TWI862019B - Therapeutic cd24 humanized monoclonal antibodies and anti-tumor translational use - Google Patents

Abstract

The invention is related to an isolated antibody, which specifically binds to CD24. The invention also provides a method for treating CD24-expressing cancer.

Description

Therapeutic CD24 humanized monoclonal antibody and anti-tumor translational applications

The present invention relates to an isolated antibody that binds to CD24 and its anti-tumor use.

CD24 is a highly glycosylated mucin-type surface membrane protein fixed by glycophosphatidylinositol (GPI). It has been determined that CD24 is highly expressed in many human malignancies, especially metastatic diseases. Breast cancer is one of the cancers with high CD24 expression.

Breast cancer is the most commonly diagnosed female cancer in Taiwan, with an estimated 16,000 new cases diagnosed each year and an estimated 2,000 deaths each year. Since 2020, female breast cancer has been ranked first among human cancers worldwide, with an estimated 2.2 million new cases each year, and has become the fifth leading cause of cancer death. In terms of genetic expression characteristics, there are at least four subtypes of breast cancer, including luminal A, luminal B, HER2+, and basal-like, and each subtype is different from the other.

According to molecular expression, the tubular A subtype tumors highly express estrogen receptor (ER) and/or progesterone receptor (PR), but do not express HER2 (ER/PR+, HER2-); tubular B subtype tumors not only express ER and/or PR, but also express HER2 (ER/PR+, HER2+); HFR2-positive subtype tumors highly express HER2, but do not express ER/PR (ER/PR-, HER2+); and basaloid subtype tumors do not express ER, PR, and HER2 (ER/PR-, HER2-), which is also called triple-negative breast cancer (TNBC).

Regardless of molecular subtype, the 5-year survival rate of early-stage breast cancer patients in Taiwan can be as high as 90%, but a 10-year follow-up study showed that breast cancer patients with ductal A/B subtype had a higher 5-year survival prognosis compared with patients with HER2-positive subtype or TNBC features. In addition, patients with TNBC features had the worst 5-year survival prognosis. In addition to conventional chemotherapy, ductal A/B subtype breast cancer patients with positive hormone receptors (BC patients) are usually treated with tamoxifen, one of the first-line endocrine therapies, to reduce the mortality rate by 31%. The use of herceptin in patients with ductal B subtype or HER2-positive subtype breast cancer with positive HER2 can reduce the risk of death by approximately 33%. Despite this, 20-30% of ductal A/B subtype breast cancer tumors are often resistant to tamoxifen treatment, and patients with HER2-positive subtypes mostly do not respond well to herceptin.

In addition, stage IV or metastatic breast cancer can spread to other organs of the body besides the breast. During the metastatic process, epithelial breast cancer cells gain invasive capabilities after undergoing epithelial-mesenchymal transition (EMT), break through the basement membrane of the terminal duct lobular unit (TDLU) and spread to nearby lymph nodes, eventually colonizing distant vital organs. Common organs for breast cancer cell spread include bones, lungs, liver, and brain. Therefore, the spread of breast cancer can significantly reduce the patient's chances of survival, and metastasis remains the leading cause of death associated with breast cancer. Other cancers such as colorectal cancer, liver cancer, kidney cancer, pancreatic cancer, ovarian cancer, and prostate cancer are also prone to metastasis.

Current breast cancer immunotherapy focuses on enhancing the anti-tumor activity of cytotoxic T cells. There are two main ways to kill cancer cells: PD-1/PD-L1 immune checkpoint blockade; and modifying the patient's T cells with chimeric antigen receptors (CARs) that have excellent tumor antigen recognition capabilities. The first method aims to enhance the anti-tumor activity of cytotoxic T cells to more effectively eliminate tumor cells. The second method focuses on improving the tumor targeting efficiency of cytotoxic T cells and prolonging the duration of cytotoxic T cell activation. However, the above treatment effects are often limited, especially in TNBC patients.

There is currently no effective targeted therapy for TNBC. Therefore, there is an urgent need to develop new effective treatments for TNBC and other breast cancer subtypes.

This study collected and analyzed 370 TNBC cases from three medical centers: Taipei Veterans General Hospital, Kaohsiung Medical University Hospital, and China Medical University Hospital. More than 80% of TNBCs have high expression of CD24, which further illustrates the clinical significance of CD24 in TNBC patients.

As used herein, "a", "said", "the", "at least one" and "one or more" can be used interchangeably.

In order to more easily understand the present invention, certain terms are first defined, and additional definitions are set forth throughout the detailed description.

The term "CD24" includes any variant or isoform expressed naturally by cells or by cells transfected with the CD24 gene.

The term "antibody" in the present invention covers complete antibodies and any antigen-binding fragments or single chains thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, which are also called complementation determining regions (CDRs). Both the VH and VL regions contain three CDRs, arranged in the following order from the amino terminus to the carboxyl terminus: CDR1, CDR2 and CDR3. VH and VL contain binding domains that interact with antigens.

The present invention provides an isolated antibody that specifically binds to CD24, the isolated antibody comprising: (a) heavy chain variable region-complementary determining region VH-CDR1, VH-CDR2 and VH-CDR3, wherein the VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and (b) light chain variable region-complementary determining region VL-CDR1, VL-CDR2 and VL-CDR3, wherein the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 12.

The present invention provides an isolated antibody that specifically binds to CD24, wherein the VH comprises an amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18, wherein the VH-CDR1 comprises an amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises an amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 comprises an amino acid sequence of SEQ ID NO: 6.

The present invention provides an isolated antibody that specifically binds to CD24, wherein the VL comprises the amino acid sequence of SEQ ID NO: 16. The VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 12.

The present invention provides an isolated antibody that specifically binds to CD24, and the isolated antibody is an isolated antibody of human CD24.

In one embodiment, the isolated antibody of the present invention is encoded by a heavy chain nucleic acid and a light chain nucleic acid, wherein the heavy chain nucleic acid and the light chain nucleic acid are nucleotide sequences shown in SEQ ID NO: 13 and SEQ ID NO: 15, respectively.

In another embodiment, the isolated antibody of the present invention is encoded by a heavy chain nucleic acid and a light chain nucleic acid, wherein the heavy chain nucleic acid and the light chain nucleic acid are the nucleotide sequences shown in SEQ ID NO: 17 and SEQ ID NO: 15, respectively.

The present invention further provides a composition for use in preparing a drug for treating cancer expressing CD24, wherein the composition comprises the above-mentioned isolated antibody and an anticancer drug.

In one embodiment of the present invention, the cancer expressing CD24 is a solid tumor.

In one embodiment of the present invention, the cancer expressing CD24 is breast cancer, liver cancer or ovarian cancer. In a preferred embodiment, the breast cancer refers to triple-negative breast cancer (TNBC).

In another embodiment, the triple-negative breast cancer is triple-negative breast cancer with lung metastasis.

In one embodiment of the present invention, the antibody can be used simultaneously with an anticancer drug. The anticancer drug comprises docetaxel, tamoxifen, herceptin or a combination thereof. Other anticancer drugs herein include chemotherapeutic agents, radiopharmaceuticals or immunotherapeutic agents. The chemotherapeutic agents herein include doxorubicin. hydrochloride), bleomycin, mitomycin, etoposide, vinblastine, vincristine, vinorelbine, paclitaxel, docetaxel, irinotecan, topotecan, hydroxyurea, cyclophosphamide, melphalan, chlorambucil, carmustine, carboplatin, cisplatin, fluorouracil, gemcitabine, capecitabine, imatinib, and goserelin acetate), but not only the above drugs.

The present invention also provides a method for detecting CD24 expression in an in vitro sample, the method comprising: (1) providing an in vitro sample of an individual; (2) contacting the in vitro sample with a capture antibody that specifically binds to CD24, wherein the capture antibody is an isolated antibody of the present invention; (3) adding a labeled detection antibody that binds to the capture antibody to form an immune complex consisting of CD24, the capture antibody and the labeled detection antibody; and (4) determining the amount of the label in the formed immune complex to determine the presence or level of CD24 in the in vitro sample.

The present invention also provides a method for detecting CD24 expression in an in vitro sample, wherein the in vitro sample is selected from at least one of blood, lymph fluid, tissue fluid, body cavity fluid, oral mucosal fluid, circulating tumor cells, or a combination thereof.

The present invention also provides a method for detecting CD24 expression in an in vitro sample, wherein the in vitro sample is blood.

The present invention also provides a method for detecting CD24 expression in an in vitro sample, wherein the blood is whole blood, plasma or serum.

The samples described in the present invention are not limited to at least one of blood, plasma, serum, lymph, tissue fluid, body cavity fluid, oral mucosal fluid, circulating tumor cells or a combination thereof.

In view of the above technical situation, the present invention provides an isolated antibody or antigen-binding fragment targeting breast cancer CD24. Heavy chain polypeptides and light chain polypeptides are also disclosed. Antibodies, antigen-binding fragments and polypeptides can be provided in isolated and/or purified forms and can be formulated into compositions suitable for research, treatment and diagnosis.

The isolated antibodies of the present invention may exist in various antibody isoforms, such as IgG1, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretory IgA, IgD and IgE. They generally include isoforms of IgG1, IgG3, IgG4 and IgM. The antibody may be complete or may include only the antigen-binding portion.

The isolated antibody of the present invention further comprises a crystallizable region fragment (Fc) region.

The isolated antibody of the present invention, wherein the Fc region is IgG, IgM, IgA, IgD, IgE antibody or any subclass thereof.

In a preferred embodiment, the Fc region in the isolated antibody of the present invention is IgG.

The present invention also provides a composition for use in the preparation of a medicament for treating cancers expressing CD24. This use is due in part to their unique specificities, such as antigenic location specificity, affinity, structure, and functional activity.

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

On the other hand, the present invention provides a pharmaceutical composition comprising the isolated antibody of the present invention and a pharmaceutically acceptable carrier and formulation. The pharmaceutical composition may also contain one or more therapeutic agents or a combination thereof, such as the anticancer drugs disclosed above.

On the other hand, the present invention also provides a reagent kit for detecting CD24 antigen or cells expressing CD24 in an in vitro sample, which comprises the isolated antibody of the present invention and instructions for use.

Other features and advantages of the present invention will be revealed from the following non-limiting detailed description and examples.

Figure 1 shows the design, expression and purification of CD24 murine fragment-crystallizable (mFc) fusion protein for mouse immunization. Figure 1A is a diagram of the construction of CD24 mFc fusion protein. Figure 1B uses Coomassie Brilliant Blue staining to check the purification of CD24 mFc fusion protein (1 = 1 μg purified CD24 3c mFc; 2 = blank sequence; 3 = 10 μg 3c protease-hydrolyzed purified CD24 3c mFc; 4 = 10 μg first flow through (FT) protein A (poly-A) resin; 5 = 10 μg second FT poly-A resin; 6 = 1 μg 3c protease). Figure 1C uses Western blot analysis to analyze the difference between anti-mouse Fc antibody and commercial CD24 antibody (1 = culture medium; 2 = FT poly-A resin; 3 = 50 ng purified CD24 mFc). Figure 1D uses FACS to analyze the ability of P-selectin Fc fusion protein to bind to 293 cells expressing recombinant human CD24 mFc. Figure 1E shows that P-selectin Fc fusion protein can specifically recognize recombinant CD24 expressed by 293 cells. Figure 1F uses purified recombinant human CD2 mFc for immunoassay. Abbreviations: GPI is glycophosphatidylinositol; FACS is fluorescence-activated cell sorting.

Figure 2 shows the screening and validation of candidate anti-CD24 monoclonal antibodies (mAbs) to generate hybridoma clones. Figure 2A analyzes the supernatants (SN) of 9 hybridoma clones by flow cytometry (FC). Figure 2B detects the supernatants of 9 hybridoma clones by ELISA. Figure 2C analyzes the purity of 9 hybridoma clone mAbs by SDS-PAGE. Figure 2D compares the MFI differences of 9 mAbs. Figure 2E compares the MFI differences of 9 mAbs at different concentrations. Abbreviation: MFI stands for mean fluorescence intensities.

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

Figure 4 shows fluorescent micrographs of 9 mAbs.

Figure 5 shows the effect of anti-CD24 mAb on cell survival of CD24-positive TNBC in an in vitro model.

Figure 6 shows the antigenic mapping of nine anti-CD24 mAbs using peptide scanning, revealing the common antibody binding region within the CD24 mature domain.

Figure 7 shows the establishment of a TNBC xenograft mouse model expressing CD24, and the sorting of CD24-positive cells by FACS.

Figure 8 shows the in vivo anti-tumor activity of candidate anti-CD24 mAbs. Figure 8A is the experimental process for testing the in vivo anti-tumor potential of 9 cloned candidate CD24 mAbs. Figure 8B is a comparative analysis of mouse tumor volume after treatment with 9 CD24 mAbs.

Figure 9 shows the anti-tumor efficacy of CD24 mAb H1 (H1) studied using the MDA-MB-468 xenograft mouse model. Figure 9A is a flowchart of the injection of MDA-MB-468 cells and antibodies into severe combined immunodeficiency (SCID) mice. Figure 9B is a flowchart of the antibody injection treatment of tumor-bearing mice. Figure 9C shows the tumor volume of tumor-bearing mice after receiving different concentrations of H1 treatment in the first week. Figure 9D shows the change in tumor volume of tumor-bearing mice after receiving different concentrations of H1 treatment within 200 days.

Figure 10 shows in vivo imaging of H1-treated tumor-bearing mice.

Figure 11 shows that H1 treatment inhibits distant lung metastasis in a primary tumor resection mouse model. Figure 11A is a flow chart of the experiment. Figure 11B is a graph showing changes in tumor volume in tumor-bearing mice. Figure 11C is a graph showing tumor volume in tumor-bearing mice at week 6. Figure 11D is a graph showing in vivo imaging and photon counting of tumor-bearing mice. Figure 11E is a graph showing lung tissue of tumor-bearing mice treated with H1 using hematoxylin-eosin staining. Figure 11F is a graph showing the survival rate of tumor-bearing mice.

Figure 12 shows that the combination of H1 and docetaxel in the treatment of CD24-positive TNBC exhibits synergistic anti-tumor effects. Figure 12A is a flowchart of the experimental design. Figure 12B shows the changes in the volume of TNBC tumors in tumor-bearing mice after different injection treatments. Figure 12C shows the survival rate of tumor-bearing mice after different injection treatments.

Figure 13 shows the biosafety assessment of lead H1.

Figure 14 shows the TNBC tumor growth analysis of H1 on IV2 CD24 positive/negative model. Figure 14A shows the TNBC tumor volume change, tumor weight and immunostaining image of IV2 CD24 positive model. Figure 14B shows the TNBC tumor volume change, tumor weight and immunostaining image of IV2 CD24 negative model.

Figure 15 shows the analysis of primary tumors removed from SCID mice after H1 injection. Figure 15A uses immunohistochemistry (IHC) to analyze the distribution of different cells in the removed tumor tissue. Figure 15B shows the number of different cells in the removed tumor tissue after H1 injection.

Figure 16 is a schematic diagram of the antibody binding region (fragment antigen binding, Fab) sequence of the main H1 deciphered.

Figure 17 shows the design, production, purification and anti-tumor validation of chimeric anti-human CD24 mAb H1 (CH-01). Figure 17A is a design diagram of CH-01. Figure 17B is a production flow chart of CH-01. Figure 17C is an in vivo imaging diagram of tumor-bearing mice within 12 weeks after injection of CH-01. Figure 17D is a photon count of tumor-bearing mice within 12 weeks after injection of CH-01. Figure 17E is a tumor volume change of tumor-bearing mice after injection of CH-01. Figure 17F is a tumor volume change of tumor-bearing mice after injection of different concentrations of CH-01.

Figure 18 shows the humanization of the lead H1 antibody. Figure 18A is a design flow chart of the humanization of the lead H1 antibody. Figure _18B is a molecular overlay of _L1H2 and the original mouse H1. Figure 18C is a vector containing a light chain or a heavy chain.

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

Figure 20 shows the comparison of anti-tumor activity of humanized HH-01-46 and CH-01 in CD24-positive TNBC xenograft mouse models. Figure 20A is the in vivo imaging of TNBC xenograft mice after injection of humanized HH-01-46 and CH-01. Figure 20B is the change in tumor volume after injection of humanized HH-01-46 and CH-01 into TNBC xenograft mice. Figure 20C is the comparison of tumor tissue and tumor size after injection of humanized HH-01-46 and CH-01 into TNBC xenograft mice.

Figure 21 shows the anti-tumor efficacy of the combination of CH-01 and docetaxel in the humanized peripheral blood mononuclear cell (PBMC) advanced severe immunodeficiency (ASID) mouse model. Figure 21A is the experimental flow chart. Figure 21B shows the changes in tumor volume of ASID mice under different treatments.

Figure 22 shows that humanized HH-01-46 promotes antibody-dependent phagocytosis and antibody-dependent cell-mediated cytotoxicity. Figure 22A is an immunostaining image. Figure 22B shows the effect of humanized HH-01-46 on the fold change of actin in different cells. Figure 22C uses antibody-dependent cell-mediated cytotoxicity (ADCC) to measure the effect of humanized HH-01-46 on antibody-dependent cell-mediated cytotoxicity.

Figure 23 shows the analysis of Akt/Erk oncogenic signaling in TNBC cells pretreated with humanized HH-01-46 and CH-01.

The following embodiments are non-limiting and merely represent various aspects and features of the present invention.

Example 1: Construction of humanized CD24 murine fragment-crystallizable (mFc) fusion protein.

Mature humanized CD24 consists of only 35 highly glycosylated amino acid residues, and CD24 is attached to the plasma membrane surface through a glycophosphatidylinositol (GPI) anchor. The fusion protein consisting of the IL2 signal peptide, CD24 mature domain and CD24 mFc fusion protein is constructed as shown in the molecular design below. Fc represents the constant fragment of mouse immunoglobulin G (IgG).

Molecular design of CD24 Fc fusion protein: (IL2 signal peptide)-SETTTGTSSNSSQSTSNSGLAPNPTNATTKAAG-(ASTGS)-Fc (i.e., high-efficiency leader peptide-CD24 mature domain-linking peptide-crystallization region fragment).

The results in Figure 1 show that a recombinant humanized CD24 mFc fusion protein was designed containing the IL2 signal peptide sequence, a unique mature domain with poor similarity between Homo sapiens and murine species, and a linker sequence containing a 3c protease cleavage site after the GPI sequence (Figure 1A).

Example 2: Separation and purification of CD24 mFc fusion protein

The CD24 Fc expression construct was transiently introduced into the Expi293F expression system using HEK293 cells. The culture medium containing the CD24 mFc fusion protein was collected and subjected to a protein A (poly-A) resin purification protocol. The purified CD24 mFc was further analyzed by SDS-PAGE combined with Coomassie Brilliant Blue staining and Western blotting. Milligram quantities of CD24 mFc fusion protein were purified from the above procedure.

The resulting humanized CD24 mFc construct was expressed in the Expi293 mammalian cell expression system. The humanized CD24 mFc fusion protein was purified from the culture medium using poly-A resin and then hydrolyzed by 3c protease (Figure 1B). The hydrolyzed and unhydrolyzed CD24 mFc fusion proteins were verified by SDS-PAGE combined with Coomassie Brilliant Blue staining (Figure 1B). CD24 mFc was also analyzed by Western blotting using anti-mouse Fc antibody and commercial anti-CD24 antibody-Beckman (Figure 1C).

The biological activity of CD24 mFc fusion protein using natural P-selectin ligand was detected by ELISA and fluorescence-activated cell sorting (FACS) assays.

P-selectin, which is expressed on activated platelets and endothelial cells, is a well-known CD24 ligand. First, 0.1, 0.5, and 1.5 μg/ml of P-selectin were applied to 96-well plates for ELISA assay. As shown in FIG. 1D , the binding between the CD24 mFc fusion protein and P-selectin increased in a dose-dependent manner. In addition, the ability of the P-selectin fusion protein to bind to 293 cells expressing recombinant human CD24 protein was confirmed by FACS analysis. For this purpose, a CD24 expression construct without mFc attached to its N-terminus was generated, and the new construct was introduced into 293T cells to express CD24 on the cell membrane. As shown in Figure 1E, P-selectin Fc fusion protein can specifically recognize recombinant CD24 expressed by 293 cells.

Example 3: Mouse immunization

Purified CD24 mFc fusion protein is used for mouse immunization. Briefly, 250 μg of purified recombinant CD24 mFc fusion protein is injected intraperitoneally into mice, and pre-immunization blood samples from 5 mice (e.g., female BALB/c; 4 to 6 weeks of age) are collected. During the immunization experiment, an appropriate volume of PBS containing 25 to 100 μg of CD24 mFc antigen is administered to each mouse. Latex containing 100 μg of CD24 mFc is injected intraperitoneally into mice using a 22-gauge needle. Potential toxicity is monitored after immunization. After the first injection, mice are further immunized twice with 50 μg of CD24 mFc every 2 weeks, for a total of three injections. After two to three weeks, blood samples are collected from immunized mice, and the antibody titers of the blood samples are measured using ELISA or FACS analysis.

Purified recombinant human CD24 fusion protein was used for mouse immunization. Pre-immunization blood samples were collected from 5 mice (female BALB/c; 4 to 6 weeks old). Mice were first immunized by intraperitoneal injection of 250 μg of purified CD24 mFc fusion protein, followed by two injections (50 μg) every 2 weeks for a total of three injections. Potential toxicity was monitored after immunization. Three weeks after the third injection of CD24 Fc, blood samples were collected from 5 mice and antibody activity was measured using Western blot or FACS analysis. As shown in Figure 1F, mouse immunization was successful, as each mouse was able to produce antibodies against human CD24 using flow cytometry (FC).

Example 4: Evaluation of immune effect by FC analysis.

CD24 positive cells were incubated with 5 mM ethylenediaminetetraacetic acid (EDTA) for 10 minutes to detach cells from the culture dish. Cells were then washed twice with 5 ml PBS to remove EDTA. 5x10 ⁵ cells were incubated with 1/20 diluted mouse serum at 4°C for 40 minutes and then treated with goat anti-mouse conjugated with PE. Samples were protected from light before FC analysis using Calibre 2 software. Data acquisition and analysis were performed using Calibur CellQuest Pro (BD) software.

Example 5: Establishment of hybridoma

Immunized mice with the best humoral responses are selected and sacrificed to isolate B cells from their spleen for subsequent fusion protocols. Fusion between myeloma cell line Sp2/0-Ag14 and B cells is performed using polyethylene glycol (PEG), followed by aminopterin selection to eliminate unfused myeloma cells. PEG acts as a fusogenic agent, fusing the plasma membranes of adjacent myeloma and/or antibody-secreting cells to produce a single cell with two or more nuclei. Unfused myeloma cells are eliminated by aminopterin due to the lack of a rescue pathway. Aminopterin is used to block the pathway by which cells synthesize nucleotides de novo and forces them to synthesize nucleotides via a rescue pathway, which myeloma cells do not have. B cells with functional rescue pathways do not survive long in culture and eventually die after two weeks. Therefore, only fusions can survive drug selection, and the obtained fusion population is then initially screened for antibody production capacity.

Mouse B cells were isolated from spleen and fused with mouse myeloma cell lines using PEG. Unfused cells were eliminated by culturing in HAT selection medium. A total of 9 positive fusion tumor clones (CD24 mAb H1-H9, H1-H9) were first isolated for further analysis. The 9 fusion tumor clones were then tested for their ability to secrete anti-CD24 mAb.

Example 6: Fusion tumor screening

One week after fusion and drug selection, the supernatants of fused cell clones were examined for the presence of anti-CD24 mAb using an ELISA assay, in which CD24 mFc was used as an antigen and an irrelevant Fc fusion protein was used as a negative antigen control group. Supernatants from clones that bound only the CD24 mFc antigen were considered potential positive clones, while those that bound both CD24 mFc and the negative antigen control group were considered false positive clones. After the initial screening, positive clones that secreted large anti-CD24 mAb titers were selected and expanded to 24-well plates. The resulting positive clones were isolated by limiting dilution, and single-cell-grown fusion tumor cells were subjected to a second round of screening to obtain fusion tumor clones with anti-CD24 mAb production capacity. The resulting fusion tumor clone was used to produce anti-CD24 mAb.

As shown in Figure 2A, FC analysis was performed on the supernatants (SN) of the clones of the nine fusion tumors. Beekman CD24 mAb was used as the positive antibody control group; the test cell lines were BT-549, MDA-MB-468, Hs-578T and MCF-7; each cell line was immersed in 10-fold diluted fusion tumor supernatant for 45 minutes and 250-fold diluted Alexa-488 α-anti-mouse antibody for 45 minutes. The analysis results showed that the clones of the nine fusion tumors were able to secrete anti-CD24 mAbs that specifically bind to CD24 on the surface of TNBC cell lines.

As shown in Figure 2B, ELISA assay also showed that the supernatants of 9 fusion tumors could specifically bind to CD24 antigen.

Example 7: Preparation of anti-CD24 mAb using the mouse ascites method

To obtain sufficient mAb, subsequent functional evaluation of anti-CD24 mAb was performed. Production of CD24 mAb in vivo is also known as the mouse ascites method.

One week before inoculation of anti-CD24 mAb-producing fusion tumor cells, BALB/c mice were first injected intraperitoneally with 1 ml of pristane. The anti-CD24 mAb-producing fusion tumor was cultured in a 175 cm ² culture flask with complete DMEM-10/HEPES/pyruvate at 37°C. The cells were then harvested and centrifuged and the supernatant removed. The cells were washed and resuspended in PBS at 2.5×10 ⁶ cells/ml. 2 ml of fusion tumor cells were injected intraperitoneally into BALB/c mice. Two weeks later, a 19G needle was inserted into the mouse peritoneal cavity to drain the mouse ascites fluid containing a high concentration of anti-CD24 mAb and collect it.

Each fusion tumor was injected into mice via peritoneal injection, and the ascites was collected and purified three weeks after injection. The collected ascites was filtered and purified by Protein A-Sepharose Fast Flow (PASFF), and then the CD24 mAb was eluted with a citric acid buffer solution with a pH value of 3.0. The final buffer was replaced with a PBS solution with a pH value of 7.0. The purity and amount of the purified CD24 mAb from each fusion tumor were analyzed by SDS-PAGE, as shown in Figure 2C. The purity of the CD24 mAb clones of the 9 fusion tumors in Figure 2C is shown in Table 1.

Table 1. Purity of CD24 mAb clones from fusion tumors

Example 8: Purification of anti-CD24 mAb from mouse ascites

Anti-CD24 mAb was purified from mouse ascites using a poly-A based capture step followed by an additional chromatography step. Briefly, ascites was applied to a poly-A column and washed with PBS. The elution buffer was then applied to the poly-A column for CD24 mAb purification. The purified CD24 mAb was passed through a 0.22 μm filter to remove potential contaminants.

Example 9: Evaluation of the sensitivity of CD24 mAb H1 (H1) by FC.

The sensitivity of nine purified CD24 mAbs was investigated. 1x10 ⁵ MDA-MB-468 cells were incubated with individual CD24 mAbs at a concentration of 50 μg/mL for 30 minutes and then treated with mouse secondary antibodies conjugated with Alexa 488 for 30 minutes. As shown in Figure 2D, H1 had the highest mean fluorescence intensities (MFI) among the nine CD24 mAbs.

The sensitivity of each CD24 mAb was further evaluated by serial dilution, followed by FC analysis. 1x10 ⁵ MDA-MB-468 cells were incubated with a series of CD24 mAbs at concentrations of 5 μg/mL, 0.5 μg/mL, and 50 μg/mL for 30 minutes, followed by treatment with mouse secondary antibodies conjugated to Alexa 488 for 30 minutes. As shown in Figure 2E, all CD24 mAbs at a concentration of 5 μg/mL were sufficient to produce significant MFIs, indicating that they bind to CD24 with high sensitivity.

Purified H1 was labeled with Cy5 fluorescent dye using the Cy5® Binding Kit (Abcam, CA, USA) according to the manufacturer’s instructions.

Example 10: Imaging of in vivo imaging system (IVIS)

Mice were intravenously injected with 100 μl of H1 labeled with Cy5 fluorescent dye, and the fluorescence signal was recorded on days 1, 3, and 7 after injection. The mice were then transferred to the imaging chamber of the IVIS Spectrum to analyze the emitted fluorescence signal at a wavelength of 647 nm (Perkin Elmer Inc., MA, USA). Fluorescence was measured and analyzed using Living Image Software 4.0 (Perkin Elmer Inc., MA, USA).

Example 11: Surface plasmon resonance (SPR) analysis

The binding affinity of each CD24 mAb clone was determined by SPR. BiacoreTM 8K, a high-throughput and high-sensitivity SPR system, was used. 10 nM recombinant CD24 mFc protein was first dissolved in NaOAc, pH 5.0, and immobilized on the metal surface of the sensor chip by amine coupling before measurement. Single cycle kinetic (SCK) procedures were performed using 5 different concentrations of CD24 mAb, and the dissociation constant Kd and association constant Ka were measured. The binding affinity (KD) of CD24 mAb is determined by the ratio of Kd and Ka. We determined the binding affinity of 8 CD24 mAbs, and the KD values ranged from 9-10M to 10-13M, which have good binding affinity. Among them, the binding affinity of H6, H7 and H8 is much higher than that of H1, H3, H4, H5 and H9 (Figure 3). In summary, all 8 clones of anti-CD24 mAb have strong binding affinity to CD24.

The CM5 series sensor chip was first docked with BiacoreTM 8K (GE Healthcare Life Sciences, NY, U.S.A.) with 0.05% P20 in PBS as the running buffer. In a NaOAc solution with a pH of 5.0, 10nM CD24 mouse fusion protein (ligand) was immobilized on the CM5 sensor chip by amine coupling at a flow rate of 10 μl/min for 30 seconds. Five concentrations of anti-CD24 mAb (7.5, 15, 30, 60, 120nM) were used as analytes for the SCK procedure with a flow rate of 50 μl/min. The binding affinity of 8 purified anti-CD24 mAbs to the CD24 mouse fusion protein ligand was examined. The association constant ka and dissociation constant kd were fitted using a 1-to-1 binding model, and the binding constant KD was calculated by the following formula:

Example 12: FACS analysis

CD24 positive cells were incubated with 5mM EDTA for 10 minutes to detach cells from the culture dish. Cells were then washed twice with 5 ml PBS to remove EDTA. 5x105 cells were incubated with mouse serum, fusion tumor supernatant, or purified CD24 mAb at a 1/20 dilution at 4°C for 40 minutes and then treated with PE-conjugated goat anti-mouse antibody. Samples were protected from light before FACS analysis with Calibre 2. Data acquisition and analysis were performed using Calibur CellQuest Pro (BD) software.

Example 13: Cell viability assay (MTS assays)

5000 cells were seeded in 96-well plates and incubated with a series of concentrations of CD24 mAb (0.312, 0.625, 1.25, 2.5, 5, 10, 20 μg/mL) and the CellTiter kit (Promega, CA, USA) was used according to the manufacturer’s instructions after three days of incubation.

Example 14: Antibody-mediated receptor internalization assay

The ability of mAbs to induce internalization of the mAb/target complex after binding to a tumor cell surface target is one of the hallmarks of an antibody's therapeutic potential. The ability of CD24 mAbs to trigger surface CD24 internalization was tested by a receptor internalization assay. MDA-MB-468 cells were stained with each of the nine CD24 mAbs for 30 minutes at 4°C, followed by staining with an Alexa 488-conjugated anti-mouse antibody for 30 minutes. The membrane localization of CD24 was observed using a fluorescent microscope at 4°C, and the CD24 internalization assay was performed after incubating the cells at 37°C for 24 hours.

As shown in Figure 4, all CD24 mAbs can trigger CD24 internalization. In addition, H1 and H9 showed better CD24 staining cell internalization intensity.

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

Example 15: Antigenic mapping

A scanning peptide library was designed and constructed based on the CD24 amino acid sequence. The overlapping peptides were offset by two residues into 12-mers, and each peptide was biotinylated for subsequent ELISA assay.

As shown in Figure 6, four overlapping peptides sharing the same amino acid sequence NSGLAP were found to react to all nine CD24 mAbs, indicating that all nine antibody clones reacted to the same CD24 epitope-determining amino acid sequence (including NSGLAP).

Example 16: Construction of hCD24 expression lentiviral vector and preparation of lentiviral particles

A fusion gene was designed that contains the coding sequence of the mouse signal peptide (interlulin-2, mIL-2) in 5 major regions, followed by the coding sequence of the mature human CD24 polypeptide. The mIL2_hCD24GPI fusion gene was synthesized by MDBio, Inc. and cloned into the lentiviral vector pLVX using specific restriction enzymes. The resulting pLVX-mIL2_hCD24GPI lentiviral plasmid was amplified using the plasmid DNA Maxiprep kit.

Lentiviral particles were prepared using the following protocol provided by the RNA Technology Platform and Gene Manipulation Core Facility (RNAi core) of Academia Sinica, Taipei, Taiwan. Briefly, 2.0x10 ⁶ 293T cells were seeded in 10 cm dishes 18 hours before transfection. Packaging plasmids of VSV-G-pseudotyped lentivirus (pCMVDR 8.91 and pMD.G) were mixed with Mirus TransIT at a predetermined ratio in OPTI-MEM transfection medium. Transfectants were added to 10 cm dishes and cells were transfected for 8 hours. After 8 hours, transfectants were removed and 10 ml of 1% BSA complete medium was added to the dish and transfected cells were incubated for 24 hours. After 24 hours, the supernatant was collected and stored at 4°C. Then, 10 ml of 1% BSA complete medium was added to the culture dish and cultured for another 24 hours. Then the new supernatant was harvested and mixed with the previously harvested supernatant. The last 20 ml of supernatant was centrifuged at 1,500 rpm for 5 minutes to remove cellular debris. The resulting lentiviral supernatant was further concentrated by mixing it with Lenti-X Concentrator solution and incubating the mixture at 4°C overnight 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 lentiviral particles were resuspended in 1 ml of complete medium and stored at -80°C.

The complete human CD24 coding sequence including the mature domain and the GPI-anchoring domain was constructed into the pLVX viral vector. pLVX lentiviral particles were prepared from 293T cells. MDA-MB-231-derived IV2 cells were then infected with viral particles for 48 hours and then drug selected in medium containing 400 μg/ml G418. Two weeks after drug selection, CD24 expression on the cell surface was analyzed using FC.

As shown in Figure 7, after drug selection, 89.4% of IV2 cells showed CD24 positivity. The CD24-positive cell population was then further sorted to obtain a cell population with a CD24 positivity rate of more than 99%. The sorted CD24-positive cell population was used to characterize the in vivo anti-tumor activity of 9 anti-CD24 mAbs.

Example 17: Evaluation of the in vivo anti-tumor efficacy of CD24 mAb using a xenograft severe combined immunodeficiency (SCID) mouse model

For the MDA-MB-468 xenograft model, 2.5x10 ⁶ cells mixed with matrigel were injected into the 4th mammary fat pad of mice three days before the monotherapy of CD24 mAb. In view of the fact that MDA-MB-468 cells only produce slow-growing tumors, in order to evaluate the long-term effect of CD24 mAb injection, the treatment regimen was designed as follows: Treatment regimen 1: Tumor-bearing mice received CD24 mAb monotherapy, with CD24 mAb intravenous injection twice a week at a dose of 5 mg/kg or 10 mg/kg for a total of 10 injections. Treatment 2: CD24 mAb was injected intravenously twice a week at a dose of 5 mg/kg or 10 mg/kg, and then suspended for two weeks. Treatment 3: Tumor-bearing mice were injected intravenously with CD24 mAb once a week at a dose of 5 mg/kg or 10 mg/kg for a total of 7 injections. Tumor growth was recorded every three days, and the mice were eventually sacrificed humanely.

For the IV2-CD24 xenograft model, 1x10 ⁶ cells mixed with matrix gel were injected into the fourth mammary fat pad of mice three days before the monotherapy of CD24 mAb. CD24 mAb was injected intravenously once a week at a dose of 5 mg/kg for a total of 8 injections within two months. Mice were humanely sacrificed and their xenograft tumors were removed and fixed in formalin solution for further examination.

As shown in Figure 8A, 9 cloned candidate CD24 mAbs were tested for their anti-tumor potential in vivo. 1x10 ⁶ IV2-CD24 cells were injected into the fourth mammary fat pad of immunized mice. Then three days later, tumor-bearing mice were treated with CD24 mAbs via tail vein injection at a dose of 10 mg/kg once a week for a total of 8 injections within eight weeks.

In the first round of experiments, H1, H2, H3, H4, and H9 were evaluated, and these CD24 mAb groups were compared with the same control group in terms of tumor volume. In the second round of experiments, a second control group was used to compare tumor volume with the H5, H6, H7, and H8 groups. As shown in Figure 8B, H1 and H9 showed the best anti-tumor efficacy, while H6 showed moderate anti-tumor efficacy. Despite their high binding affinity to CD24, H2, H3, H4, H5, H7, and H8 did not show significant anti-tumor potential.

Example 18: Evaluation of the in vivo anti-tumor efficacy of CD24 mAb using a xenograft SCID mouse model

For the primary tumor resection model, 1x10 ⁶ luciferase-labeled IV2-CD24 (IV2-CD24-luc) cells were mixed with matrix gel and injected into the fourth mammary fat pad of mice. The tumor was resected at the sixth week when the tumor diameter reached approximately 1 cm. One week after primary tumor resection, CD24 mAb monotherapy was performed by intravenous injection. Mice were injected with PBS or CD24 mAb at a dose of 10 mg/kg once a week for a total of 6 injections. Lung metastasis was detected in mice using IVIS. Briefly, mice were injected intravenously with 0.2 ml of 5 mg/ml luciferin and anesthetized with isoflurane gas. The mice were placed in an IVIS chamber to detect luminescence signals from the mouse lungs. The photon signals were calculated using living image software.

The anti-tumor efficacy of H1 in different TNBC xenograft models using MDA-MB-468 cells was evaluated in Figure 9. 2.5x10 ⁶ MDA-MB-468 cells were injected into the fourth mammary fat pad of SCID mice, and tumor-bearing mice received three rounds of CD24 mAb treatment at two different doses of 5 mg/kg and 10 mg/kg (Figure 9A and Figure 9B).

As shown in Figure 9C, tumor-bearing mice in the three groups had similar tumor volumes in the first week.

As shown in Figure 9D, administration of 5 mg/kg and 10 mg/kg of H1 can effectively inhibit the growth of primary tumors in the MDA-MB-468 xenograft mouse model.

In summary, the anti-tumor activity of the tested mAbs was demonstrated. H1 showed the best potential and was selected as the lead antibody.

Then, the next experiments were to confirm that targeting of the selected CD24 mAb to the tumor site could lead to tumor shrinkage.

1 mg of H1 was covalently coupled to Cy5 fluorescence dye using an amine coupling kit. 1x10 ⁶ MDA-MB-468 cells were orthotopically transplanted into SCID mice to establish xenograft tumors. Two weeks after tumor inoculation, 100 μg of Cy5-labeled H1 was injected into tumor-bearing mice via the tail vein. Two days later, mice were anesthetized with isoflurane and placed in an IVIS chamber with oxygen supply, and the fluorescence signal was measured using the IVIS imaging system.

As shown in Figure 10, Cy5-labeled H1 was detected at the tumor site imaged by IVIS, indicating that H1 has tumor targeting ability.

Example 19: Distant lung metastasis in a primary tumor resection mouse model

As shown in Figure 11, ^1x106 IV2-CD24 cells were injected into the 4th mammary fat pad of SCID mice, and the primary tumor was excised when it reached 1 cm in diameter. As shown in Figures 11B and 11C, the tumor growth curves showed that eight tumor-bearing mice carried similar tumor burdens. After the primary tumors of the mice were excised, the mice were randomly divided into two groups (samples per group = 4). One week after the primary tumor was excised, the mice were injected with 10 mg/kg H1 through the tail vein once a week for a total of four injections. Lung metastasis status was monitored using IVIS at week 11 after the fourth mAb injection.

As shown in Figure 11D, mice injected with CD24 mAb showed reduced lung metastasis compared to control mice. Moreover, as shown in Figure 11E, no lung metastasis was observed in the lung tissue of mice treated with CD24 mAb by HE staining (hematoxylin-eosin staining). Three mice received CD24 mAb injection at the end of the experiment, and all survived (Figure 11F).

Example 20: Cell culture

MDA-MB-468 and IV2 cells were maintained in complete DMEM medium containing 10% fetal bovine serum (FBS), 2mM L-glutamine, and 1% penicillin/streptomycin. Stable cell lines expressing CD24 were maintained in complete medium containing 400 μg/mL G418. Stable cell lines expressing GFP-luciferase were cultured in 5 μg/mL puromycin. All cell lines were maintained in a humidified incubator at 37°C and 5% CO ₂ .

Example 21: Combination therapy of anti-CD24 mAb and docetaxel

According to previous clinical trials, immune checkpoint inhibitors (ICIs) are often used in combination with standard chemotherapy drugs, showing further improvement in cancer patients compared with chemotherapy alone. Therefore, the synergistic anti-tumor effect of the combination therapy of H1 and docetaxel was evaluated in a xenograft mouse model.

The experimental design is shown in Figure 12A. First, 2x10 ⁶ MDA-MB-231-IV2 cells expressing CD24 were injected into the fourth mammary fat pad of SCID mice one week before treatment. 2 mg/kg H1 was injected via peritoneal injection once a week for a total of 8 injections. 5 mg/kg docetaxel was injected intravenously one day after receiving CD24 mAb for a total of 4 injections.

The treatment groups were designed as follows: tumor-bearing mice were injected intravenously with (1) PBS (as control group) (sample number = 4), (2) H1 (sample number = 5), (3) docetaxel (sample number = 5), and (4) H1 plus docetaxel (as combination therapy group) (sample number = 5) weekly for six weeks. 10 mg/kg H1 and 2.5 mg/kg docetaxel were used for combination therapy. Tumor growth was monitored and recorded during six weeks.

As shown in Figure 12B, compared with H1 or docetaxel monotherapy, H1 plus docetaxel treatment observed significant synergistic anti-tumor effects in the early stage of treatment and continued to the end of the study. In this embodiment, the tumor growth pattern was 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 time was 106.8 days in the control group and 97.4 days in the docetaxel group. Neither the H1 group nor the combination therapy group reached the median survival time. Compared with the control group and the docetaxel group, both monotherapy and combination therapy of H1 significantly prolonged the survival of tumor-bearing mice. In addition, mice receiving combination therapy showed the best survival benefit.

Three days before the combination therapy, ^1x106 IV2-CD24 cells mixed with matrix gel were injected into the fourth mammary fat pad of mice. The difference between H1 plus docetaxel treatment and PBS plus docetaxel treatment in the combination therapy was evaluated.

Example 22: Evaluation of the potential toxicity of H1

The toxic potential of H1 can be assessed by liver function, kidney function, and human erythrocyte binding affinity. Human whole blood from healthy donors was diluted 1:100 and stained with H1, followed by anti-mouse fluorescein fluorescent (FITC) staining.

As shown in Table 2, the liver and kidney functions of the H1-treated group did not exceed the normal range of the following indicators (ALT (GPT): liver function index (alanine aminotransferase); BUN: serum urea nitrogen (blood urea nitrogen); creatinine: creatinine).

Table 2. Evaluation and analysis of H1

As shown in Figure 13, H1 did not bind to human red blood cells, indicating that H1 is highly specific and has no cross-reaction with human red blood cells.

To further confirm the anti-tumor activity of H1, the CD24 level of the tumor was further considered. This example evaluates the anti-tumor effect of H1 in CD24-negative and CD24-positive TNBC xenograft mouse models.

As shown in Figure 14A, H1 significantly inhibited the growth of CD24-positive tumors. However, Figure 14B showed that H1 had limited anti-tumor effects on CD24-negative tumors, indicating that the anti-tumor activity of H1 is completely dependent on the CD24 level of the tumor.

Example 23: Immunohistochemistry (IHC) of CD24 in clinical samples

To elucidate the possible mechanism of H1's intrinsic antitumor activity, this example examined the tumor proliferation status of primary tumors excised from H1-treated or PBS-treated SCID mice. IHC was used to analyze the distribution of myeloid-derived suppressor cells (MDSC), tumor-infiltrating macrophages (TIM), and natural killer cells (NK).

The intensity of CD24 expression in IHC-stained tissue samples was evaluated and scored by two independent researchers and reviewed by a board-certified pathologist. Immunostaining was graded into three categories: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong.

As shown in Figures 15A and 15B, the number of tumor-infiltrating CD11b-positive MDSCs was significantly reduced and the number of F4/80-positive tumor-infiltrating macrophages was significantly increased in H1-treated mice compared with PBS-treated mice. In addition, H1-treated tumors were severely surrounded and invaded by F4/80-positive macrophages. In addition, the number of tumor-infiltrating CD68+M1 macrophages was significantly increased in H1-treated tumors compared with PBS-treated tumors. At the same time, as shown in Figures 15A and 15B, similar numbers of CD206-positive M2 macrophages were shown in PBS-treated and H1-treated tumors. In addition, similar numbers of CD206-positive M2 macrophages were observed in PBS-treated and H1-treated tumors. Similar numbers of Ki67-positive tumor cells were observed in PBS-treated and H1-treated tumors. The above results all indicate that H1 has no effect on tumor cell proliferation. And the above results indicate that in the anti-tumor effect of H1, the role of tumor-killing macrophages is more significant than the inhibition of cell growth.

Example 24: Sequencing of VH and VL of H1 by 5' Rapid Amplification of cDNA End (RACE) and next generation sequencing (NGS)

As shown in Figure 16, the sequencing process starts with total RNA extracted from a fusion tumor clone that secretes H1. The antibody cDNA library was established using modified RACE and Fc-specific reverse transcription primers. The antibody cDNA was prepared by PCR amplification using universal forward and reverse primers. The antibody cDNA library was then subjected to RNA sequencing analysis. The CDR features in the antibody binding region (fragment antigen binding, Fab) domain of the H1 clone were analyzed using the online CDR prediction algorithm AbodyBuilder. The sequence identification of the three individual CDRs of VH and VL is shown in Table 3.

Table 3. Sequence identification of three individual CDRs of VH and VL

The generation, production, and anti-tumor validation of the chimeric anti-human CD24 mAb H1 (CH-01) were then evaluated.

The Fab of H1 was first subcloned into an expression vector upstream of the human IgG1 Fc sequence. CH-01 with the human IgG1 Fc domain was produced in 100 ml ExpiCHO cell culture (Figure 17A and Figure 17B), and the antibody was purified and validated using SDS-PAGE combined with Coomassie Brilliant Blue staining. The anti-tumor efficacy of CH-01 was then tested using the CD24-positive MDA-MB-468 orthotopic mouse model.

As shown in Figures 17C and 17D, tumor-bearing mice treated with CH-01 showed a decrease in photon counts during CD24 mAb treatment compared to the IgG control group. And three mice in the CD24 mAb-treated group had almost no detectable luminescence signal 3 months after injection of CH-01. Similarly, direct measurement of tumor volume in Figure 17E also showed that the tumors in the CD24 mAb-treated group were significantly smaller than those in the control group. In addition, the anti-tumor activity of CH-01 was further confirmed in a second CD24-positive TNBC xenograft model using IV2-CD24TNBC cells. As shown in Figure 17F, when CH-01 was used at a dose of 0.5 mg/kg or 5 mg/kg, it effectively inhibited tumor growth.

Example 25: Humanization of anti-CD24 mAb

Computer-based simulations were used to calculate the molecular dynamics (MD) trajectory of mouse H1 and predict the MD trajectory of humanized CD24 mAb, and the root-mean-square deviation (RMSD) value of the humanized CD24 mAb structure was calculated.

As shown in Figure 18, the difference of atomic RMSD was further adjusted to obtain weighted RMSD (wRMSD). Compared with other predictions, the wRMSD values of the two combinations containing 2 humanized heavy chains (SEQ ID NO: 14 or SEQ ID NO: 18 ₎ and 1 humanized light chain (SEQ ID NO: 16) ( _L1H1 : 2.111; _L1H2 : _2.056 ) were closest to the wRMSD value of mouse H1 in Figure 18A ( _H1 : _1.556 ). The molecular superposition of _L1H2 and mouse H1 is shown in Figure 18B. The molecular superposition also shows that the MD trajectory of the Fab domain of CD24 mAb HH-01-46 (HH-01-46) ( _{L1H2 )} is very similar to the MD trajectory of the Fab domain of H1. Finally, the combination of humanized light chain _L1 and humanized heavy chain _H2 was selected as the lead humanized HH-01-46, and the antibody sequence was cloned into the expression vector pFUSE-hIgG1Fc, as shown in Figure 18C.

In another embodiment, pFUSE-Ig expression plasmids containing humanized heavy chain and humanized light chain, respectively, were transfected into Expi293 cells to produce recombinant humanized anti-CD24 mAb. Humanized CD24 mAb was purified using a poly-A column and verified by Coomassie Brilliant Blue staining, as shown in FIG. 19A , respectively verifying the antigen binding sensitivity and specificity of HH-01-46 using CD24-positive MDA-MB-468 and CD24-negative MDA-MB-231.

As shown in Figure 19B, HH-01-46 has similar antigenic sensitivity and specificity to its parental CH-01. The binding affinity of humanized HH-01-46 was determined by surface plasmon resonance (SPR). Multiple cycle kinetics (MCK) analysis showed that the KD constant of HH-01-46 was 92.9nM, indicating that HH-01-46 has similar affinity to the parental CD24 mAb, with a KD constant of 69.5nM (Figure 19C). FC analysis showed that humanized HH-01-46 can bind to a series of TNBC cell lines, and it shows similar binding strength to the parental CH-01 (Figure 19D)

The anti-tumor effect of HH-01-46 was then evaluated in a CD24-positive TNBC xenograft mouse model. SCID mice were inoculated with ^2x106 GFP-Luc-labeled IV2-CD24 cells in the fourth mammary fat pad and then treated with HH-01-46 intravenously once a week for 4 weeks.

As shown in Figures 20A and 20B, based on IVIS analysis, the anti-tumor efficacy of HH-01-46 was as good as that of the parent CH-01, inhibiting 95% of tumor growth, as shown in Figure 20C.

In one embodiment of CH-01, a model was established using human peripheral blood mononuclear cells (PBMCs) to reconstruct the anti-tumor efficacy shown in the immune mouse model.

Example 26: PBMCs reconstructed immune mouse model

The advanced severe immunodeficiency (ASID) mouse model (NOD.Cg-Prkdcscid Il2rgtm 1 Wjl/YckNarl) was used. First, 40 ml of whole blood was drawn from a healthy donor and diluted with 1X PBS at a ratio of 1:1. Then PBMCs were separated using density gradient centrifugation. Finally, 2.5x10 ⁷ PBMCs were obtained from 40 ml of whole blood. Next, each ASID mouse was injected with 1x10 ⁷ PBMCs 7 days before tumor inoculation. As shown in Figure 21B, after reconstruction with PBMCs, ASID mice were injected with 2.5x10 ⁵ IV2-CD24 cells on day 7. PBMC humanized ASID mice received 5 mg/kg CH-01 monotherapy, 5 mg/kg docetaxel monotherapy, and CH-01 plus docetaxel combination therapy on days 9, 14, 21, and 27, respectively. The flow chart of the PBMC humanized ASID mouse trial is shown in Figure 21A. Tumor growth was recorded until day 21 during the trial. As shown in Figure 21B, both CH-01 monotherapy and docetaxel monotherapy induced tumor regression in tumor-bearing ASID mice in the early stage of treatment. The antitumor activity of the combination therapy was compared with single drug treatment, and the results are shown in Figure 21B. The combination therapy of CH-01 plus docetaxel had the best tumor inhibition effect.

In addition, the role of HH-01-46 in promoting macrophage phagocytosis, including inducing NK cell activation and tumor killing ability, was also evaluated. As shown in Figure 22A, CD24-positive TNBC cells incubated with monocyte-derived human macrophages in the presence of HH-01-46 effectively promoted antibody-dependent phagocytosis. As shown in Figure 22B, HH-01-46 treatment also activated NK92MI cells to secrete tumor killing cytokines IFN-γ and IL-12 in a time-dependent manner. In addition, as shown in Figure 22C, antibody-dependent cell-mediated cytotoxicity (ADCC) assays using human PBMC and NK92MI indicated that HH-01-46 could effectively promote antibody-dependent cell-mediated cytotoxicity.

In addition, 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 5 μg/mL 24 hours before EGF stimulation.

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.

Although the present invention has been described and illustrated in sufficient detail for those skilled in the art to make and use it, various substitutions, modifications and improvements should be apparent without departing from the spirit and scope of the present invention.

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the above-mentioned objects and obtain the above-mentioned objects and advantages, as well as those inherent therein. The above-mentioned processes and methods for producing them represent preferred embodiments, are exemplary, and do not limit the scope of the invention. Those skilled in the art will think of modifications and other uses thereof. Such modifications are included in the spirit of the invention and are limited by the scope of the claims.

TWI862019B_112126897_SEQL.xml

Claims (17)

An isolated antibody that specifically binds to CD24, the isolated antibody comprising: (a) heavy chain variable region-complementary determining region VH-CDR1, VH-CDR2 and VH-CDR3, wherein the VH-CDR1 comprises the amino acid sequence of SEQ ID NO: 2, the VH-CDR2 comprises the amino acid sequence of SEQ ID NO: 4, and the VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 6; and (b) Light chain variable region - complementary determining regions VL-CDR1, VL-CDR2 and VL-CDR3, wherein the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 10, and the VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 12. The isolated antibody as described in claim 1, wherein the VH comprises the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 18. The isolated antibody as described in claim 1, wherein the VL comprises the amino acid sequence of SEQ ID NO: 16. The isolated antibody as described in claim 1 further comprises an Fc region. The isolated antibody as described in claim 4, wherein the Fc region is an IgG, IgM, IgA, IgD, IgE antibody or any subclass thereof. The isolated antibody as described in claim 5, wherein the Fc region is IgG. A pharmaceutical composition comprising an isolated antibody as described in claim 1 and a pharmaceutically acceptable carrier or formulation. A composition for preparing a drug for treating cancer expressing CD24, wherein the composition comprises the isolated antibody as described in claim 1 and an anticancer drug. The use as described in claim 8, wherein the anticancer drug comprises docetaxel, tamoxifen, herceptin or a combination thereof. The use as described in claim 8, wherein the cancer is a solid tumor. The use as described in claim 8, wherein the cancer is breast cancer, liver cancer or ovarian cancer. The use as described in claim 11, wherein the breast cancer is triple-negative breast cancer (TNBC). The use as described in claim 12, wherein the triple-negative breast cancer is triple-negative breast cancer with lung metastasis. A method for detecting CD24 expression in an in vitro sample, the method comprising: (1) Providing an in vitro sample from an individual; (2) contacting the in vitro sample with a capture antibody that specifically binds to CD24, wherein the capture antibody is the isolated antibody as described in claim 1; (3) adding a labeled detection antibody, which binds to the capture antibody to form an immune complex consisting of CD24, capture antibody and labeled detection antibody; and (4) Determine the amount of marker in the immune complex formed to determine the presence or level of CD24 in the in vitro sample. The method as described in claim 14, wherein the in vitro sample is selected from at least one of blood, lymph fluid, tissue fluid, body cavity fluid, oral mucosal fluid, circulating tumor cells, or a combination thereof. The method as described in claim 15, wherein the in vitro sample is blood. The method as described in claim 16, wherein the blood is whole blood, plasma or serum.