Jurkat cells, indicating that pairing of PP6373 hole and OKT3 knob is optimal for the knob-and-hole strategy.

[0153] In another embodiment, the bispecific antibody is generated by a tandem repeat of two single-chain binding motifs. Again, the activity of two constructs with different binding motifs in the opposite order, PP6373-OKT3 and OKT3-PP6373, as shown in SEQ ID NOs: 23 and 24, respectively, was compared. As shown in Figure 16, the construct with the PP6373 single chain at the N-terminus (PP6373-OKT3, SEQ ID NO: 23) shows higher bispecific activity.

[0154] To determine whether the bispecific antibody has anti-tumor cell activity, the lung cancer cell line H727 was co-cultured with T cells activated with anti-CD3 and anti-CD28 for 2 days. First, we tested whether cancer cells could specifically induce cytokine production. As shown in Figure 17, important cytokines are induced by the bispecific antibody but not by OKT3-Fc of PP6373-Fc. More importantly, the bispecific antibody does not induce cytokine production unless both T cells and tumor cells coexist. These data indicate that the bispecific antibody causes T cell activation by engaging both T cells and tumor cells.

[0155] Concurrently with the cytokine release assay, cytotoxicity against tumor cells was also assessed based on bead-based counting of viable dye-labeled tumor cells by flow cytometry. As shown in Figure 18, the bispecific antibody induces tumor cell loss only in the presence of T cells.

[0156] In yet another embodiment, the bispecific antibody may be produced by FIT-Ig technology. Briefly, a bispecific antibody comprises a VL 6373 -CL-VH OKT3 -CH1-Fc (SEQ ID NO: 25), VH 6373 -CH1 (SEQ ID NO: 26), and VL OKT3 The bispecific antibody FIT-Ig was formed by co-expression of three constructs each encoding OKT3-CL (SEQ ID NO: 27). As shown in FIG. 19, the FIT-Ig antibody showed good bispecific binding activity to both OKT3 and SA-N-CD24. In addition to binding, it was also found that this bispecific antibody induced a significant cytokine response (FIG. 20) and cytotoxicity against tumor cells (FIG. 21). Furthermore, this bispecific antibody (FIT-Ig) showed high thermal stability compared to the previous bispecific antibodies PP6373-OKT3 and OKT3-PP6373 (FIG. 22).

[0157] Example 7 Use of PP6373 on chimeric antigen receptor (CAR) modified T cells (CAR-T) for cancer treatment The anti-CD24 antibody reacts with a broad spectrum of cancer cells and can be used to generate chimeric antigen receptors to confer anti-cancer activity to T cells. In one embodiment, the PP6373 single chain Fv sequence (SEQ ID NO: 28) or other anti-CD24 mAb single chain (αCD24SC) is inserted into a CAR-T vector known in the art, as shown in Figure 23. The construct is then inserted into a genetic vector known in the art, including those derived from retrovirus, lentivirus, adeno-associated virus, or adenovirus vectors.

[0158] To test the activity of CAR, PBMCs from healthy donors were enriched to obtain T cells using the Pan T Cell Isolation Kit, human (Miltenyl Biotec) (day 0). Human pan T cells were stimulated with anti-CD3 and anti-CD28 for 24 h and cultured with IL-2 for 2 days. Activated T cells were mock treated (control T) or infected with lentivirus carrying CD24-CAR (day 2). To test the antitumor activity of CAR-T, control T cells or CD24 CAR-T cells were co-cultured overnight with CellTraceViolet (Thermo Fisher)-labeled tumor cells. Tumor cell lysis was measured by staining with Fixable Viability DyeeFluor™ 660 (eBioscience) and calculated by the following formula:

[0159] Lysis % = (% dead cells - % autolysed cells) / (1 - % autolysed cells)

[0160] As shown in Figure 24, CD24 CAR-T exhibits potent cytotoxicity against the lung cancer cell line A549 within a wide range of effector to target cell ratios (E / T).

[0161] To test whether CAR-T is activated by cancer cells, 4×104 CAR-T cells or control T cells were incubated with A549 tumor cells overnight and IFNγ was measured in the supernatant. As shown in Figure 25, CAR-T, but not control T cells, produced IFNγ in response to A549 tumor cell stimulation. CD24 is widely expressed across multiple cancer types. As shown in Figure 26, CD24 CAR-T shows broad cytotoxicity against many cancer types, including lung cancer, breast cancer, prostate cancer, cervical cancer, neuroblastoma, and glioma.

[0162] Taken together, the data of the present disclosure indicate that the antibody-based CD24CAR-T of the present disclosure has great potential for cancer treatment, including but not limited to brain cancer, head and neck cancer, sarcoma, lung cancer, gastrointestinal cancer, breast cancer, testicular cancer, prostate cancer, pancreatic cancer, liver cancer, or hematological malignancies.

[0163] Example 8 Humanization of PP6373 for cancer treatment The structure of pdb4PB0 was used as a model structure to construct the PP6373 Fv homology model. Both VH and VL share more than 90% homology with 4PB0. A search of the human Ig database identified the human germline V-region sequence IGHV3-73*01 and J-region sequence IGHJ4*01 as suitable structures and were used as the human acceptor framework for the CDR region of the heavy chain (Onc-1 VH). The human germline V-region IGKV2-29*02 and J-region sequence IGKJ4*01 were applied as the human acceptor framework for the CDR region of the light chain (Onc-1 VL). Four VH and four VL sequences were designed (SEQ ID NOs: 29-36). The new products improve the humanization score from 73% to more than 83% in VH and from 80% to more than 83% in VL. Structural alignment of the PP6373 murine Fv and the humanized version of the Fv of PP6373 (hu-VHv1VLv1, SEQ ID NOs: 29 and 33) showed a high degree of similarity (Figure 27).

[0164] To select the best HuVH and HuVL combination for CD24 binding, the different combinations were co-transfected into 293 cells for 72 hours. Two ELISAs were then performed using expression media. ELISA1: 96-well plates were coated with purified goat anti-human polyclonal IgG (GAH), and after blocking, expression media or purified control IgG was added, and goat anti-human IgG-HRP was used as detection antibody. ELISA2: 96-well plates were coated with CD24-GST protein, and after blocking, expression media or purified control IgG was added, and goat anti-human IgG-HRP was used as detection antibody. If the binding of the chimeric PP6373 antibody is considered 100% in both ELISAs, the different VH and VL combinations showing different degrees of binding will be compared to the chimeric antibody combination and ranked by relative binding (selected leads from prescreening will be compared again after purification). The first round of prescreening data is summarized in Figure 28. The data from this experiment suggested that a) L3 has a high binding capacity per unit protein making it the lead, and b) H1L3 (SEQ ID NOs: 29 and 35), H2L3 (SEQ ID NOs: 30 and 35), H3L3 (SEQ ID NOs: 31 and 35), and H4L3 (SEQ ID NOs: 32 and 35) are the four humanized leads for PP6373.

[0165] To test whether the lead antibodies H2L3 and H3L3 retained the ability to bind to tumor cells, the humanized antibodies were biotinylated along with PP6373. As shown in Figure 29, PP6373 showed better binding to the two human cancer cells tested, while both H2L3 and H3L3 showed low IC 50 shows strong binding in the nM range.

[0166] ADCC assays were performed using PBLs (Figures 30, 31) or NK cells purified from PBLs (Figure 32) as effectors and A549 cells as target cells. Surprisingly, H2L3 and H3L3 bind poorly to tumor cells (Figure 29), but are more potent effectors in ADCC when low antibody concentrations are used (Figure 30). As expected, defucosylated PP6373 (d6373) is more potent in ADCC (Figures 31, 32).

[0167] Taken together, the data of this disclosure demonstrated that the humanized clone of PP6373 exhibits significant binding to human cancer cells and surprisingly strong ADCC activity. In one embodiment, the antibody may be used to treat cancer. In another embodiment, the humanized antibody may be used as a key component of a bispecific antibody. To investigate this activity, two constructs containing H3 and L3 were generated to produce a bispecific antibody based on FIT-Ig technology. The sequences of the humanized FIT-Ig antibody are shown in SEQ ID NOs: 37 and 38 and are used in conjunction with SEQ ID NO: 27. In addition, several mutations were made to optimize the humanized FIT-Ig sequence. They are shown in SEQ ID NOs: 39-41. Specifically, all three sequences contain a signal sequence at the N-terminus for protein purification and synthesis. In SEQ ID NO: 39, a mutation (D to A) was introduced in the Fc region to block ADCC. In SEQ ID NO: 27, one extra R induced by a restriction enzyme site during construction is present between VLOKT3 and CL. In SEQ ID NO:41, the extra R was deleted.

[0168] In yet another embodiment, humanized antibodies may be used as key components of CAR-T for cancer treatment, using methods known in the art.

[0169] Example 9 Anti-CD24 antibodies with glycosylated epitopes do not bind to normal cells that highly express CD24 An important requirement for antibody-based immunotherapeutics is minimal reactivity to normal tissues. Because CD24 is abundantly expressed on hematopoietic cells, especially granulocytes, B cells, some red blood cells, and some monocytes, PP6373 and its two humanized clones, H2L3 and H3L3, were compared with ML5, a conventional anti-CD24 mAb. As shown in Figure 33, ML5 exhibits strong binding to cells that normally express high levels of CD24, whereas H2L3 and H3L3 do not bind to B cells or red blood cells, and barely bind to granulocytes. This result indicates minimal binding to other cell types, such as macrophages and some non-B lymphocytes. Exemplary aspects of the invention are described below. <1> A composition comprising an antibody that binds to an epitope that is glycan-shielded on non-cancerous cells but exposed on cancer cells. <2> the antibody binds to CD24, <1> The composition described in <3> The antibody binds to a peptide comprising the sequence set forth in SEQ ID NO: 48. <1> The composition described in <4> The antibody comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:1 and a light chain variable region comprising the sequence shown in SEQ ID NO:2. <1> The composition described in <5> The antibody comprises a heavy chain variable region comprising any one of the sequences shown in SEQ ID NOs: 3 to 10 and a light chain variable region comprising any one of the sequences shown in SEQ ID NOs: 11 to 16. <1> The composition described in <6> The antibody comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO:6 and a light chain variable region comprising the sequence shown in SEQ ID NO:16. <5> The composition described in <7> The antibody comprises a heavy chain variable region comprising any one of the sequences shown in SEQ ID NOs: 29 to 32 and a light chain variable region comprising any one of the sequences shown in SEQ ID NOs: 33 to 36. <1> The composition described in <8> The antibody comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 30 and a light chain variable region comprising the sequence shown in SEQ ID NO: 35. <7> The composition described in <9> The antibody comprises a heavy chain variable region comprising the sequence shown in SEQ ID NO: 31 and a light chain variable region comprising the sequence shown in SEQ ID NO: 35. <7> The composition described in <10> <1> ~ <9> and a second antibody domain comprising a second antibody or an antigen-binding fragment thereof. <11> the second antibody domain attracts immune effector T cells to the cancer cells for cancer immunotherapy; <10> 2. The bispecific antibody according to claim 1 . <12> the second antibody or antigen-binding fragment thereof binds to CD3; <10> 2. The bispecific antibody according to claim 1 . <13> The sequence shown in SEQ ID NO: 17 and the sequence shown in SEQ ID NO: 18 are included. <10> 2. The bispecific antibody according to claim 1 . <14> Contains any one of the sequences shown in SEQ ID NOs: 23 to 27 and SEQ ID NOs: 37 to 41. <10> 2. The bispecific antibody according to claim 1 . <15> the second antibody or antigen-binding fragment thereof binds to a TCR-α chain, a TCR-β chain, a TCR-γ chain, or a TCR-δ chain; <10> 2. The bispecific antibody according to claim 1 . <16> It has antibody-mediated cell-mediated cytotoxicity (ADCC) activity. <1> ~ <15> 1. The antibody or bispecific antibody according to any one of claims 1 to 9. <17> Designed to have enhanced ADCC activity, <16> The antibody or bispecific antibody described in claim 1. <18> It has antibody-mediated cellular phagocytosis (ADCP) activity. <1> ~ <17> 1. The antibody or bispecific antibody according to any one of claims 1 to 9. <19> Designed to have enhanced ADCP activity, <18> The antibody or bispecific antibody described in claim 1. <20> <4> ~ <9> 2. A chimeric antigen receptor comprising a single chain antibody comprising the composition according to any one of claims 1 to 11. <21> Comprising the sequence shown in SEQ ID NO: 28, <20> The chimeric antigen receptor described in. <22> <1> ~ <21> and a second anti-cancer therapeutic agent. <23> <1> ~ <22> 2. A method of treating cancer in a patient in need of cancer treatment comprising administering to the patient an antibody, bispecific antibody, chimeric antigen receptor or composition according to any one of claims 1 to 11. <24> The cancer is lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, kidney cancer, testicular cancer, prostate cancer or neuroblastoma; <23> The method according to <25> A method for treating an adverse effect associated with a therapy involving an anti-CD24 antibody or cells expressing a receptor that binds to CD24 in a patient in need of such treatment, comprising administering to the patient a composition comprising the sequence set forth in SEQ ID NO:48. <26> 11. A method of treating or preventing cancer in a patient in need thereof comprising administering to said patient a composition comprising SEQ ID NO:48. <27> <1> 2. A method for diagnosing malignant tissue or metastatic lesions comprising the use of an antibody as described in claim 1. <28> <1> 23. A method for identifying circulating cancer cells comprising the use of an antibody as described above. <29> In the manufacture of a medicament for treating cancer, <1> ~ <22> 2. Use of the antibody, bispecific antibody, chimeric antigen receptor or composition according to any one of claims 1 to 11. <30> The cancer is lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, ovarian cancer, renal cancer, testicular cancer, prostate cancer or neuroblastoma; <29> The method according to <31> Use of a composition comprising the sequence shown in SEQ ID NO: 48 in the manufacture of a medicament for treating adverse effects associated with the therapeutic use of an anti-CD24 antibody or a cell expressing a receptor that binds to CD24. <32> Use of a composition comprising SEQ ID NO: 48 in the manufacture of a medicament for treating or preventing cancer.

Claims

1. (a) a heavy chain variable region in the sequence shown in SEQ ID NO:1 and a light chain variable region in the sequence shown in SEQ ID NO:12; (b) a heavy chain variable region in the sequence shown in SEQ ID NO:3 and a light chain variable region in the sequence shown in SEQ ID NO:12; (c) a heavy chain variable region in the sequence set forth in SEQ ID NO: 7 and a light chain variable region in the sequence set forth in SEQ ID NO: 14; (d) a heavy chain variable region in the sequence set forth in SEQ ID NO: 9 and a light chain variable region in the sequence set forth in SEQ ID NO: 15; or (e) a heavy chain variable region in the sequence set forth in SEQ ID NO:9 and a light chain variable region in the sequence set forth in SEQ ID NO:14; An anti-CD24 antibody comprising:

2. A pharmaceutical composition for treating cancer comprising the anti-CD24 antibody of claim 1.

3. The pharmaceutical composition according to claim 2, wherein the cancer is lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer or neuroblastoma.

4. 1. An anti-CD24 single chain antibody comprising, from N-terminus to C-terminus, a heavy chain variable region, a linker, and a light chain variable region, wherein the heavy chain variable region is the heavy chain variable region in the sequence shown in SEQ ID NO:6, the linker comprises the sequence shown in SEQ ID NO:49, and the light chain variable region is the light chain variable region in the sequence shown in SEQ ID NO:

16.

5. 5. The anti-CD24 single chain antibody of claim 4, comprising the sequence shown in SEQ ID NO:

17.

6. A pharmaceutical composition for treating cancer, comprising the anti-CD24 single chain antibody of claim 4 or claim 5. Pharmaceutical composition.

7. The pharmaceutical composition according to claim 6, wherein the cancer is lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer or neuroblastoma.

8. A chimeric antigen receptor (CAR) comprising, from the N-terminus to the C-terminus, an anti-CD24 single chain antibody of claim 4, a hinge region, a transmembrane domain, and an intracellular T cell signaling domain.

9. The CAR of claim 8, wherein the hinge region and the transmembrane domain are derived from human CD28, and the intracellular T cell signaling domain comprises the intracellular domain of human CD28, the intracellular domain of human 4-1BB, and the intracellular domain of human T cell receptor CD3ζ domain.

10. The CAR of claim 9, wherein the anti-CD24 single chain antibody comprises the sequence shown in SEQ ID NO:

28.

11. A CAR-T cell comprising the CAR according to any one of claims 8 to 10.

12. A pharmaceutical composition for treating cancer, comprising the CAR-T cell of claim 11.

13. The pharmaceutical composition of claim 12, wherein the cancer is lung cancer, ovarian cancer, breast cancer, liver cancer, brain cancer, cervical cancer, renal cancer, testicular cancer, prostate cancer or neuroblastoma.

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