Therapeutic CD24 humanized monoclonal antibodies and Anti-tumor translational use

Abstract

The present invention provided an isolated antibody, which specifically binds to human CD24, wherein the isolated antibody comprises: (a) a heavy chain variable region-complementarity determining region 1 (VH-CDR1), a VH-CDR2 and a VH-CDR3, wherein the VH-CDR1 consists of the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 consists of the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 consists of the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable region-complementarity determining region 1 (VL-CDR1), a VL-CDR2 and a VL-CDR3, wherein the VL-CDR1 consists of the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 consists of the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 consists of the amino acid sequence of SEQ ID NO: 12. The present invention also provides a method for treating CD24 expressing cancer.

Description

THERAPEUTIC CD24 HUMANIZED MONOCUONAE ANTIBODIES AND ANTI-TUMOR TRANSLATIONAL USEThis application contains disclosure of nucleotide and / or amino acid sequences which are included in a sequence listing, and the XML file name is 3906-CMU- PCT-SequenceListing, created on January 25, 2024, and the size is 18KB.FIELD OF THE INVENTION

[0001] The present invention relates to an isolated antibody that binds to CD24 and an anti-tumor method thereof.BACKGROUND OF THE INVENTION

[0002] CD24 is a highly glycosylated mucin-type surface membrane protein anchored via glycosylphosphatidylinositol (GPI). CD24 has been identified to be highly expressed in a number of human malignancies, especially in metastatic diseases. One of the cancers with high CD24 expression is breast cancer.

[0003] Breast cancer (BC) is the most frequently diagnosed female cancer in Taiwan, with an estimated 16,000 new cases being diagnosed per year and approximately 2,000 patients are expected to die per year. Worldwide, female breast cancer has been the first leading human cancer since 2020 with an estimated 2,200,000 new diagnoses per year and has become the fifth leading cause of cancer death. There are at least four subtypes of breast cancer (luminal A, luminal B, HER2 positive and basal-like) in terms of gene expression signatures that make individual subtype unique from one another.

[0004] Stratified by molecular expressions, luminal A subtype tumors havehigh expression of estrogen receptor and / or progesterone receptor and no expression of HER2 (ER or PR+, HER2-) and luminal B subtype not only have ER and / or PR expression but also have HER2 expression (ER or PR+ HER2+). HER2 subtype have high expression of HER2 and no expression of ER / PR (HER2+ ER / PR-). Basal-like tumors, on the other hand, do not have ER, PR and HER2 expression (ER-PR-HER2-). This subtype of breast cancer is also called triple-negative breast cancer (TNBC).

[0005] In Taiwan, regardless of molecular subtypes, 5-year survival of early-stage breast cancer patients can be up to 90%, however, a 10-year followup study showed that luminal subtype (A and B) breast cancer patients have better prognosis in 5-year survival than the patients with ER-PR-HER2+ or with TNBC signature. Moreover, patients with TNBC signature have the worst prognosis in 5-year survival. In addition to conventional chemotherapy, hormone receptor-positive (luminal A / B) BC patients usually receive Tamoxifen treatment, one of the first-line endocrine therapy, and could further reduce mortality by 31%. The use of trastuzumab (Herceptin) in HER2 -positive (luminal B and HER2) BC patients could lower the risk of death by around 33%. Despite that, 20-30% of luminal BC tumors are often resistant to Tamoxifen treatment and a significant number of HER2 patients respond poorly to Herceptin.

[0006] In addition, stage IV breast cancer or metastatic breast cancer can spread beyond the breast to other organs of the body. During metastasis, epithelial breast cancer cells undergo epithelial-mesenchymal transition (EMT) to acquire invasive ability to break through the basement membrane of theterminal duct lobular unit and spread to the nearby lymph nodes and ultimately colonize the distant vital organs. The common organs that breast cancer cells spread include bones, lung, liver, and brain. As a result, the spread of breast cancer drastically decreases the survival probability of patients and metastasis is still the major cause of breast cancer-related death. Other cancers such as colorectal cancer, liver cancer, kidney cancer, pancreatic cancer, ovarian cancer and prostate cancer are also susceptible to metastasis.

[0007] Current immunotherapies of breast cancer focus on enhancing cytotoxic T cell’s anti-tumor activity to kill cancer cells using two major approaches: The PD-1 / PD-L1 immune checkpoint blockade; and modification of patient’s T cells with chimeric antigen receptor (CAR) with excellent tumor antigen recognition. The first approach aims to enhance the anti-tumor activity of cytotoxic T cells to effectively eliminate out tumor cells more effectively and the second approach focuses on increasing the tumor targeting efficiency of cytotoxic T cells as well as prolonging the duration of cytotoxic T cell activation. However, the efficacy is often limited, especially in the TNBC patients.

[0008] Presently, there is no effective targeted treatment for TNBC. Accordingly, there exists an urgent need for the development of new effective treatments for TNBC and for other subtypes of breast cancer as well.

[0009] According to the statistics of the present invention, 370 TNBC cases from three medical centers, Taipei Veteran General Hospital, Kaohsiung Medical University Hospital and China Medical University Hospital, were collected. There are over 80% of TNBC express high levels of CD24 expression, furtherindicating the clinical significance of CD24 in TNBC population.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 shows the design, expression, and purification of CD24 murine fragment-crystallizable (mFc) fusion protein for mouse immunization. FIG 1A shows the construction diagram of CD24 mFc fusion protein. (GPI: glycophosphatidylinositol) Figure IB uses Coomassie brilliant blue staining to examine the purification of CD24 mFc fusion protein (1=1 pg purified CD24 3c mFc; 2=blank; 3=10 pg 3c protease hydrolyzed purified CD24 3c mFc; 4=10 pg first flow-through (FT) protein A (poly- A) resin; 5 = 10 pg of second FT poly-A resin; 6 = 1 pg of 3c protease). Figure 1C shows the analysis of differences between anti-mouse Fc antibodies and commercial CD24 antibodies using Western blotting (1 = culture medium; 2 = FT poly-A resin; 3 = 50 ng of purified CD24 mFc). Figure ID shows the ability of P-selectin Fc fusion protein binding to 293 cells expressing recombinant human CD24 mFc by fluorescence- activated cell sortin (FACS). Figure IE shows that P-selectin Fc fusion protein can specifically recognize recombinant CD24 expressed in 293 cells. Figure IF shows an immunoassay using purified recombinant human CD2 mFc.

[0011] Figure 2 shows the screening and validation of the candidate anti- CD24 monoclonal antibody (mAb)-producing hybridoma clones. Figure 2Auses flow cytometry (FC) to analyze the supernatant (SN) of 9 hybridoma clones. Figure 2B detects the supernatants of 9 hybridoma clones by ELISA. Figure 2C analyzes the purity of 9 hybridoma clones mAbs by SDS-PAGE. Figure 2D compares the differences of mean fluorescence intensities (MFI) of 9 mAbs. Figure 2E compares the differences of MFI of 9 mAbs at differentconcentrations.

[0012] Figure 3 shows the single cycle kinetics analysis of the binding affinity of in-house prepared CD24 mAb using surface plasmon resonance (SPR) technique.

[0013] Figure 4 shows the fluorescence microscopy of 9 mAbs.

[0014] Figure 5 shows the effect of anti-CD24 mAb on cell viability of CD24-positive TNBC in an in vitro model.

[0015] Figure 6 shows the epitope mapping of 9 anti-CD24 mAbs using the peptide scanning approach reveals a common antibody binding region within the CD24 mature domain.

[0016] Figure 7 shows the establishment of a CD24-expressing TNBC xenograft mouse model for the characterization of in vivo antitumor activity of 9 anti-CD24 mAbs.

[0017] Figure 8 shows the in vivo antitumor activity of candidate anti- CD24 mAbs. Figure 8A shows an experimental procedure for testing the in vivo anti-tumor potential of candidate CD24 mAbs of 9 clones. Figure 8B shows a volumes comparative analysis of murine tumor after the 9 CD24 mAbs treatment respectively.

[0018] Figure 9 shows the antitumor efficacy of CD24 mAb Hl (Hl) using an MDA-MB-468 xenograft mouse model. Figure 9A shows the procedure of injecting MDA-MB-468 cells and injecting antibodies into severe combined immunodeficiency (SCID) mice. Figure 9B shows the procedure of tumorbearing mice with antibody injection treatment. Figure 9C shows the tumor volume of tumor-bearing mice treated with different concentrations of Hl in thefirst week. Figure 9D shows the tumor volume change of tumor-bearing mice treated with different concentrations of Hl within 200 days.

[0019] Figure 10 shows the in vivo image of tumor-bearing mice with Hl treatment.

[0020] Figure 11 illustrates that Hl administration inhibits distant lung metastasis in a primary tumor resection mouse model. Figure 11A shows the procedure of the test. Figure 11B shows a diagram of the tumor volume change in tumor-bearing mice. Figure 11C shows the tumor volume of tumor-bearing mice at the sixth week. Figure 11D shows the in vivo image and photon counting diagram of tumor-bearing mice. Figure HE uses hematoxylin-eosin (HE) staining to analyze the lung tissue of tumor-bearing mice with Hl treatment. Figure 1 IF shows the survival rate of tumor-bearing mice.

[0021] Figure 12 illustrates the combinatorial treatment of CD24-positive TNBC with Hl and docetaxel exhibits a synergistic anti-tumor effect. Figure 12A shows the procedure of the experimental design. Figure 12B shows the tumor volume change in TNBC in tumor-bearing mice with different injection treatments. Figure 12C shows the survival rate of tumor-bearing mice with different injection treatments.

[0022] Figure 13 shows the biosafety assessment of the lead Hl .

[0023] Figure 14 shows that Hl selectively inhibits CD24-positive TNBC tumor growth. Figure 14A shows the changes in TNBC tumor volume, tumor weight and immunostaining in the IV2 CD24 positive model. Figure 14B shows the changes in TNBC tumor volume, tumor weight and immunostaining in the IV2 CD24 negative model.

[0024] Figure 15 shows the analysis of primary tumor resected from the SCID mice with Hl treatment. Figure 15Auses immunohistochemistry (IHC) to analyze the distribution of different cells in the resected tumor tissue. Figure 15B shows the number of different cells in the resected tumor tissue with Hl treatment.

[0025] Figure 16 shows a schematic of deciphering the fragment antigen binding (Fab) sequences of the lead Hl.

[0026] Figure 17 shows the design, production, purification and antitumor validation of chimeric CD24 mAb Hl (CH-01). Figure 17A shows the design of CH-01. Figure 17B shows the production procedure of CH-01. Figure 17C shows the in vivo image of tumor-bearing mice with CH-01 treatment within 12 weeks. Figure 17D shows the photon counts of tumor-bearing mice with CH-01 treatment within 12 weeks. Figure 17E shows the tumor volume change of tumor-bearing mice with CH-01 treatment. Figure 17F shows the tumor volume change of tumor-bearing mice with different concentrations of CH-01 treatment.

[0027] Figure 18 shows the antibody humanization of the lead Hl . Figure 18A shows the antibody humanization design of lead Hl . Figure 18B shows a molecular overlay graph of L1H2 and original murine Hl . Figure 18C shows a vector containing either a light chain or a heavy chain.

[0028] Figure 19 shows the production and functional validation of the humanized mAb HH-01-46 (HH-01-46). Figure 19A tests the purification of HH-01-46 using Coomassie Brilliant Blue staining. Figure 19B shows the sensitivity and specificity of HH-01-46 and parental CH-01 against CD24- positive MDA-MB-468. Figure 19C determines the binding affinity of HH-01-46 using surface plasmon resonance (SPR). Figure 19D uses FC to analyze the binding intensity of HH-01-46 and the parental CH-01 to a series of TNBC cell lines.

[0029] Figure 20 shows the comparison of anti-tumor activity between HH- 01-46 and CH-01 in a CD24-positive TNBC xenograft mouse model. Figure 20A shows in vivo image of TNBC xenograft mice with HH-01-46 and CH-01 treatment. Figure 20B shows the tumor volume changes in TNBC xenograft mice with HH-01-46 and CH-01 treatment. Figure 20C shows a comparison of tumor tissue and tumor amount in TNBC xenograft mice with HH-01-46 and CH-01 treatment.

[0030] Figure 21 shows the combination CH-01 and docetaxel displaying promising anti-tumor efficacy in a PBMC-humanized ASID mouse model. Figure 21 A shows the experimental procedure. Figure 2 IB shows the tumor volume changes in ASID mice with different treatments.

[0031] Figure 22 shows that HH-01-46 promotes Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity. Figure 22 A shows the image of immunostaining. Figure 22B shows the effect of HH-01-46 on the fold change of actin in different cells. Figure 22C tests HH-01-46 promoting antibody-dependent cell-mediated cytotoxicity using antibody-dependent cell- mediated cytotoxicity (ADCC) assay.

[0032] Figure 23 uses the pretreatment of TNBC cells with HH-01-46 and CH-01 to analyze the oncogenic signal of Akt / Erk.

[0033] Figure 24 shows that HH-01-46 therapy overcomes Herceptin resistance in a BT474 xenograft mouse model. Figure 24A shows the schematicof selecting Herceptin resistant BT474 cells. Figure 24B shows the evaluation of Herceptin resistance in BT-474-HR subline using MTS assay. Figure 24C shows the procedure of the test. Figure 24D shows the tumor growth kinetics of BT474-HR-bearing mice receiving IgG and HH-01-46 therapy, respectively.DETAILED DESCRIPTION OF THE INVENTION

[0034] As used herein, “a,” “an,” “the,” “at least one,” and “one or more” are used interchangeably.

[0035] In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0036] The term “CD24” includes any variants or isoforms which are naturally expressed by cells or are expressed by cells transfected with the CD24 gene.

[0037] The term “antibody” as referred here includes intact antibodies and any antigen binding fragment or single chain thereof. Each heavy chain comprised a heavy chain variable region (VH) and a heavy chain constant region. Each light chain comprised a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR). Both the VH and VL regions consists of three CDRs, arranged from amino-terminus to carboxy-terminus in the following order: CDR1, CDR2 and CDR3. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0038] The present invention provided an isolated antibody, whichspecifically binds to human CD24, wherein the isolated antibody comprises: (a) a heavy chain variable region-complementarity determining region 1 (VH- CDR1), a VH-CDR2 and a VH-CDR3, wherein the VH-CDR1 consists of the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 consists of the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 consists of the amino acid sequence of SEQ ID NO: 6; and (b) a light chain variable regioncomplementarity determining region 1 (VL-CDR1), a VL-CDR2 and a VL- CDR3, wherein the VL-CDR1 consists of the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 consists of the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 consists of the amino acid sequence of SEQ ID NO: 12.

[0039] The present invention also provided an isolated antibody, wherein the VH consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, and wherein the VH-CDR1 consists of the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 consists of the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 consists of the amino acid sequence of SEQ ID NO: 6.

[0040] The present invention also provided an isolated antibody, wherein the VL consists of the amino acid sequence of SEQ ID NO: 16, and wherein the VL-CDR1 consists of the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 consists of the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 consists of the amino acid sequence of SEQ ID NO: 12.

[0041] In one embodiment, the isolated antibody is encoded by a heavy chain nucleic acid and a light chain nucleic acid, and the heavy chain nucleic acid and the light chain nucleic acid are the nucleic acid sequence of SEQ ID NO: 13 and SEQ ID NO: 15, respectively.

[0042] In another embodiment, the isolated antibody is encoded by heavy chain nucleic acid and light chain nucleic acid, and the heavy chain nucleic acid and light chain nucleic acid are the nucleic acid sequence of SEQ ID NO: 17 andSEQ ID NO: 15, respectively.

[0043] The present invention provides an isolated antibody, wherein the isolated antibody is an isolated antibody of human CD24.

[0044] The present invention provides an isolated antibody, which further comprises a Fc region.

[0045] In one embodiment, the Fc region is an IgG, IgM, IgA, IgD, IgE antibody, or any subclass thereof. In a preferred embodiment, the Fc region is an IgG.

[0046] The present invention also provides a pharmaceutical composition comprises the isolated antibody of the present invention and a pharmaceutical acceptable carrier or excipient.

[0047] The present invention further provided a method of treating CD24 expressing cancer in a subject comprising administering to the subject with CD24 cancer the isolated antibody of the present invention.

[0048] In one embodiment, which further comprises administering one or more additional anti-cancer therapy or anti-cancer drugs.

[0049] In one embodiment, the isolated antibody could be co-administered simultaneously with an anticancer agent, docetaxel, tamoxifen, Herceptin, or other breast cancer drug, and combination thereof. The anticancer agent herein comprises a chemotherapeutic agent, a radiotherapeutic agent or an immunotherapeutic agent. The chemotherapeutic agents herein include,doxorubicin HCL, bleomycin, mitomycin, etoposide, vinblastine, vincristine, vinorelbin, paclitaxel, docetaxel, irinotecan, topotecan, hydroxyurea, cyclophosphamide, melphalan, chlorambucil, carmustine, carboplatin, cisplatin, fluorouracil, gemcitabine, capecitabine, imatinib and goserelin acetate, but not always limited thereto.

[0050] In a preferred embodiment, the one or more additional anti-cancer therapy or anti-cancer drugs are chemo therapy, chemo drug, tamoxifen, Herceptin or combination thereof.

[0051] In one embodiment, the CD24 expressing cancer comprises breast cancer, liver cancer, or ovarian cancer. In a preferred embodiment, the breast cancer referred to a triple-negative type breast cancer.

[0052] In another embodiment, the triple-negative type breast cancer is a lung metastasis triple-negative breast cancer.

[0053] The present invention also provided a method for detecting expression of CD24 in an in vitro sample comprising: (a) providing an in vitro sample from a subject; (b) contacting the in vitro sample with a capture antibody that specifically binds to the CD24, wherein the capture antibody is the isolated antibody of the present invention; (c) adding a labeled detection antibody that binds to the capture antibody to form an immune complex consisting of the CD24, the capture antibody and the labeled detection antibody; and (d) measuring an amount of the immune complex to determine the presence or level of the CD24 in the in vitro sample.

[0054] In one embodiment, the in vitro sample is selected from at least one of blood, lymphatic fluid, tissue fluid, body cavity fluid, oral mucosal fluid,circulating tumor cell or combination thereof.

[0055] In a preferred embodiment, the in vitro sample is blood. And in a more preferred embodiment, the blood is whole blood, plasma or serum.

[0056] The present invention also provides a use of a composition for preparing a drug for treating CD24 expressing cancer, wherein the composition includes the isolated antibody of the present invention and an anti-cancer drug. This use is due in part to their unique specificities, such as epitope specificity, affinity, structure, and functional activity.

[0057] In a preferred embodiment, the anti-cancer comprises docetaxel, tamoxifen, Herceptin or combination thereof.

[0058] In one embodiment, the cancer is a solid tumor.

[0059] In one embodiment, the cancer is selected from breast cancer, liver cancer, or ovarian cancer. In a preferred embodiment, the cancer is a triplenegative type breast cancer. In a more preferred embodiment, the triple-negative type breast cancer is a lung metastasis triple-negative breast cancer.

[0060] On the other hand, the isolated antibody of the present invention can be co-administered with one or more therapeutic agents or a combination thereof, for example, together to form a pharmaceutical composition or administered separately. Such therapeutic agents may be one or more additional anticancer drugs, chemotherapeutic drugs, radiopharmaceuticals, immune checkpoint inhibitors, or combinations thereof.

[0061] On the other hand, the present invention provides a pharmaceutical composition, which includes an isolated antibody of the present invention and a pharmaceutically acceptable carrier and excipient. The pharmaceuticalcomposition may also include one or more therapeutic agents or a combination thereof, such as the anti-cancer drugs disclosed above.

[0062] On the other hand, the present invention also provides a kit for detecting CD24 antigen or cells expressing CD24 in in vitro samples, which includes the isolated antibody of the present invention and instructions for use.EXAMPLE

[0063] The following examples are non-limited and are merely representative of various aspects and features of the present invention

[0064] EXAMPLE 1 : construction of human CD24 murine fragment- crystallizable (mFc) fusion protein

[0065] The mature human CD24 consists of only 35 amino acid residues that are heavily glycosylated and CD24 is attached to the surface of plasma membrane by a glycosyl phosphatidylinositol (GPI) anchor. AFc fusion protein consisting of IL2 signal peptide, mature domain of CD24 and murine Fc were constructed as shown in the following molecular design. The Fc stands for the constant fragment of mouse immunoglobulin G (IgG).

[0066] The molecular design of CD24-Fc fusion protein: (IL2 signal peptide)-SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG-(ASTGS)-Fc (namely, efficient leader peptide-the mature domain of CD24-linker peptide-Fc).

[0067] As shown in Figure 1A-E, a recombinant human CD24 murine Fc (mFc) fusion protein that contains IL2 signal peptide sequences was designed. The unique mature domain which shared poor similarity between Homo sapiens and murine species, and the linker sequence containing 3c protease cleavage site following glycosyl phosphatidylinositol (GPI) sequences (Figure 1A).

[0068] EXAMPLE 2: purification of the mFc fusion protein

[0069] The CD24-Fc expression construct was transiently introduced into Expi293F expression system using HEK293 cells. The culture medium containing CD24 mFc fusion protein was collected and subjected to protein A resin purification protocol. The purified CD24 mFc was further confirmed by SDS-PAGE coupled with Coomassie blue staining and western blotting. Milligram levels of CD24 mFc fusion protein was purified from the above procedures.

[0070] The resulting human CD24 mFc construct was expressed in Expi293 mammalian cell expression system and human CD24 mFc fusion protein was purified from the culture medium using protein A resin followed by 3c protease digestion (Figure IB). The resulting digested and undigested CD24 mFc protein were validated by SDS-PAGE coupled with Coomassie blue staining (Figure IB) and CD24 mFc was also analyzed by western blotting using anti-mouse Fc antibody and commercial anti-CD24 antibody, respectively (Figure 1C).

[0071] The bioactivity of CD24 mFc fusion protein using its naturally- occurring ligand, P-selectin, by ELISA and FACS assay was tested.

[0072] P-selectin expressed on activated platelets and endothelial cells is the well-known ligand for CD24. First, 0.1, 0.5, 1.5 ug / mL of P-selectin were coated on the 96-well for the ELISA assay. As shown in Figure ID, the binding between CD24 mFc fusion protein and P selectin was increased in a dosedependent manner. Further, P-selectin Fc fusion protein was confirmed for its ability to bind to the recombinant human CD24 protein-expressing 293 cells viaFACS analysis. To do so, CD24 expression construct without mFc attached to its N-terminus was generated and introduced this new construct into 293 T cells to express the membrane-bound CD24. As show in Figure IE, P-selectin Fc fusion protein could specifically recognize the recombinant CD24 expressed on 293 cells.

[0073] EXAMPLE 3: immunization of mice

[0074] Purified CD24 mFc fusion protein was used for immunization of mice. In brief, mice were primed by intraperitoneal injection with 250 ug of the purified recombinant CD24 mFc fusion protein. The pre-immune blood samples were collected from 5 mice (e.g., female BALB / c; 4 to 6 weeks old). For the immunization, the appropriate volume of PBS containing 25 to 100 pg CD24 mFc antigen per mouse was administered. The emulsion containing 100 ug CD24 mFc was injected into mice intraperitoneally using a 22-gauge needle. The potential toxicity was monitored after immunization. Mice were further immunized twice with 50 pg of CD24 mFc every 2 weeks after first injection for a total of 3 injections. After 2 to 3 weeks, blood samples were collected from the immunized mice to measure antibody titer using ELISA or FACS analysis.

[0075] The purified recombinant human CD24 fusion protein was used for the immunization of mice. The pre-immune blood samples were collected from 5 mice (female BALB / c; 4- to 6-weeks old) prior to the immunization. The mice were firstly immunized via intraperitoneal injection with 250 ug of the purified CD24 mFc fusion protein followed by two subsequent injections (50 ug) every 2 weeks for a total of 3 injections. The potential toxicity was monitored after immunization. Three weeks after third injection of CD24-Fc, the blood sampleswere collected from 5 mice to measure antibody activity using western blotting or FACS analysis. As show in Figure IF, the immunization of the mice was successful in that each mouse was able to produce antibody against human CD24 using flow cytometry.

[0076] EXAMPLE 4: Evaluation of immunization efficacy by flow cytometry analysis

[0077] CD24-positive cells were incubated with 5 mM EDTA for 10 minutes to detach cells from the petri dish. Cells were then washed with 5 ml PBS twice to remove EDTA. 5 x 105cells were incubated with mouse serum at 1 / 20 dilution at 4°C for 40 min followed by treatment with goat anti-mouse conjugated with PE. The samples were shielded from light before subjecting to Flow cytometry analysis using Caliber 2. Calibur CellQuest Pro (BD) software was applied for data acquisition and analysis.

[0078] EXAMPLE 5: Establishment of hybridoma

[0079] The immunized mouse with the best humoral response was chosen and sacrificed to isolate B cells from the spleen for the subsequent fusion protocol. The fusion between the myeloma cell lines Sp2 / 0-Agl4 and B cells was performed using polyethylene glycol (PEG) followed by aminopterin selection to eliminate the unfused myeloma cells. PEG acts as a fusing agent that fuses plasma membranes of adjacent myeloma and / or antibody-secreting cells, producing a single cell with two or more nuclei. The unfused myeloma cells were eliminated by aminopterin due to the lack of a salvage pathway. Aminopetrin is used to block the de novo nucleotide pathway of cells and forces them to go by salvage pathway to synthesize nucleotides, which myeloma cellsdo not have. B cells that have a functional salvage pathway do not live long in culture and eventually die after two weeks. Therefore, only the hybrids can survive the drug selection and the resulting colonies of hybrids were subjected to initial screening for the antibody production capability.

[0080] Mouse B cells were isolated from the spleen and fused with mouse myeloma cell line using polyethylene glycol (PEG). The unfused cells were eliminated by culturing in HAT selective medium. A total of nine positive hybridoma clones (CD24 mAb H1-H9, H1-H9) were first isolated for further analysis. The resulting nine hybridoma clones were validated for their ability to secret anti-CD24 mAbs.

[0081] EXAMPLE 6: hybridoma screening

[0082] One week after the fusion and drug selection, the supernatant from colonies of hybrid cells were examined for the presence of anti-CD24 mAb using ELISA assay where CD24-mFc served as the antigen and an unrelated Fc fusion protein served as the negative control antigen. The supernatant from colonies that only bind to the CD24-mFc antigen were considered potential positive colonies and those that bind to both CD24-mFc and the negative control antigen were considered false positive colonies. Following the initial screening, the positive colonies that secret substantial titer of anti-CD24 mAb were chosen and expanded to 24-well plates. The resulting positive clones were separated by limiting dilution and the second-round screening of hybridoma grown from a single cell was performed to obtain the hybridoma clones that have anti-CD24 mAb producing ability. The resulting hybridoma clones were used in the production of anti-CD24 mAb.

[0083] As shown in Figure 2A of the flow cytometry analysis of the supernatant (SN) of nine hybridoma clones, all of them were able to secret the anti-CD24 mAbs that specifically binds to the surface CD24 of TNBC cell lines.

[0084] As shown in Figure 2B, ELISA assay also showed that nine hybridoma SNs could specifically bind CD24 antigen.

[0085] EXAMPLE 7: production of anti-CD24 mAbs by mouse ascites method

[0086] In order to obtain a sufficient amount of mAbs to carry on the subsequent functionality evaluation of anti-CD24 mAbs. In vivo monoclonal antibody production, also known as the mouse ascites method was carried out.

[0087] One week prior to inoculation with anti-CD24 mAb-producing hybridoma cells, BALB / c mice were first primed by intraperitoneal injection of 1ml Pristane. The anti-CD24 mAb -producing hybridomas were grown in a 175 cm2culture flask in complete DMEM-10 / HEPES / pyruvate at 37 °C. Cells were harvested and centrifuged and the supernatant was removed. Cells were washed and re-suspended at 2.5 xlO6cells / ml in PBS. BALB / c mice were intraperitoneally injected with 2 ml of the hybridoma cells. After two weeks, mouse ascites was harvested by inserting the 19G needle into the mouse abdominal cavity to drain the ascites fluid that contains a high concentration of anti-CD24 mAb.

[0088] Each hybridoma was i.p. injected into the mouse and the ascitic fluid was harvested and purified three weeks post injection. The harvested ascitic fluid was filtered and purified by Protein A-Sepharose Fast Flow (PASFF) and mAbs were later eluted with a pH 3.0 citric acid buffer. The finalbuffer was exchanged to a pH 7.0 PBS solution. The purified CD24 mAbs from each hybridoma were resolved in SDS-PAGE to show its purity (Figure 2C). The CD24 mAbs purity of the nine hybridoma clones in Figure 2C is shown in Table 1.

[0089] Table 1. The purity of CD24 mAbs in nine hybridoma clones

[0090] EXAMPLE 8: purification of anti-CD24 mAb from mouse ascitic fluid

[0091] Anti-CD24 mAb was purified from the mouse ascites using a protein A-based capture step followed by additional chromatography steps. Briefly, ascites was applied to the protein A column followed by PBS wash. The elution buffer was then applied to the protein A column used for CD24 Mab purification. The purified mAbs were passed through the 0.22 pm filter to remove potential contaminants.

[0092] EXAMPLE 9: Evaluation of sensitivity of Hl -01 mAb (Hl) by flow cytometry

[0093] The sensitivity of nine purified mAbs was investigated. 1 x 105MDA-MB-468 cells were incubated with individual mAb at a concentration of 50 pg / ml for 30 min followed by a 30 min treatment of mouse secondary antibody conjugated with Alexa 488. As shown in Figure 2D, Hl mAb had the highest mean fluorescence intensities (MFIs) among the nine mAbs.

[0094] The sensitivity of each mAb was further assessed by serial dilutionfollowed by flow cytometry analysis of the mAb. 1 x 105MDA-MB-468 cells were incubated with a series of mAb concentrations ranging from 50 pg / ml, 0.5 pg / ml to 5ng / mL for 30 min followed by 30 min treatment of Alexa 488 conjugated anti-mouse secondary antibody. As shown in Figure 2E, all mAbs, at a concentration of 5 ng / ml was sufficient to give a substantial MFI, indicating their high sensitivity is binding to CD24.

[0095] The purified Hl -01 mAb was labeled with Cy5 fluorescence dye using Cy5® conjugation kit (Abeam, CA, U.S.A.) according to the instruction of manufacturer.

[0096] EXAMPLE 10: in vivo imaging system (IVIS) imaging

[0097] Mice were intravenously injected with 100 ul of Cy5-labeled Hl -01 mAb and fluorescence signals were recorded on day 1, 3 and 7 post injection. Mice were transferred to the imaging chamber of IVIS Spectrum to analyze the emitting fluorescence signals at a wavelength of 647 nm (Perkin Elmer Inc., MA, USA). The fluorescence was measured and analyzed using Living Image Software 4.0 (Perkin Elmer Inc., MA, USA).

[0098] EXAMPLE 11 : surface plasmon resonance (SPR) analysis

[0099] The binding affinity of each clone of mAb was determined by Surface Plasmon Resonance (SPR). BiacoreTM 8K, a high-throughput and high- sensitivity SPR system was used. First, 10 nM recombinant CD24 mFc protein was dissolved in NaOAc pH 5.0 and immobilized on the metal surface of the sensor chip by amine coupling prior to the measurement. Single cycle kinetic program was carried out using five different concentrations of CD24 mAbs and the dissociation constant Kd and association constant Ka were measured. Thebinding affinity (KD) of CD24 mAbs was determined by the ratio of Kd and Ka. We have determined the binding affinity of eight CD24 mAbs to have a great binding affinity with KD values ranging from 10-9 to 10-13 M. Among them, H6, H7 and H8 gave much higher binding affinity than Hl, H3, H4, H5 and H9 (Figure 3). Taken together, all 8 clones of the anti-CD24 mAbs have great binding affinity toward CD24.

[0100] The series CM5 sensor chip was first docked into BiacoreTM 8K (GE Healthcare Life Sciences, NY, U.S.A.) with 0.05% P20 in PBS as the running buffer. 10 nM CD24 mFc (ligand) in NaOAc solution (pH 5.0) was immobilized onto the CM5 sensor chip by amine coupling method for 30 seconds with a flow rate of lOuL / min flow rate. Single cycle kinetic (SCK) program was carried out using five concentrations of anti-CD24 mAbs (7.5, 15, 30, 60, 120 nM) as the analyte. The flow rate was 50 ul / min. Eight purified anti- CD24 mAbs were examined for the binding affinity toward the ligand CD24 mFc. The association constant ka and the dissociation constant kd were fitted using 1 to 1 binding model and the binding constant KD is calculated by the equation shown below:

[0102] EXAMPLE 12: fluorescence activated cell sorting (FACS) analysis

[0103] CD24-positive cells were incubated with 5 mM EDTA for 10 minutes to detach cells from the petri dish. Cells were then washed with 5 ml PBS twice to remove EDTA. 5 x 105cells were incubated with either mouse serum, the SN of hybridoma or purified CD24 mAb at 1 / 20 dilution at 4°C for 40 min followed by treatment with goat anti-mouse conjugated with PE. Thesamples were shielded from light before subjecting to FACS analysis using Caliber 2. Calibur CellQuest Pro (BD) software was applied for data acquisition and analysis.

[0104] EXAMPLE 13: MTS assays

[0105] 5000 cells were seeded in the 96-well plate and incubated with a range of concentration of CD24 MAb (20, 10, 5, 2.5, 1.25, 0.625, 0.312 ug / ml) and the cell viability was measured after three days of culture using CellTiter kit (Promega, CA, U.S.A.) according to manufacturer’s instruction.

[0106] EXAMPLE 14: Antibody-mediated receptor internalization assay

[0107] The ability of a monoclonal antibody to induce mAb / target complex internalization upon mAb binding to the surface target of tumor cells is one of the hallmarks for antibody therapeutic potential. The ability of CD24 mAbs to trigger surface CD24 internalization was tested through receptor internalization assay. MDA-MB-468 cells were stained with nine mAbs individually for 30 min at 4°C followed by staining with Alexa 488-conjugated anti-mouse Ab for 30 min. Membrane localization of CD24 was observed by fluorescent microscopy at 4 °C and CD24 internalization was measured 24 hours after 37 °C incubation of the cells.

[0108] As shown in Figure 4, all mAbs were capable of triggering CD24 internalization. Moreover, Hl and H9 gave the better intracellular intensity of the internalized CD24 staining.

[0109] As shown in Figure 5, all CD24 mAbs did not affect TNBC cell growth in the in vitro model.

[0110] EXAMPLE 15 : epitope mapping

[0111] A scanning peptide library was designed and constructed according to the CD24 amino acid sequence. Overlapping peptides are 12-mers offset by two residues and each peptide were then biotinylated for the subsequent ELISA assay.

[0112] As shown in Figure 6, four overlapping peptides which share the same amino acid sequence NSGLAP were found to be responsive to all nine CD24 mAbs, suggesting that all nine antibody clones react to the same CD24 epitope amino acid sequence including NSGLAP.

[0113] EXAMPLE 16: construction of hCD24-expressing lentivirus vector and preparation of lentivirus particles

[0114] A fusion gene containing the coding sequences of mouse interlulin- 2 (mIL-2) signaling peptide in the 5 prime regions followed by the sequence coding for the mature human CD24 peptide was designed. The mIL2_hCD24GPI fusion gene was synthesized by MDBio, Inc. and cloned into the lentiviral vector pLVX using specific restriction enzymes. The resulting pLVX-mlL2_hCD24GPI lentiviral plasmid was amplified using plasmid DNA Maxiprep kit.

[0115] Lentivirus particles were prepared using the following protocol provided by the RNAi Core at Academia Sinica, Taipei, Taiwan. Briefly, 2.0 x 106293T cells were seeded on 10-cm culture dish 18 hours prior to transfection. The package plasmid of VSV-G-pseudotyped lentivirus (pCMVDR 8.91 and pMD.G) along with pLVX-mlL2_hCD24GPI lentiviral plasmid were mixed with Minis TransIT at a predefined ratio in OPTI-MEM transfection medium. The transfectant was added in 10-cm culture dish and the cells were transfectedfor 8 hours. The transfectant was removed after 8 hours and 10 ml 1% BSA completed medium was added to the culture dish and the transfected cells were incubated for 24 hours. The supernatant was harvested after 24 hours and stored at 4°C and another 10 ml 1% BSA completed medium was added to the culture dish and the cells were cultured for another 24 hours. The new supernatant was then harvested and combined with the previously harvested supernatant. The final 20 ml supernatant was centrifuged at 1,500 rpm for 5 minutes to remove cell debris. The resulting lentiviral supernatant was further concentrated by mixing it with Lenti-X Concentrator solution and the mixture was incubated overnight at 4 °C to fully precipitate the viral particles. The mixture was centrifuged at 2,000 g for 30 minutes at 4°C to remove the supernatant. The concentrated lentivirus particles were re-suspended in 1ml complete medium and stored at -80°C.

[0116] Full human CD24 coding sequence containing the mature domain and GPI anchor domain was constructed into the pLVX viral vector. pLVX lentiviral particles were prepared from 293T cells. Next, MDA-MB -231 -derived IV2 cells were infected with the virus particles for 48 hours followed by drug selection in culture medium containing 400 pg / ml G418. Two weeks after drug selection, cells were analyzed for surface CD24 expression using flow cytometry.

[0117] As shown in Figure 7, after drug selection, 89.4% of IV2 cells were shown CD24 positive. Then, the CD24-positive population was further sorted to obtain the cell population with over 99% CD24 positive.

[0118] EXAMPLE 17: evaluation of in vivo antitumor efficacy of theCD24 mAb using xenograft SCID mouse model

[0119] For MDA-MB-468 xenograft model, 2.5 x 106cells mixed with matrigel were injected into the 4th mammary fat pad of mice three days prior to CD24 mAb monotherapy. Given that MDA-MB-468 cells only produced slow- growing tumors and in order to evaluate the long-term effect of CD24 mAb administration, the treatment protocol was designed as follows. Treatment 1: Tumor-bearing mice received CD24 mAb monotherapy was carried out by i.v. injection of CD24 mAb twice a week at a dosage of 5 mg / Kg or 10 mg / Kg for a total of 10 injections. Treatment 2: Repeat the treatment 1 cycle followed by a two-week pause. Treatment 3: Tumor-bearing mice were i.v. injected with CD24 mAb once a week at a dosage of 5 mg / kg or 10 mg / kg for a total of 7 injections. Tumor growth was recorded every three days and mice were sacrificed at a humane endpoint.

[0120] For IV2-CD24 xenograft model, 1 x 106cells mixed with matrigel were injected into the 4th fat pad of mice three days prior to CD24 mAb monotherapy. CD24 mAb was administered via i.v. injection once a week at a dosage of 5 mg / kg for a total of eight injections over two months. Mice were sacrificed at a humane endpoint and xenograft tumors were removed and fixed in a formalin solution for further examinations.

[0121] As shown in Figure 8 A, nine clones of candidate mAbs were tested for their in vivo antitumor potential. 1 x 106IV2-CD24 cells were injected into mammary fat pads of SCID mice. After three days, the tumor-bearing mice then received mAb treatment through tail vein injection at a dosage of lOmg / kg once a week for a total of eight injections over the course of eight weeks.

[0122] In the first round of the experiment, Hl, H2, H3, H4 and H9 were evaluated, and therefore the tumor sizes of those mAb group were compared to that of the same control group. In the second round of the experiment, the tumor sizes of the second control group were used to compared with the tumor sizes of H5, H6, H7 and H8 group. As shown in Figure 8B, Hl and H9 mAbs showed the most promising antitumor efficacy and H6 displayed moderate antitumor efficacy. While H2, H3, H4, H5, H7 and H8 did not show significant antitumor potential despite their high binding affinity toward CD24.

[0123] EXAMPLE 18: evaluation of in vivo antitumor efficacy of CD24 mAb using xenograft SCID mouse model

[0124] For the primary tumor resection model, 1 x 106luciferase-tagged IV2-CD24 (IV2-CD24-luc) cells mixed with matrigel were injected into the 4th mammary fat pad of mice and tumors were surgically removed when reached about 1 cm in diameter in week six. One week after primary tumor removal, CD24 mAb monotherapy was carried out by i.v. injection of mice with PBS or CD24 mAb at a dosage of 10 mg / kg once a week for a total of six injections. The status of lung metastasis in mice was detected by IVIS. Briefly, mice were i.v. injected with 0.2 ml 5 mg / ml luciferin followed by isoflurane gas anesthesia. Mice were placed in the IVIS detection chamber to detect the luminescent signal coming from the mouse lung. The photon signals were calculated using living image software.

[0125] The antitumor efficacy of Hl in a different TNBC xenograft model using MDA-MB-468 cells was evaluated in Figure 9. 2.5 x 106MDA-MB-468 cells were injected into the mammary fat pads of SCID mice and the tumor-bearing mice was received three rounds of mAb treatment using two different dosages, 5 mg / kg and 10 mg / kg, respectively. (Figure 9A and Figure 9B).

[0126] As shown in Figure 9C, the tumor-bearing mice among three groups had similar tumor size on week one.

[0127] As shown in Figure 9D, administration of Hl at a dosage of 5 mg / kg and 10 mg / kg could both effectively inhibit the primary tumor growth in the MDA-MB-468 xenograft mouse model.

[0128] Taken together, the antitumor activity of the tested mAbs was shown. Hl exhibited the best potential and was chosen as the lead antibody.

[0129] Then, the following experiment was to confirm the targeting effect on the tumor site to cause the tumor regression of the selected CD24 mAb.

[0130] 1 mg of Hl mAb was covalently conjugated to Cy5 fluorescence dye using an amine coupling kit. 1 x 106MDA-MB-468 cells were orthotopically transplanted to SCID mice to establish xenograft tumors. Two weeks after tumor inoculation, 100 pg Cy5-labeled Hl was then injected into the tumor-bearing mice via tail vein. Two days later, mice were anesthetized with isoflurane and placed in the IVIS chamber supplied with oxygen, and the fluorescence signal was measured using IVIS imaging system.

[0131] As shown in Figure 10, the Cy 5 -labeled Hl was detected in the tumor site in IVIS image, indicating its ability on tumor targeting.

[0132] EXAMPLE 19: distant lung metastasis in a primary tumor resection mouse model

[0133] As shown in Figure 11, 1 x 106IV2-CD24 cells were injected into4th mammary fat pad of SCID mice and the primary tumors were resected whenthey reached 1 cm in diameter. As shown in Figure 11B and Figure 11C, tumor growth curve showed that eight tumor-bearing mice carried similar tumor load. After primary tumor resection, mice were randomized into two groups (n=4 per group). One week after primary tumor removal, mice were injected with lOmg / kg Hl via tail vein once every week for a total of four injections. The lung metastasis status was monitored on week 11 after forth mAb injection using IVIS.

[0134] As shown in Figure 11D, mice that received mAb injection showed reduced lung metastasis compared with the control mice. And also, as shown in Figure HE, mice treated with mAb had no observable metastasis in the HE staining of mouse lung tissue. Three mice received mAb injection at the end of the experiment all survived (Figure 1 IF).

[0135] EXAMPLE 20: cell culture

[0136] MDA-MB-468 and IV2 cells were maintained in the complete DMEM medium containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1% penicillin / streptomycin. CD24-expressing stable cell lines were maintained in the complete medium containing 400 pg / ml G418. GFP-Luciferase- expressing stable cell lines were cultured in 5pg / ml puromycin. All cell lines were maintained in the 37°C humidified incubator supplied with 5% CO2.

[0137] EXAMPLE 21: anti-CD24 mAb and docetaxel Combination therapy

[0138] According to the previous clinical trials, immune checkpoint inhibitor (ICIs) is often used in combination with standard chemo drugs and showed further beneficial effect on cancer patients as compared to thechemotherapy alone. Therefore, the synergistic anti-tumor effect of Hl plus docetaxel treatment were evaluated in a xenograft mouse model.

[0139] The experimental design was shown in Figure 12A. First, 2 x 106CD24-expressing MDA-MB-231-IV2 cells were injected into the mammary fat pads of SCID mice one week prior to the treatment. 2 mg / kg Hl was administered via i.p. injection every week for a total of eight injections. 5mg / kg docetaxel was given through i.v. injection separately one day after mAb administration for a total of four injections.

[0140] The treatment groups were designed as follows: Tumor-bearing mice were i.v. injected with (1) PBS (n=4), (2) Hl (n=5), (3) docetaxel (n=5) and (4) Hl plus docetaxel (n=5) weekly for six weeks, respectively. 10 mg / kg Hl and 2.5 mg / kg docetaxel were used in the combination therapy. Tumor growth was monitored and recorded over six weeks.

[0141] As shown in Figure 12B, significant synergistic anti-tumor effect of Hl plus docetaxel regimen was observed as compared with either the Hl or docetaxel monotherapy at the early phase of treatment and lasted through the study endpoint. Tumor growth was recorded and stopped on the day of the first mouse death. In addition, as shown in Figure 12C, Kaplan-Meier survival analysis showed that the median survival was 106.8 days in the control group and 97.4 days in the docetaxel group. The median survival was not reached in both Hl group and combinatorial treatment group. Hl monotherapy and combination therapy could both significantly prolong the survival of tumorbearing mice as compared to the control group and docetaxel group. Further, mice received combination therapy showed the best survival benefit.

[0142] 1 x 106IV2-CD24 cells mixed with matrigel were injected into the 4th mammary fat pads of mice three days prior to combination therapy. The combination therapy evaluated Hl plus docetaxel compared with PBS plus docetaxel.

[0143] EXAMPLE 22: assessment of the potential toxicity of Hl

[0144] The potential toxicity of the Hl was evaluated by the liver function, kidney function and the red blood cell binding affinity. The human whole blood collected from the healthy donor was stained with Hl at a dilution factor of 1:100 followed by anti- mouse FITC staining.

[0145] As shown in the Table 2, the liver function and kidney function did not exceed the normal range of the indicators below.

[0147] As shown in the Figure 13, none of Hl bound to human red blood cells, suggesting that the Hl are highly specific and have no cross-reactivity to human RBC.

[0148] To further confirm the antitumor activity of Hl, the tumor’s CD24 level was further considered. Such antitumor effect of Hl in a CD24-negative and CD24-positive TNBC xenograft mouse model were evaluated.

[0149] As shown in Figure 14 A, Hl significantly inhibited the tumor growth of CD24-postive tumors while had limited antitumor effect on CD24- negative tumors in Figure 14B, indicating that the antitumor activity of Hl is solely dependent on tumor’s CD24 level.

[0150] EXAMPLE 23: immunohistochemistry staining (IHC) of CD24 in clinical specimens

[0151] To clarify the possible action mechanism of the antitumor activity of our in-house Hl, tumor proliferation status was examined in the primary tumors resected from SCID mice treated with Hl or PBS. IHC analysis was applied to investigate the distribution of myeloid-derived suppressor cells (MDSCs), tumor-infiltrating macrophages (TIMs) and Natural Killer (NK) cells.

[0152] The intensity of CD24 expression in tissue samples of IHC staining was evaluated and scored by two independent researchers and reviewed by a board-certified pathologist. The immunostaining scores were categorized into three groups: 0 = none; 1 = weak; 2 = moderate; and 3 = strong.

[0153] As shown in Figure 15A and Figure 15B, the number of tumorinfiltrating CDllb-positive MDSCs were dramatically reduced and F4 / 80- positive tumor-infiltrating macrophages were significantly increased in tumors resected from mice injected with Hl as compared to those resected from mice injected with PBS. Besides, Hl-treated tumors were heavily surrounded and invaded by F4 / 80-postitive macrophages. Further, a significantly increased number of tumor-infiltrated CD68+ Ml macrophages was found in Hl-treated tumors as compared to those of PBS-treated ones. Meanwhile, as shown in figure 15A and 15B, similar number of CD206-positive M2 macrophages inPBS- and Hl-treated tumors were shown. In addition, a similar number of CD206-positive M2 macrophages was observed in PBS- and Hl-treated tumors. In addition, the number of Ki67-positive tumor cells was similar between PBS- and Hl-treated tumors, suggesting that Hl had no effect on tumor cell proliferation. These results indicated that tumor-killing macrophages plays a more significant role than inhibition of cell growth in the antitumor effect of Hl.

[0154] EXAMPLE 24: sequencing of variable heavy (VH) and variable light (VL) domain of Hl by 5’ Rapid Amplification of cDNA End (RACE) and Next Generation Sequencing

[0155] As shown in Figure 16, the sequencing procedure started with the total RNAs extraction from Hl -secreting hybridoma clone. The antibody cDNA library was established using a modified 5’ Rapid Amplification of cDNA End (RACE) method with antibody constant region (Fc)-specific RT primer. The antibody cDNA was prepared by amplifying cDNA with PCR reaction using universal forward primer and reverse primer. Antibody cDNA library was then subjected to RNAseq analysis. The Complementarity-Determining Regions (CDRs) features in the Fab domain of Hl clone were analyzed using an on-line CDR prediction algorithm AbodyBuilder. The sequence identification of the three individual CDR of VH and VL is shown in Table 3.

[0156] Table 3. Sequence identification of the three individual CDR of the VH and VL.

[0157] Then, the generation, production and anti-tumor validation of the chimeric anti- human CD24 mAb (CH-01) were evaluated.

[0158] The fragment antigen binding region (Fab) of Hl was firstly subcloned into an expression vector upstream of the human IgGl Fc sequences. The chimeric anti- human CD24 mAb (CH-01) with human IgGl Fc domain was produced in the 100 ml ExpiCHO cell culture (Figure 17A and Figure 17B) and antibody was purified and verified using SDS-PAGE coupled with Coomassie blue staining. The anti-tumor efficacy of CH-01 was then tested using CD24-positive MDA-MB-468 orthotopic mouse model.

[0159] As shown in Figure 17C and Figure 17D of IVIS analysis, tumorbearing mice receiving CH-01 treatment showed reduced photon counts over the course of mAb treatment as compared to the IgG control group and three mice in mAb group showed almost undetectable luminescent signal 3 months after CH-01 injection. Similarly, direct measurement of tumor size showed the same result in Figure 17E. In addition, antitumor activity of CH-01 was further confirmed in the second CD24-positive TNBC xenograft model using IV2- CD24 TNBC cells. As shown in Figure 17F, CH-01 could effectively suppress tumor growth when used at a dosage of either 0.5 mg / kg or 5 mg / kg.

[0160] EXAMPLE 25: humanization of anti-CD24 mAb HH-01-46 (HH-01-46)

[0161] The computer-based simulation was applied to calculate the molecular dynamics (MD) trajectories of mouse Hl and the predicted HH-01- 46, and the value of root-mean-square deviation (RMSD) of the Ab structure was calculated.

[0162] As shown in Figure 18, the differences of atoms the RMSD was further adjusted to obtain the weighted RMSD (wRMSD). Two combinations including two humanized heavy chains and one humanized light chain with the wRMSD value (LiHi: 2.111; LiH2:2.056) were closest to the wRMSD value of the original mouse Hl (Hi : 1.556) as compared to other predictions (Figure 18A). The molecular superimposition of L|H2and the original mouse Hl was shown in Figure 18B. The molecular superimposition also demonstrated that the MD trajectories of the Fab domain of HH-01-46 (LIH2) was very similar to that of the Fab domain of CH-01. In the end, the combination of the humanized light chain Li and the humanized heavy chain Hi were selected to be the lead HH-01- 46 and the antibody sequences were cloned into expression vector pFUSE- hlgGlFc as shown in Figure 18C.

[0163] In another embodiment, pFUSE-Ig expression plasmids containing the humanized heavy chain and light chain, respectively, were transfected into Expi293 cells to produce the recombinant humanized anti-CD24 mAb. The humanized mAb was purified using protein A column and verified by Coomassie blue staining, which was shown in Figure 19 A. The antigen binding sensitivity and specificity of HH-01-46 using CD24-positive MDA-MB-468 andCD24-negative MDA-MB-231 were verified, respectively.

[0164] As shown in FIG 19B, HH-01-46 has similar antigen sensitivity and specificity as compared to its parental mAb CH-01 , the binding affinity of the HH-01-46 was determined by surface plasmon resonance (SPR). Multiple cycle Kinetics (MCK) analysis indicated that a KD constant of 92.9 nM was measured for HH-01-46, showing that HH-01-46 possesses similar affinity to the parent mAb with a KD constant of 69.5 nM (Figure 19C). Flow cytometry analysis showed that HH-01-46 could bind to a range of TNBC cell lines including metaplastic BC cell lines and it showed similar binding intensity with the parental mAb CH-01 (Figure 19D).

[0165] Then, the anti-tumor effect of HH-01-46 in a CD24-positive TNBC xenograft mouse model was evaluated. SCID mice were inoculated with 2 x 106of GFP-Luc-labeled IV2-CD24 cells in the fat pad followed by administration of HH-01-46 treatment via i.v injection once a week for 4 weeks.

[0166] As shown in Figure 20A and Figure 20B, the anti-tumor efficacy of HH-01-46 was as good as the parental CHOI, suppressing 95% of tumor growth from IVIS analysis. The corresponding image was shown in Figure 20C.

[0167] In one embodiment of CH-01, anti-tumor efficacy was shown in a model using human peripheral blood mononuclear cells (PBMCs)-reconstituted ASID mouse model.

[0168] EXAMPLE 26: PBMCs-reconstituted ASID mouse model

[0169] ASID (NOD.Cg-Prkdcscid I12rgtmlWjl / YckNarl) mice model was applied. First, 40 ml of whole blood were drawn from a healthy donor and was diluted with IX PBS at 1: 1 ratio. PBMCs were then isolated using densitygradient centrifugation. Finally, 2.5 x 10 PBMCs from 40 ml whole blood were obtained. Next, each ASID mouse was infused with 1 x 10 PBMCs for 7 days prior to tumor inoculation. As shown in Figure 2 IB, after PBMC reconstitution, ASID mice were injected with 2.5 x 105IV2-CD24 cells on day 7. PBMC- humanized ASID mice were then received 5mg / kg CH-01 monotherapy, 5mg / kg docetaxel, and CH-01 plus docetaxel, respectively, on day 9, day 14, day 21 and day 27. Schematic of PBMC-humanized ASID mouse was shown in Figure 21 A. Tumor growth was recorded through day 21. As shown in Figure 2 IB, CH-01 monotherapy and docetaxel monotherapy both induced tumor regression in tumor-bearing ASID mice at the early phase of treatment. The anti-tumor activity in combinational treatment was compared to the single agent therapy and the results are shown in Figure 2 IB. CH-01 plus docetaxel had the best tumor inhibitory effect.

[0170] In addition, the effect of HH-01-46 on promoting macrophage phagocytosis including inducing NK cell activation and tumor-killing ability were evaluated. As shown in Figure 22A, incubation of CD24-positive BC cells with human macrophage derived from monocytes in the presence of HH-01-46 could effectively promote Ab-dependent phagocytosis. As shown in Figure 22B, HH-01-46 treatment could also activate NK92MI cells to secrete tumor-killing cytokines IFN-y and IL- 12 in a time-dependent manner. Moreover, as shown in Figure 22C, ADCC assay using human PBMC and NK92MI indicated that HH- 01-46 could effectively promote Ab-dependent cell-mediated cytotoxicity.

[0171] Besides, the Akt / Erk oncogenic signaling was analyzed in HH-01-46 or CH-01. First, MDA-MB-468 cells were pretreated with HH-01-46 or CH-01 at a dose of 5pg / ml 24 hours prior to EGF stimulation.

[0172] As shown in Figure 23, pretreatment of TNBC cells with HH-01-46 or CH-01 anti-CD24 mAb significantly reduced EGF-induced Akt and Erk activation.

[0173] EXAMPLE 27: HH-01-46 shows great anti-tumor activity in the Herceptin resistant BT474 xenograft model

[0174] In our previous cohort study investigating the expression in BC patients with different subtypes, we found that patients with HER2 subtype exhibit strong CD24 expression, and therefore could possibly benefit from anti- CD24 therapy. In the context of HER2 -positive breast cancer therapy, it has been observed that a substantial proportion, approximately 40%, of patients undergoing Herceptin treatment encounter the emergence of resistance to this targeted therapy. This notable phenomenon warrants comprehensive investigation to elucidate the underlying molecular mechanisms and potential avenues for overcoming or circumventing resistance, thus optimizing the clinical management of HER2-positive breast cancer patients. In this study, we examined the potential of anti-CD24 therapy as an advancing precision medicine approach targeting HER2 -positive patients. Our investigation aims to shed light on the efficacy and mechanisms of action of this novel therapeutic strategy in combating Herceptin resistance and enhancing treatment outcomes.

[0175] As illustrated in Figure 24 A, we initiated the development of a Herceptin-resistant cell line from the HER2-positive BT474 cell line by subjecting it to five rounds of drug selection using 100 pg / mL Herceptin (Trastuzumab). Following this selection process, the surviving clones underwentevaluation to assess their resistance to Herceptin treatment, and the results were compared with those of the parental BT474 cells. As depicted in Figure 24B, the resulting BT474-HR subline displayed notable resistance to Herceptin treatment when compared to the parental BT474 cells. Subsequently, the BT-474-HR cells were utilized to establish a Herceptin-resistant breast cancer (BC) xenograft mouse model. As showed in Figure 24C, 2 x 106cells were orthotopically injected into the 4th fat pad of SCID mice, followed by HH-01-46 treatment on day 7, day 14, and day 22. The analysis of tumor growth kinetics revealed that mice receiving HH-01-46 therapy exhibited a significant reduction in tumor growth compared to those receiving control IgG (Figure 24D).

[0176] These findings indicate the promising potential of anti-CD24 mAb as a novel therapeutic intervention for managing Herceptin-resistant breast cancer. The utilization of anti-CD24 therapy presents a new and hopeful avenue for advancing precision medicine approaches, specifically targeting HER2- positive patients. Further exploration and validation of anti-CD24 mAb therapy in clinical settings could pave the way for more personalized and effective treatments for HER2 -positive breast cancer patients.

[0177] While the invention has been described and exemplified in sufficient details for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of this invention.

[0178] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The processes and methods for producing them are representative of preferred embodiments, are exemplary, andare not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. 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INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..18 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier>MNSDQualifier id="q2"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>ggaggatatagctggcac< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="2"><INSDSeq><INSDSeq_length>6< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..6< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q4"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>GGYSWH< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="3"><INSDSeq><INSDSeq_length>45< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..45 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q6"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>tatatccactacagcggagacaccaaatacaacccccccctgaaa< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="4"><INSDSeq><INSDSeq_length>15< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..15 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q8"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>YIHYSGDTKYNPPLK< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="5"><INSDSeq><INSDSeq_length>24< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..24< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="qlO"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>ggcaccagaaactccctggactac< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="6"><INSDSeq><INSDSeq_length>8< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..8< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="ql2"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>GTRNSLDY< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="7"><INSDSeq><INSDSeq_length>33< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..33 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="ql4"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>tgcgccgtgagcggatatagcattaccggagga< / INSDSeq_sequence>< / INSDSeq>< / SequenceData>< SequenceData sequenceIDNumber=" 8 " ><INSDSeq><INSDSeq_length> 11 < / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..11 < / INSDFeature_location><INSDFeature_quals><INSDQualifier>< INSDQualifier_name > mol_type < / IN SD Qualifier_name ><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="ql6"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>NCKSSQSLLYS< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="9"><INSDSeq><INSDSeq_length>18< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..18 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="ql8"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>atccactacagcggcgac< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="10"><INSDSeq><INSDSeq_length>6< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..6< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q20"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>LLIYLS< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber=" 11 " ><INSDSeq><INSDSeq_length>24< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..24< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q22"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>ggcaccagaaacagcctggactac< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="12"><INSDSeq><INSDSeq_length>8< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location>1..8< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q24"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>IYPLTFGQ< / INSDSeq_sequence>< / INSDSeq>< / SequenceData>< SequenceData sequenceIDNumber=" 13 " ><INSDSeq><INSDSeq_length>348< / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..348 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q26"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>gacgtgcagctgcaggagagcggacccggactggtgaaacccagcgacacactgagcctgacat gcgccgtgagtggatatagcattaccggaggatatagctggcactggattagacagccccccggcaagggactggagtggattggct atatccactacagcggagacaccaaatacaacccccccctgaaaagcagagtgaccatgagccgggacacctccaagaaccagttc agcctgaaactgagcagcgtgaccgccctggacaccgccgtgtactactgcgccagaggcaccagaaactccctggactactgggg ccagggaacactggtgaccgtgagc< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber="14"><INSDSeq><INSDSeq_length>106< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..106< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q28"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>DVQLQESGPGLVKPSDTLSLTCAVSGYSITGGYSWHWIRQPPG KGLEWIGYIHYSGDTKYNPPLKSRVTMSRDTSKNQFSLKLSSVTALDTAVYYCARGT RNSLDY< / INSDSeq_sequence>< / INSDSeq>< / SequenceData>< SequenceData sequenceIDNumber=" 15 " ><INSDSeq><INSDSeq_length>339< / INSDSeq_length>ctgcaagagcagccagagcctgctgtacagcaacgaccagaaaaactacctggcctggtaccagcagaaacccggccagcccccc aaactgctgatctacctgagcagtacccgggaaagcggcgtgcccgacagattcagcggcagcggcagcggaacagacttcaccct gaccatcagcagcctgcaggctgaggacgtggccgtgtactactgccagcagtacttcatttatcccctgacattcggacagggcacc aagctggagatcaag< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber=" 16" ><INSDSeq><INSDSeq_length> 113< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..113< / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q32"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>DIVMTQSPDSLAVSLGERATINCKSSQSLLYSNDQKNYLAWYQ QKPGQPPKLLIYLSSTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYFIYPL TFGQGTKLEIK< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber=" 17" ><INSDSeq><INSDSeq_length>351 < / INSDSeq_length><INSDSeq_moltype>DNA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..351 < / INSDFeature_location><INSDFeature_quals><INSDQualifier><INSDQualifier_name>mol_type< / INSDQualifier_name><INSDQualifier_value>other DNA< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q34"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>gacgtgcagctgcaggagagcggacccggactggtgaaacccagcgagacactgagcctgacat gcgccgtgagcggatatagcattaccggaggatatagctggcactggattagacagccccccggcaagggactggagtggattggct atatccactacagcggcgacaccaaatacaacccccccctgaaaagcagagtgaccatcagtcgggacacctcaaagaaccagttca gcctgaagctgtcatccgtgaccgccgccgacaccgccgtgtactactgcgccagaggcaccagaaacagcctggactactggggc cagggcacactggtgaccgtgagcagc< / INSDSeq_sequence>< / INSDSeq>< / SequenceData><SequenceData sequenceIDNumber=" 18" ><INSDSeq><INSDSeq_length> 117< / INSDSeq_length><INSDSeq_moltype>AA< / INSDSeq_moltype><INSDSeq_division>PAT< / INSDSeq_division><INSDSeq_feature-table><INSDFeature><INSDFeature_key>source< / INSDFeature_key><INSDFeature_location> 1..117< / INSDFeature_location><INSDFeature_quals><INSDQualifier>< INSDQualifier_name > mol_type < / IN SD Qualifier_name ><INSDQualifier_value>protein< / INSDQualifier_value>< / INSDQualifier><INSDQualifier id="q36"><INSDQualifier_name>organism< / INSDQualifier_name><INSDQualifier_value>synthetic construct< / INSDQualifier_value>< / INSDQualifier>< / INSDFeature_quals>< / INSDFeature>< / INSDSeq_feature-table><INSDSeq_sequence>DVQLQESGPGLVKPSETLSLTCAVSGYSITGGYSWHWIRQPPG KGLEWIGYIHYSGDTKYNPPLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARGT RNSLDYWGQGTLVTVSS< / INSDSeq_sequence>< / INSDSeq>

Claims

What is claimed is1. An isolated antibody, which specifically binds to human CD24, wherein the isolated antibody comprises:(a) a heavy chain variable region-complementarity determining region 1 (VH-CDR1), a VH-CDR2 and a VH-CDR3, wherein the VH-CDR1 consists of the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 consists of the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 consists of the amino acid sequence of SEQ ID NO: 6; and(b) a light chain variable region-complementarity determining region 1 (VL- CDR1), a VL-CDR2 and a VL-CDR3, wherein the VL-CDR1 consists of the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 consists of the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 consists of the amino acid sequence of SEQ ID NO: 12.

2. The isolated antibody of claim 1, wherein the VH consists of the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18.

3. The isolated antibody of claim 1, wherein the VL consists of the amino acid sequence of SEQ ID NO: 16.

4. The isolated antibody of claim 1, which further comprises a Fc region.

5. The isolated antibody of claim 4, wherein the Fc region is an IgG, IgM, IgA, IgD, IgE antibody, or any subclass thereof.

6. The isolated antibody of claim 5, wherein the Fc region is an IgG.

7. A pharmaceutical composition comprises the isolated antibody of claim 1 and a pharmaceutical acceptable carrier or excipient.

8. A method of treating CD24 expressing cancer in a subject comprisingadministering to the subject with CD24 cancer the isolated antibody of claim 1.

9. The method of claim 8, which further comprises administering one or more additional anti-cancer therapy or anti-cancer drugs.

10. The method of claim 9, wherein the one or more additional anti-cancer therapy or anti-cancer drugs are chemo therapy, chemo drug, tamoxifen, Herceptin or combination thereof.

11. The method of claim 10, wherein the cancer is a solid tumor.

12. The method of claim 8, wherein the cancer is selected from breast cancer, liver cancer, or ovarian cancer.

13. The method of claim 12, wherein the breast cancer is a triple-negative type breast cancer.

14. The method of claim 13, wherein the triple-negative type breast cancer is a lung metastasis triple-negative breast cancer.

15. A method for detecting expression of CD24 in an in vitro sample comprising:(a) providing an in vitro sample from a subject;(b) contacting the in vitro sample with a capture antibody that specifically binds to the CD24, wherein the capture antibody is the isolated antibody of claim 1;(c) adding a labeled detection antibody that binds to the capture antibody to form an immune complex consisting of the CD24, the capture antibody and the labeled detection antibody; and(d) measuring an amount of the immune complex to determine the presence or level of the CD24 in the in vitro sample.

16. The method of claim 15, wherein the in vitro sample is selected from at least one of blood, lymphatic fluid, tissue fluid, body cavity fluid, oral mucosal fluid, circulating tumor cell or combination thereof.

17. The method of claim 16, wherein the in vitro sample is blood.

18. The method of claim 17, wherein the blood is whole blood, plasma or serum.

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