Bispecific antibody fusion proteins targeting cd24 and cd47 and methods of making and using the same

Abstract

The application discloses a bispecific antibody fusion protein targeting CD24 and CD47 and a preparation method and application thereof. The bispecific antibody fusion protein comprises a CD24 antibody heavy chain, a CD24 antibody light chain and a CD47-Fc binding peptide, wherein the constant region of the CD24 antibody heavy chain is connected with the Fc segment of the CD47-Fc binding peptide through a "lock and key structure", and the fusion protein can be combined with CD47 and CD24 simultaneously. The bispecific antibody fusion protein has the functions of anti-CD24 and CV1-hFc, and can block the proliferation of tumor cells mediated by CD47 and CD24. In addition, the bispecific antibody fusion protein PPAB001 can maximize the traditional monoclonal antibody structure like a traditional IgG molecule, and due to the presence of the Fc segment, the bispecific antibody fusion protein can be purified through a Protein A column affinity chromatography method, and is beneficial to large-scale production and purification.

Description

Technical Field

[0001] This invention relates to the field of tumor immunology, and more specifically, to a bispecific antibody fusion protein targeting CD24 and CD47, its preparation method, and its application. Background Technology

[0002] Malignant tumors are a serious threat to human health, ranking second among all causes of death. In recent years, the incidence of malignant tumors has increased significantly. At the same time, their treatment outcomes are poor, they are prone to metastasis in advanced stages, and their prognosis is unfavorable. Currently, conventional clinical treatments such as radiotherapy, chemotherapy, and surgery have greatly alleviated pain, but these therapies still have significant side effects and limitations, making it difficult to further improve their efficacy.

[0003] The development and progression of tumors is a multifactorial, multi-mechanism, and multi-factor process. CD47, or integrin-associated protein (IAP), is expressed on the cell surface and can bind to signal-regulatory protein α (SIRPα) on the macrophage surface, thereby phosphorylating the intracytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) of SIRPα. This recruits the phosphatase SHP-1 and sends an inhibitory signal to macrophages, warning them to "don't eat me." The widespread expression of CD47 on the surface of tumor cells inhibits the phagocytic effect of macrophages on tumor cells. Based on this, researchers have developed CD47 antagonists that can activate the phagocytic effect of macrophages. CD47 antagonists activate macrophage phagocytosis by blocking the binding of CD47 and SIRPα, enhancing the anti-tumor immune response, and ultimately inhibiting tumor growth.

[0004] CD24, also known as a heat-stable antigen (HSA), is a short peptide containing 31 amino acids. As a cell adhesion molecule, CD24 exhibits highly variable glycosylation that is cell-type dependent. Highly glycosylated CD24 is anchored to lipid rafts within the cell membrane via phosphatidylinositol (GPI). Studies have shown that CD24 is overexpressed on the surface of various tumor cells, including breast, ovarian, and lung cancer. It can bind to the inhibitory receptor Siglec-10 expressed on the surface of macrophages, sending a "don't eat me" signal that limits macrophage phagocytosis. This immunosuppressive signal often works through complementarity with the classic CD47 signaling pathway, thus CD24 may be a novel target for tumor immunotherapy. Further research has revealed that anti-CD24 antibodies can effectively activate macrophage phagocytic activity. Therefore, CD24 is not only a newly discovered immune checkpoint but also a potential target for anti-tumor drugs. In addition, the study found that CD24 molecules are not expressed on the surface of human red blood cells, so CD24-targeted drugs do not pose a potential risk of blood toxicity.

[0005] Therefore, if CD47 and CD24 can be blocked simultaneously, not only can the invasion and metastasis of tumors caused by CD24 signal activation be inhibited, but the tumor suppressor signals mediated by CD47 and CD24 will also be blocked, activating the immune response against the tumor. Thus, it may have a better synergistic effect in inhibiting tumor growth and proliferation than blocking CD24 or CD47 alone, which will be of great significance for the treatment of tumors.

[0006] However, the combined use of monoclonal antibodies has several limitations. First, there are still no detailed reports on the safety and efficacy of using two antibodies in clinical practice, which may pose safety risks. In addition, the combined use of two antibodies also has drawbacks such as regulatory disorders and high cost, which limit its clinical application.

[0007] Therefore, constructing a bispecific antibody fusion protein that can simultaneously block two targets using genetic engineering technology, thereby blocking both CD24 and CD47 and thus more effectively inhibiting tumor growth and proliferation, has become an urgent technical problem to be solved. Summary of the Invention

[0008] The purpose of this invention is to provide a bispecific antibody fusion protein targeting CD24 and CD47, its preparation method, and its application.

[0009] To achieve the objectives of this invention, in a first aspect, this invention provides a bispecific antibody fusion protein CD47 / CD24-BsAb-Ig (denoted as PPAB001) that targets CD24 and CD47, wherein the fusion protein comprises a CD24 antibody heavy chain, a CD24 antibody light chain, and a CD47-Fc binding peptide;

[0010] The constant region of the CD24 antibody heavy chain is connected to the Fc segment of the CD47-Fc binding peptide through a "lock-and-key structure".

[0011] The fusion protein can bind to both CD47 and CD24 simultaneously.

[0012] Further, the CD24 antibody light chain contains the amino acid sequence shown in SEQ ID NO:14, the CD24 antibody heavy chain contains the amino acid sequence shown in SEQ ID NO:16, and the CD47-Fc binding peptide contains the amino acid sequence shown in SEQ ID NO:18.

[0013] Secondly, the present invention provides a method for preparing the fusion protein, comprising the following steps:

[0014] S1. Construct vectors containing the nucleotide sequences shown in SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17 respectively, and co-transfect them into host cells for culture to screen cell clones that stably express the target fusion protein;

[0015] S2. The cell culture is isolated and purified to obtain the target fusion protein.

[0016] Furthermore, the vector can be the eukaryotic expression vector pcDNA3.1.

[0017] Furthermore, the host cell may be a CHO-K1 cell.

[0018] Furthermore, cell clones that stably express the target fusion protein were screened using a selective medium containing 600 μg / mL G418 and 250 μg / mL Zeocin.

[0019] Thirdly, the present invention provides a nucleic acid molecule encoding the fusion protein.

[0020] Fourthly, the present invention provides biological materials containing the nucleic acid molecules, including but not limited to recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

[0021] Fifthly, the present invention provides a composition containing the fusion protein, wherein the composition optionally includes an antitumor agent.

[0022] The antitumor agent is selected from at least one of the following A to E:

[0023] A. Cytotoxic drugs, including: (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrosamines, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxalate platinum; mitomycin C (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and alimta; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; nucleotide reductase inhibitors such as hydroxyurea. (HU) etc.; DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz (Gemz); (3) Drugs that act on nucleic acid transcription: drugs that selectively act on DNA templates and inhibit DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, scintillans, etc.; (4) Drugs that act on microtubule synthesis, such as paclitaxel, texole, vinblastine, vinorelbine, podophyllodes, homoharringtonine; (5) Other cytotoxic drugs, such as asparaginase, which mainly inhibit protein synthesis;

[0024] B. Hormonal drugs, including: (1) anti-estrogens, such as tamoxifen, droloxifene, exemestane, etc.; (2) aromatase inhibitors, such as ammonia glutide, lantron, letrozole, reninide, etc.; anti-androgens: (3) flutamethasone RH-LH agonists / antagonists, such as phenadol, phenadol, etc.

[0025] C. Biological response modifiers: including: (1) tumor interferons that mainly inhibit the body's immune function, such as interleukin-2; (2) thymosin peptides;

[0026] D. Monoclonal antibodies, such as cetuximab (C225); trastuzumab; and bevacizumab.

[0027] E. Others include some drugs whose mechanisms are currently unclear and require further research: including (1) cell differentiation inducers, such as retinoids; (2) cell apoptosis inducers.

[0028] In a sixth aspect, the present invention provides the use of the fusion protein or the composition in the preparation of antitumor drugs.

[0029] The tumors include, but are not limited to, breast cancer, ovarian cancer, colon cancer, and lung cancer, with breast cancer and ovarian cancer being preferred.

[0030] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0031] This invention uses genetic engineering technology to construct a bispecific antibody fusion protein CD47 / CD24-BsAb-Ig that can simultaneously block two targets. It includes a heavy chain on the anti-CD24 side, a light chain on the anti-CD24 side, and an Fc fusion protein on the anti-CD47 side. This bispecific antibody fusion protein can block both CD24 and CD47, thereby more effectively inhibiting tumor growth and proliferation.

[0032] The bispecific antibody fusion protein prepared using the method provided in this invention possesses both anti-CD24 and CV1-hFc functions, and can block the proliferation of CD47 and CD24-mediated tumor cells. Furthermore, like traditional IgG molecules, this bispecific antibody fusion protein retains the structure of traditional monoclonal antibodies to the greatest extent possible. Due to the presence of the Fc fragment, it can be purified using conventional Protein A column affinity chromatography, facilitating large-scale production and purification. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of the bispecific antibody fusion protein and the results of gel filtration chromatography purification in a preferred embodiment of the present invention; wherein, A is anti-CD24 protein, B is bispecific antibody fusion protein PPAB001, C is CV1-hFc protein, and D is the analysis results of the molecular weight of each component.

[0034] Figure 2 This is an SDS-PAGE verification experiment of the bispecific antibody fusion protein in a preferred embodiment of the present invention; from left to right, the first lane is the protein molecular weight control, the second lane is the CV1-hFc protein, the third lane is the anti-CD24 protein, and the fourth lane is the bispecific antibody fusion protein PPAB001.

[0035] Figure 3 The results of the bispecific antibody fusion protein binding activity with CD24 and CD47 are shown in the preferred embodiment of the present invention.

[0036] Figure 4 The results of flow cytometry verification of the binding of the bispecific antibody fusion protein to CD24 and CD47 double-positive cells in a preferred embodiment of the present invention are shown.

[0037] Figure 5 This is an immunofluorescence experiment demonstrating the activation of macrophages by a bispecific antibody fusion protein, as described in a preferred embodiment of the present invention. In this experiment, A represents the immunofluorescence observation of macrophage phagocytosis of breast cancer cells (fluorescence microscope magnification 400x), and B represents the statistical analysis of the phagocytosis rate of breast cancer cells.

[0038] Figure 6This is a preferred embodiment of the present invention, which describes an experiment on the inhibition of tumor formation in 4T-1 cells of tumor-bearing mice by a bispecific antibody fusion protein.

[0039] Figure 7 This invention relates to a preferred embodiment of an experiment demonstrating the inhibition of tumor formation in SK-OV-3 cells of tumor-bearing mice by a bispecific antibody fusion protein.

[0040] Figure 8 The effect of bispecific antibody fusion protein on tumor formation in MFC cells of tumor-bearing mice is described in a preferred embodiment of the present invention. Detailed Implementation

[0041] This invention provides a bispecific antibody fusion protein against CD47 and CD24 and its preparation method. The bispecific antibody fusion protein can inhibit CD24 and antagonize CD47, and can be used to prepare anti-tumor drugs.

[0042] The present invention adopts the following technical solution:

[0043] In a first aspect, the present invention provides a bispecific antibody fusion protein CD24 / CD47-BsAb-Ig (i.e., PPAB001) that is anti-CD47 and CD24, wherein the bispecific antibody fusion protein comprises a heavy chain on the anti-CD24 side, a light chain on the anti-CD24 side, and an Fc fusion protein on the anti-CD47 side.

[0044] The heavy chain on the anti-CD24 side, the light chain on the anti-CD24 side, and the Fc fusion protein on the anti-CD47 side can be combined by forming disulfide bonds in CHO cells to obtain a bispecific antibody fusion protein that can inhibit CD24 and antagonize CD47.

[0045] To prevent self-linking or combination of the anti-CD24 side antibody or the anti-CD47 side fusion protein during the formation of the above-mentioned components, this invention performs site-directed mutagenesis on the CH3 region of the constant region in the heavy chain of the anti-CD24 side and the Fc fusion protein of the anti-CD47 side, specifically as follows: One mutation is introduced in the CH3 region of the constant region of the anti-CD24 side heavy chain, including the conversion of threonine to tryptophan at position 249 in SEQ ID NO:2. Three mutations are introduced in the Fc region of the anti-CD47 side of PPAB001, including the conversion of threonine to serine at position 152 in SEQ ID NO:12, the conversion of leucine to alanine at position 154, and the conversion of tyrosine to valine at position 193. After mutation, the Fc segment of the anti-CD47 side and the constant region of the heavy chain of the anti-CD24 side can form a "lock-in-hole" binding pattern through these amino acids, greatly increasing the probability of correct pairing and reducing the possibility of self-pairing.

[0046] Based on the above adjustments, the bispecific antibody fusion protein specifically includes the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:16 and SEQ ID NO:18.

[0047] Secondly, the present invention provides a method for preparing the aforementioned bispecific antibody fusion protein, comprising the following steps:

[0048] S1. Construct vectors containing the nucleotide sequences shown in SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17 respectively, and co-transfect them into host cells using liposomes for culture, and screen cell clones that stably express bispecific antibody fusion proteins;

[0049] S2. The cell culture was isolated and purified to obtain the bispecific antibody fusion protein PPAB001.

[0050] It should be noted that the vector described in this invention can be a vector conventionally used in the art, such as pCDNA3.1, pDHFF, etc. The expression vector includes a fusion DNA sequence linked with suitable transcription and translation regulatory sequences.

[0051] Alternatively, as in a specific embodiment of the present invention, the eukaryotic expression vector pcDNA3.1 may be used.

[0052] Furthermore, the host cell used to express the bispecific antibody fusion protein can be a prokaryotic cell, such as DH5α, BL21(DE3), TG1, etc.; it can also be a mammalian or insect host cell, such as COS, CHO, NSO, sf9 and sf21, etc., which are all applicable to this invention.

[0053] Preferably, the present invention selects CHO-K1 cells (ATCC) as host cells for the expression of bispecific antibody fusion proteins.

[0054] Furthermore, the host cells are cultured under conventional conditions in the field, such as using DMEM medium containing 10% serum.

[0055] Further, in S2, cell clones stably expressing the bispecific antibody fusion protein are screened using a selective medium containing 600 μg / mL G418 and 250 μg / mL Zeocin. Using the above method, the bispecific antibody fusion protein can be purified into a substantially homogeneous substance, for example, appearing as a single band on SDS-PAGE electrophoresis. Furthermore, the bispecific antibody fusion protein disclosed in this invention can be separated and purified using affinity chromatography. Depending on the characteristics of the affinity column used, conventional methods such as high-salt buffer or pH adjustment can be used to elute the bispecific antibody fusion protein bound to the affinity column.

[0056] This invention performed affinity testing on PPAB001, revealing that it perfectly retained the affinity for both anti-CD24 and CV1-hFc. Further experiments using PPAB001, including tumor cell proliferation, in vivo tumor inhibition, and macrophage phagocytosis promotion, showed that the bispecific antibody fusion protein PPAB001 disclosed in this invention simultaneously possesses anti-CD24 and CV1-hFc functions, and can block CD47 and CD24-mediated tumor cell proliferation. Furthermore, this bispecific antibody fusion protein PPAB001, like traditional IgG molecules, retains the traditional monoclonal antibody structure to the greatest extent. Due to the presence of the Fc fragment, it can be purified using conventional Protein A column affinity chromatography, facilitating large-scale production and purification. Experiments show that, at the same dosage, the bispecific antibody fusion protein PPAB001 has significantly better anti-tumor therapeutic effects than anti-CD24.

[0057] Thirdly, based on the aforementioned technical solutions, host cells capable of simultaneously expressing the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:16, and SEQ ID NO:18, as well as vector combinations including those containing the nucleotide sequences shown in SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17 respectively, are also within the scope of protection of this invention.

[0058] Fourthly, the present invention also provides a composition containing the aforementioned bispecific antibody fusion protein PPAB001.

[0059] The present invention also provides the use of the aforementioned bispecific antibody fusion protein PPAB001 or a composition containing it in the preparation of antitumor drugs.

[0060] The bispecific antibody fusion protein PPAB001 disclosed in this invention, when combined with pharmaceutically acceptable excipients, forms a pharmaceutical formulation composition that provides more stable therapeutic efficacy. These formulations ensure the conformational integrity of the amino acid core sequence of the fully human antibody disclosed in this invention, while also protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination, or oxidation). Typically, liquid formulations are stable for at least one year at 2-8°C, and lyophilized formulations remain stable for at least six months at 30°C. The formulation can be a commonly used pharmaceutical preparation such as a suspension, injection, or lyophilization, preferably an injection or lyophilized formulation. For the injection or lyophilized formulation of the fully human antibody disclosed in this invention, pharmaceutically acceptable excipients include one or a combination of surfactants, solution stabilizers, isotonic modifiers, and buffers. Surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); poloxamer (e.g., poloxamer). 188); Triton; Sodium dodecyl sulfate (SDS); Sodium lauryl sulfate; Tetradecyl, linoleic, or octadecyl sarcosine; Pluronics; MONAQUAT™, etc., the amount added should minimize the granulation tendency of the bispecific antibody fusion protein. The solution stabilizer can be a sugar, including reducing sugars and non-reducing sugars; an amino acid, including monosodium glutamate or histidine; an alcohol, including one or a combination of triols, higher sugar alcohols, propylene glycol, and polyethylene glycol. The amount of solution stabilizer added should be such that the final formulation remains stable within the time that a person skilled in the art would consider to be stable. The isotonic adjuster can be one of sodium chloride and mannitol. The buffer can be one of TRIS, histidine buffer, and phosphate buffer.

[0061] The above-mentioned formulation is a composition containing a bispecific antibody fusion protein, which has shown significant anti-tumor effects after administration to animals, including humans. Specifically, it is effective in the prevention and / or treatment of tumors and can be used as an anti-tumor drug.

[0062] The antitumor drug described in this invention refers to a drug that inhibits and / or treats tumors, and may include delaying the development of symptoms associated with tumor growth and / or reducing the severity of these symptoms. It further includes alleviating existing symptoms associated with tumor growth and preventing the occurrence of other symptoms, and reducing or preventing metastasis.

[0063] In this invention, the bispecific antibody fusion protein PPAB001 and compositions containing it are administered to animals, including humans. The dosage varies depending on the patient's age and weight, disease characteristics and severity, and route of administration. The results of animal experiments and various circumstances can be referenced, and the total dosage should not exceed a certain range.

[0064] The bispecific antibody fusion protein PPAB001 and compositions containing it provided by this invention can also be administered in combination with other antitumor drugs for the treatment of tumors. These antitumor drugs include:

[0065] 1. Cytotoxic drugs

[0066] (1) Drugs that act on the chemical structure of DNA: alkylating agents such as nitrogen mustard, nitrosamines, and methanesulfonates; platinum compounds such as cisplatin, carboplatin, and oxalate platinum; mitomycin C (MMC); (2) Drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine); purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; nucleotide reductase inhibitors such as hydroxyurea (HU); D NA polymerase inhibitors such as cytarabine (Ara-C) and Gemz; (3) Drugs that act on nucleic acid transcription: drugs that selectively act on DNA templates and inhibit DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, sclerosomycin, etc.; (4) Drugs that mainly act on microtubule synthesis: paclitaxel, texotetra, vinblastine, vinorelbine, podophyllodes, homoharringtonine; (5) Other cytotoxic drugs: asparaginase mainly inhibits protein synthesis;

[0067] 2. Hormones: Anti-estrogens: tamoxifen, droloxifene, exemestane, etc.; Aromatase inhibitors: ammoniaglutide, lantron, letrozole, reninide, etc.; Anti-androgens: flutamethasone RH-LH agonists / antagonists: phenadol, phenadol, etc.

[0068] 3. Biological response modifiers: mainly inhibit tumor interferon, interleukin-2, and thymosin through the body's immune function;

[0069] 4. Monoclonal antibodies: Cetuximab (C225); Trastuzumab; Avastin;

[0070] 5. Others include some drugs whose mechanisms are currently unclear and require further research; cell differentiation inducers such as retinoids; and apoptosis inducers.

[0071] The bispecific antibody fusion protein PPAB001 and its composition provided by the present invention can be used in combination with one or more of the above-mentioned anti-tumor drugs.

[0072] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are performed under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 2001), Molecular Cloning (Sambrook, J., Fritsch, E.F. and Maniais, T., 1989): A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, or as recommended by the manufacturer's instructions.

[0073] The terms and abbreviations used in this invention are as follows:

[0074] PPAB001: A bispecific antibody fusion protein targeting CD47 and CD24;

[0075] anti-CD24: A monoclonal antibody against CD24;

[0076] CV1-hFc: A fusion protein of a crystallizable fragment of a signal regulatory protein-α mutant;

[0077] CD47: Integrin-associated protein;

[0078] IgG: Immunoglobulin G;

[0079] ELISA: Enzyme-linked immunosorbent assay;

[0080] SDS-PAGE: Sodium dodecyl sulfonate-polyacrylamide gel electrophoresis.

[0081] Example 1: Cloning of human antibody light and heavy chain constant regions and Fc region genes

[0082] Lymphocytes from healthy individuals were isolated using lymphocyte separation medium. Total RNA was extracted using Trizol reagent (Invitrogen). Primers were designed to amplify the heavy and light chain constant region genes of IgG1 antibody based on sequences reported in the literature (Cell, 1980, 22: 197-207) and (Nucleic Acids Research, 1982, 10: 4071-4079). All PCR reactions were hot-started, with the following conditions: 94℃ for 5 min; 94℃ for 45 s, 60℃ for 45 s, 72℃ for 70 s, 30 cycles; 72℃ for 10 min. The PCR products were purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector (Promega). Sequencing confirmed the correct clone. SEQ ID NO:1 and SEQ ID NO:2 are the nucleotide and amino acid sequences of the heavy chain constant region (CH), respectively. SEQ ID NO:3 and SEQ ID NO:4 are the nucleotide and amino acid sequences of the light chain constant region (CL), respectively. SEQ ID NO:11 and SEQ ID NO:12 are the nucleotide and amino acid sequences of the constant region Fc segment (Fc), respectively. The correct clones are designated as pGEM-T / CH, pGEM-T / CL, and pGEM-T / Fc.

[0083] Example 2: Construction of bispecific antibody fusion protein PPAB001

[0084] Specific construction method: anti-CD24 was obtained by screening with hybridoma cells and humanizing the constant region of the antibody. The heavy and light chain variable regions of anti-CD24 were synthesized in its entirety; the sequence of CV1 can be found in Science 341, 88 (2013). The heavy chain variable region of anti-CD24 was linked to the heavy chain constant region of human IgG1 antibody to form the anti-CD24 side heavy chain gene of PPAB001. CV1 was cloned by PCR and linked to the Fc segment of the heavy chain constant region of human IgG1 antibody to form the anti-CD47 side fusion protein gene of PPAB001. The light chain variable region of anti-CD24 was linked to the light chain constant region of human IgG1 antibody using overlap PCR to form the anti-CD24 side light chain gene of PPAB001. The above heavy and light chain genes and fusion protein genes were respectively inserted into the eukaryotic expression vector pcDNA3.1 (Invitrogen product). The plasmids were transfected together into CHO-K1 cells (ATCC) using liposomes, and cell clones stably expressing the bispecific antibody fusion protein were screened using selective medium containing 600 μg / mL G418 and 250 μg / mL Zeocin. The bispecific antibody fusion protein PPAB001 was purified from the cell culture supernatant using affinity chromatography with a Protein A column.

[0085] The construction and expression process of the bispecific antibody fusion protein PPAB001 is as follows:

[0086] The variable regions of the heavy and light chains on the anti-CD24 side of PPAB001 and the CV1 region on the anti-CD47 side are PPAB001VH, PPAB001VL, and PPAB001CV1, respectively. After synthesis by overlap PCR, the target bands were recovered and cloned into the pGEM-T vector. Positive clones were screened and sequenced to obtain pGEM-T / PPAB001VH (nucleotide and amino acid sequences are shown in SEQ ID NO:5 and SEQ ID NO:6), pGEM-T / PPAB001VL (nucleotide and amino acid sequences are shown in SEQ ID NO:7 and SEQ ID NO:8), and pGEM-T / PPAB001CV1 (nucleotide and amino acid sequences are shown in SEQ ID NO:9 and SEQ ID NO:10), respectively. PCR was performed using a high-fidelity amplification system from Roche, with the following reaction conditions: 95℃ for 5 min; 94℃ for 50 s, 55℃ for 45 s, 72℃ for 55 s, 30 cycles; 72℃ for 10 min.

[0087] The pGEM-T / PPAB001VH gene was ligated to the heavy chain constant region gene in pGEM-T / CH using overlap PCR and cloned into the pGEM-T vector. After selecting the correct clone, the target fragment was digested with HindIII and EcoRI, purified and recovered by agarose gel electrophoresis, and ligated with the digested plasmid pCDNA3.1(+) using T4 DNA ligase to construct the eukaryotic expression vector pCDNA3.1 / ZEO(+)(PPAB001VHCH) (nucleotide and amino acid sequences are shown in SEQ ID NO:15 and SEQ ID NO:16).

[0088] The pGEM-T / PPAB001CV1 gene was ligated to the Fc segment gene in pGEM-T / Fc using overlap PCR and cloned into the pGEM-T vector. After selecting the correct clone, the target fragment was digested with HindIII and EcoRI, purified and recovered by agarose gel electrophoresis, and ligated with the digested plasmid pCDNA3.1(+) using T4 DNA ligase to construct the eukaryotic expression vector pCDNA3.1 / ZEO(+)(PPAB001CV1-hFc) (nucleotide and amino acid sequences are shown in SEQ ID NO:17 and SEQ ID NO:18).

[0089] The PPAB001VL gene in pGEM-T / PPAB001VL was ligated to the light chain constant region gene in pGEM-T / CL using overlap PCR and cloned into the pGEM-T vector. After selecting the correct clone, it was digested with HindIII and EcoRI, and the target fragment was purified and recovered by agarose gel electrophoresis. It was then ligated with the same digested plasmid pCDNA3.1(+) using T4 DNA ligase to construct the eukaryotic expression vector pCDNA3.1 / ZEO(+)(PPAB001VLCL) (nucleotide and amino acid sequences are shown in SEQ ID NO:13 and SEQ ID NO:14).

[0090] Inoculate 3 × 10⁶ cells / mL into a 3.5 cm tissue culture dish. 5 CHO-K1 cells were cultured to 90%-95% confluence and then transfected: 9 μg of plasmid (3 μg each of pCDNA3.1(+)pCDNA3.1 / ZEO(+)(PPAB001VHCH), pCDNA3.1 / ZEO(+)(PPAB001VLCL), and pCDNA3.1 / ZEO(+)(PPAB001CV1-hFc)) and 20 μL of Lipofectamine 2000 Reager were added. Invitrogen [product of Invitrogen] was dissolved separately in 500 μL of serum-free DMEM medium and allowed to stand at room temperature for 5 minutes. The two liquids were then mixed and incubated at room temperature for 20 minutes to allow the DNA-liposome complex to form. During this time, 3 mL of serum-free DMEM medium was used to replace the serum-containing medium in the culture dish. The formed DNA-liposome complex was then added to the plate, and the cells were incubated in a CO2 incubator for 4 hours. Afterward, 2 mL of complete DMEM medium containing 10% serum was added, and the plate was incubated in a CO2 incubator for further incubation. 24 hours after transfection, the cells were replaced with selection medium containing 600 μg / mL G418 and 250 μg / mL Zeocin to screen for resistant clones. The high-expression clones obtained from the screening were expanded in serum-free medium, and the bispecific antibody fusion protein PPAB001 was isolated and purified using a Protein A affinity column (GE product). PPAB001 was dialyzed against PBS and finally quantified by UV absorption. The structure of PPAB001 is shown below. Figure 1 As shown in Figure B, the left side represents the heavy and light chains of the anti-CD24 side of PPAB001, and the right side represents the Fc fusion protein of the anti-CD47 side of PPAB001 (CV1). The structure of the anti-CD24 protein is shown below. Figure 1 As shown in Figure A, the structure of the CV1-hFc protein is as follows: Figure 1 As shown in C.

[0091] Experimental Example 1: Gel filtration chromatography purification of bispecific antibody fusion protein

[0092] After equilibrating a Superose 12 10 / 300GL column (GE product) with 40 mL of equilibration buffer (10 mmol / L tris(hydroxymethyl)aminomethane hydrochloride (pH 7.4), 150 mmol / L sodium chloride), the concentrated anti-CD24, PPAB001, and CV1-hFc proteins were loaded separately for gel filtration chromatography purification to analyze the molecular weight of each component. The results are as follows: Figure 1 As shown in D, the antibody anti-CD24, due to its largest molecular weight, was eluted first, followed by the bispecific antibody fusion protein PPAB001, and finally the fusion protein CV1-hFc.

[0093] Experiment Example 2: Verification Experiment of the Purity of Bispecific Antibody Fusion Protein

[0094] A polyacrylamide gel containing SDS and 10% acrylamide was prepared. The fusion protein CV1-hFc, the antibody anti-CD24, and the bispecific antibody fusion protein PPAB001 were loaded onto the gel for electrophoresis to analyze the purity of each component. The results are as follows: Figure 2 As shown, the purity of PPAB001 is above 90%.

[0095] Experiment 3: Detection of the binding activity of the bispecific antibody fusion protein PPAB001 to CD24 and CD47.

[0096] CD24 (R&D product) was diluted to 2 μg / mL with 0.05 mmol / L sodium carbonate-sodium bicarbonate buffer (pH 9.6), 50 μL / well, and incubated overnight at 4°C. After blocking with 10% skim milk at room temperature for 2 h, different concentrations of bispecific antibody fusion proteins, CV1-hFc and anti-CD24, 50 μL / well were added, with 3 parallel wells for each concentration, and incubated at room temperature for 2 h. The supernatant was discarded, and the samples were washed three times with PBS. HRP-labeled CD47 diluted to the titer was added, 50 μL / well, and incubated at room temperature for 45 min. After thorough washing with PBS, TMB was used for color development, and the absorbance (A450) was measured at 450 nm using a microplate reader (BIO-Rad, USA). The experimental results are as follows. Figure 3As shown in the figure, the vertical axis represents the optical density (or absorbance) at 450 nm, and the horizontal axis represents the logarithm of the antibody concentration. CV1-hFc and anti-CD24 can only bind to CD47 or CD24, so their readings are close to 0. The PPAB001 group, however, shows a concentration-dependent effect, with the OD value increasing with increasing antibody concentration. This experiment confirms that PPAB001 can bind to both CD47 and CD24. The experimental results show that because the bispecific antibody fusion protein PPAB001 can bind to both CD24 and CD47, it exhibits a concentration-dependent effect with an S-curve. Anti-CD24 and CV1-hFc, because they can only bind to CD24 or CD47, have no binding activity. These experimental results indicate that the constructed bispecific antibody fusion protein PPAB001 retains the binding activity of both parental anti-CD24 and CV1-hFc.

[0097] Experiment 4: Flow Cytometry Analysis

[0098] CD24 and CD47 double-positive breast cancer BT-474 cells were resuspended in 2% FCS-PBS to form 1×10⁻⁶ cells. 6 Cells were incubated at 4°C for 1 hour with different dilutions of PPAB001, anti-CD24, CV1-hFc, and control IgG, and washed twice with 2% FCS-PBS. Then, FITC-labeled goat anti-human IgG (H+L) secondary antibody was added and incubated at 4°C for 1 hour. After washing twice with 2% FCS-PBS, the cells were analyzed by flow cytometry to calculate the percentage of positive cells and compare the relative affinities of each antibody. The experimental results are as follows: Figure 4 As shown in the figure, the horizontal axis represents the antibody concentration, and the vertical axis represents the average fluorescence intensity. Flow cytometry results showed that PPAB001 binds to CD47 and CD24 double-positive cells in a concentration-dependent manner, exhibiting an S-curve. Anti-CD24 and CV1-hFc also bind to CD47 and CD24 double-positive cells in a concentration-dependent manner, but with lower average fluorescence intensities. The control group consisted of cells with added irrelevant IgG. The experimental results indicate that PPAB001 can bind to BT-474 cells that are double-positive for CD24 and CD47, meaning that PPAB001 can simultaneously bind to both CD24 and CD47 on the cell surface.

[0099] Experiment Example 5: In vitro experiment on the promotion of macrophage phagocytosis of tumor cells by bispecific antibody fusion protein PPAB001.

[0100] Mouse peritoneal macrophages were prepared by perfusion of the peritoneal cavity with serum-free culture medium. The specific method was as follows: Three NOD-SCID mice were intraperitoneally injected with 1-2 mL of thioglycolate medium 5 days prior to treatment; 5 days later, the mice were euthanized by cervical dislocation. 5 mL of serum-free RPMI-1640 medium containing penicillin and antibiotics was injected into the peritoneal cavity using a syringe; the abdomen was gently massaged with a cotton ball for 1-2 minutes, and the cell suspension was aspirated; after centrifugation at 1000 rpm for 5 min, the cells were washed once with serum-free RPMI-1640 medium, centrifuged again, and resuspended in RPMI-1640 medium containing 10% fetal bovine serum for culture; then, the phagocytic effect was observed using a fluorescence microscope, specifically by: [further details on the phagocytic effect and phagocytosis]. 4 Mouse macrophages derived from the peritoneum were seeded in 24-well plates at a density of cells / well and cultured overnight; 5 × 10⁶ cells / well were then cultured. 6 BT474, BT474R, or SK-OV-3 cells were labeled with carboxyfluorescein acetoacetate (CFSE). The culture medium for macrophages was replaced with serum-free medium, and the cells were starved for 2 hours. 2 × 10⁶ cells were added to each well. 5 CFSE-labeled tumor cells were then added; the following antibody groups were added, three replicates per group, with a final concentration of 10 μg / mL: (1) IgG group; (2) CV1-hFc group; (3) anti-CD24 group; (4) PPAB001 group, and incubated at 37℃ for 2-3 h. The cells were washed three times with culture medium, observed and photographed under a confocal fluorescence microscope, and the percentage of CFSE-positive cells per 100 macrophages (three fields of view) was calculated. The experimental results are as follows: Figure 5 As shown in the figure, A represents the significantly enhanced phagocytic efficiency of macrophages against breast cancer cells after treatment with PPAB001 compared to the CV1-hFc or anti-CD24 monoclonal antibody treatment groups; B represents statistical data showing a significant difference in the number of cells phagocytosed by the PPAB001 treatment group compared to the CV1-hFc or anti-CD24 groups. The experimental results indicate that PPAB001 can effectively promote the phagocytosis of tumor cells by macrophages compared to the control groups CV1-hFc and anti-CD24.

[0101] Experiment Example 6: In vivo inhibition of tumor cell growth by bispecific antibody fusion protein PPAB001

[0102] Female NOD-SCID mice were subcutaneously inoculated with 4T-1 breast cancer cells, SK-OV-3 ovarian cancer cells, or MFC gastric cancer cells, respectively. Tumors were cultured until the tumor tissue reached 80-120 mm in size. 3The mice were intravenously injected with the following four groups, six mice in each group: ① human IgG control group; ② anti-CD24; ③ CV1-hFc; ④ PPAB001. Injections were given twice a week for one week. During the administration, tumor growth was observed every two days, and tumor size was measured to evaluate the antitumor effect of PPAB001.

[0103] The experimental results are as follows: Figures 6-8 As shown, Figure 6 The horizontal axis represents the time after tumor inoculation in mice bearing 4T-1 tumors, the vertical axis represents the tumor volume, and the arrows represent the time between two drug injections. This indicates that after a period of treatment with the drug PPAB001, compared with the unrelated IgG treatment group, the anti-CD24 group, or the CV1-hFc group, the PPAB001 group showed a significant tumor regression trend, and the difference was statistically significant. Figure 7 The horizontal axis represents the time after tumor inoculation in SK-OV-3 tumor-bearing mice, the vertical axis represents the tumor volume, and the arrows represent the time between two drug injections. This indicates that after a period of treatment with the drug PPAB001, compared with the unrelated IgG treatment group, the anti-CD24 group, or the CV1-hFc group, the PPAB001 group showed a significant tumor regression trend, and the difference was statistically significant. Figure 8 The horizontal axis represents the time after tumor inoculation in MFC-bearing mice, and the vertical axis represents tumor volume. The arrows indicate the time between two drug injections. This shows that after a period of treatment with PPAB001, the tumors in the PPAB001 group did not regress compared to the unrelated IgG treatment group, the anti-CD24 group, or the CV1-hFc group. These experimental results indicate that PPAB001 can effectively inhibit the growth of breast and ovarian cancer tumors in tumor-bearing mice, and its tumor-suppressing effect is significantly stronger than that of the anti-CD24 group, the CV1-hFc group, and the IgG control group. However, PPAB001 cannot inhibit the growth of gastric cancer tumors in tumor-bearing mice.

[0104] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A bispecific antibody fusion protein targeting CD24 and CD47, characterized in that, The fusion protein comprises a CD24 antibody heavy chain, a CD24 antibody light chain, and a CD47-Fc binding peptide; Among them, the constant region of the CD24 antibody heavy chain is connected to the Fc segment of the CD47-Fc binding peptide through a "lock-and-key structure". The fusion protein can bind to both CD47 and CD24 simultaneously. The amino acid sequence of the CD24 antibody light chain is shown in SEQ ID NO:14, the amino acid sequence of the CD24 antibody heavy chain is shown in SEQ ID NO:16, and the amino acid sequence of the CD47-Fc binding peptide is shown in SEQ ID NO:

18.

2. The method for preparing the fusion protein according to claim 1, characterized in that, Includes the following steps: S1. Construct vectors expressing the nucleotide sequences shown in SEQ ID NO:13, SEQ ID NO:15 and SEQ ID NO:17 respectively, and co-transfect them into host cells for culture to screen cell clones that stably express the target fusion protein; S2. The cell culture is isolated and purified to obtain the target fusion protein.

3. The method according to claim 2, characterized in that, The vector is the eukaryotic expression vector pcDNA3.

1.

4. The method according to claim 2, characterized in that, The host cell is a CHO-K1 cell.

5. The method according to any one of claims 2 to 4, characterized in that, Cell clones that stably express the target fusion protein were screened using a selective medium containing 600 μg / mL G418 and 250 μg / mL Zeocin.

6. A nucleic acid molecule encoding the fusion protein of claim 1.

7. A biomaterial containing the nucleic acid molecule of claim 6, characterized in that, The biological materials are recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria, or transgenic cell lines.

8. A composition comprising the fusion protein of claim 1, wherein the composition further comprises an antitumor agent.

9. The composition according to claim 8, characterized in that, The antitumor agents are selected from nitrogen mustard, cisplatin, carboplatin, oxalate platinum, methotrexate, Alimta, fluorouracil, 6-mercaptopurine, 6-TG, hydroxyurea, cytarabine, gemcitabine, tamoxifen, droloxifen, exemestane, aminoglutethimide, lantron, letrozole, reninide, enolide, phenazone, cetuximab, Herceptin, bevacizumab, and interleukin-2.

10. The use of the fusion protein of claim 1 or the composition of claim 8 or 9 in the preparation of an antitumor drug; The tumors were selected from ovarian cancer, breast cancer, and gastric cancer.

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