Scube2 neutralizing antibodies and medical uses thereof

Abstract

The present application provides a SCUBE2 neutralizing antibody and its medical use. Specifically, the present application provides an antibody capable of competitively binding to SCUBE2 protein. The antibody of the present application has high affinity, can bind to the CUB functional region of SCUBE2 protein, competitively block the binding of Hedgehog signal ligand (such as SHH protein) to the CUB domain in SCUBE2 protein, and thereby inhibit the process of breast cancer bone metastasis mediated by Hedgehog signal. The antibody has the prospect of treating breast cancer bone metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to SCUBE2 neutralizing antibodies and their pharmaceutical uses. Background Technology

[0002] Breast cancer, the most common cancer among women worldwide, is a leading cause of cancer death, posing a significant threat to women's health. Compared to primary tumors, distant metastasis is the primary cause of death in breast cancer patients. Metastatic organs include bone, liver, brain, and lungs, with bone being the most common site. Clinically, over 70% of patients with advanced breast cancer are diagnosed with bone metastases, leading to severe bone pain, fractures, and other fatal complications. Currently, the main treatment for breast cancer bone metastases is to inhibit osteoclast-induced bone resorption, using drugs such as bisphosphonates and denosumab. However, these drugs only slow the progression of metastasis and do not effectively improve overall survival.

[0003] SCUBE2 is an evolutionarily conserved protein containing nine EGF-like repeat sequences, three cysteine-rich repeat components, and a CUB region. SCUBE2 is a crucial protein in Hedgehog signaling, responsible for cleaving and releasing Hedgehog signaling ligands anchored to the cell membrane, such as SHH, thereby allowing free SHH to bind to the Hedgehog receptor and initiate downstream Hedgehog signaling. Studies have found that the CUB region of SCUBE2 plays a key role in the cleavage and release of SHH protein; truncated proteins lacking the CUB region cannot bind to SHH protein and cannot release it from the cell membrane. Previous studies have found that SCUBE2 plays an important role in breast cancer bone metastasis. SCUBE2 promotes the release of SHH from breast cancer cells, thereby helping cancer cells colonize the bone microenvironment. Therefore, targeting SCUBE2 protein for functional inhibition is a potential treatment for breast cancer bone metastasis. Currently, there are no marketable inhibitory molecules or drugs targeting SCUBE2.

[0004] Therefore, there is a need in this field to develop a novel therapeutic agent that targets SCUBE2 for breast cancer bone metastases. Summary of the Invention

[0005] The purpose of this invention is to provide a novel therapeutic drug that targets SCUBE2 in the treatment of breast cancer bone metastases.

[0006] In a first aspect of the invention, an antibody is provided that binds to the SCUBE2 protein, the antibody specifically binding to the CUB domain of the SCUBE2 protein, and the antibody competitively blocking the binding of the Hedgehog signaling ligand to the SCUBE2 protein.

[0007] The amino acid sequence of the CUB domain is shown in SEQ ID NO:2.

[0008] In another preferred embodiment, the competitive blocking means reducing the binding rate of the Hedgehog signaling ligand to the SCUBE2 protein by 50%, preferably by 70%, and more preferably by 90%.

[0009] In another preferred embodiment, the antibody comprises a heavy chain and a light chain.

[0010] The variable region of the heavy chain has a complementary determinant region (CDR) selected from the following group:

[0011] VH-CDR1 as shown in SEQ ID NO:9, 15 or 20,

[0012] VH-CDR2 shown in SEQ ID NO:10, 16 or 21, and

[0013] VH-CDR3 as shown in SEQ ID NO: 11, 17 or 22;

[0014] Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group:

[0015] VL-CDR1 shown in SEQ ID NO:12, 18 or 23,

[0016] VL-CDR2 shown in SEQ ID NO:13 or 24, and

[0017] VL-CDR3 as shown in SEQ ID NO:14, 19 or 25;

[0018] Furthermore, any amino acid sequence in the aforementioned CDR sequence may also include a derivative sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid, such that the derivative antibody composed of the heavy and light chains containing the derived CDR sequence can retain SCUBE2 binding affinity.

[0019] In another preferred embodiment, the heavy chain variable region of the antibody has a complementarity-determining region (CDR) selected from the group consisting of:

[0020] The amino acid sequences of VH1 are as shown in SEQ ID NO:9 for VH-CDR1, SEQ ID NO:10 for VH-CDR2, and SEQ ID NO:11 for VH-CDR3.

[0021] The amino acid sequences of VH2 are as shown in SEQ ID NO:15 (VH-CDR1), SEQ ID NO:16 (VH-CDR2), and SEQ ID NO:17 (VH-CDR3); or

[0022] The amino acid sequences of VH3 are as shown in SEQ ID NO:20 (VH-CDR1), SEQ ID NO:21 (VH-CDR2), and EQ ID NO:22 (VH-CDR3).

[0023] In another preferred embodiment, the light chain variable region of the antibody has a complementarity-determining region (CDR) selected from the group consisting of:

[0024] The amino acid sequences of VL1 are as shown in SEQ ID NO:12 (VL-CDR1), SEQ ID NO:13 (VL-CDR2), and SEQ ID NO:14 (VL-CDR3);

[0025] The amino acid sequences of VL2 are as shown in SEQ ID NO:18 (VL-CDR1), SEQ ID NO:13 (VL-CDR2), and SEQ ID NO:19 (VL-CDR3); and

[0026] The amino acid sequences of VL3 are as shown in SEQ ID NO:23 (VL-CDR1), SEQ ID NO:24 (VL-CDR2), and SEQ ID NO:25 (VL-CDR3).

[0027] In another preferred embodiment, the heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0028] VH-CDR1 shown in SEQ ID NO:9, VH-CDR2 shown in SEQ ID NO:10, and VH-CDR3 shown in SEQ ID NO:11;

[0029] Furthermore, the light chain variable region includes the following three complementary determinant regions (CDRs):

[0030] VL-CDR1 shown in SEQ ID NO:12, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:14.

[0031] In another preferred embodiment, the heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0032] VH-CDR1 shown in SEQ ID NO:15, VH-CDR2 shown in SEQ ID NO:16, and VH-CDR3 shown in SEQ ID NO:17;

[0033] Furthermore, the light chain variable region includes the following three complementary determinant regions (CDRs):

[0034] VL-CDR1 shown in SEQ ID NO:18, VL-CDR2 shown in SEQ ID NO:13, and VL-CDR3 shown in SEQ ID NO:19.

[0035] In another preferred embodiment, the heavy chain variable region includes the following three complementary determinant regions (CDRs):

[0036] VH-CDR1 shown in SEQ ID NO:20, VH-CDR2 shown in SEQ ID NO:21, and VH-CDR3 shown in EQ ID NO:22;

[0037] Furthermore, the light chain variable region includes the following three complementary determinant regions (CDRs):

[0038] VL-CDR1 shown in SEQ ID NO:23, VL-CDR2 shown in SEQ ID NO:24, and VL-CDR3 shown in SEQ ID NO:25.

[0039] In another preferred embodiment, the variable region of the heavy chain has the amino acid sequence shown in SEQ ID NO:3, 5 or 7.

[0040] In another preferred embodiment, the heavy chain further includes a heavy chain constant region.

[0041] In another preferred embodiment, the heavy chain constant region is of human or mouse origin.

[0042] In another preferred embodiment, the variable region of the light chain has the amino acid sequence shown in SEQ ID NO:4, 6 or 8.

[0043] In another preferred embodiment, the light chain further includes a light chain constant region.

[0044] In another preferred embodiment, the light chain constant region is of human or mouse origin.

[0045] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3, 5 or 7; and / or the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4, 6 or 8.

[0046] In another preferred embodiment, the amino acid sequence of the heavy chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO:3, 5, or 7 in the sequence listing.

[0047] In another preferred embodiment, the amino acid sequence of the light chain variable region has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO:4, 6, or 8 in the sequence listing.

[0048] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4.

[0049] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:6.

[0050] In another preferred embodiment, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:8.

[0051] In another preferred embodiment, the heavy chain variable region of the antibody further includes a human-derived frame region, and / or the light chain variable region of the antibody further includes a human-derived frame region.

[0052] In another preferred embodiment, the heavy chain variable region of the antibody further includes a mouse-derived frame region, and / or the light chain variable region of the antibody further includes a mouse-derived frame region.

[0053] In another preferred embodiment, the antibody is selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, or combinations thereof.

[0054] In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.

[0055] In another preferred embodiment, the antibody is a partially or fully humanized, or fully human, monoclonal antibody.

[0056] In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

[0057] In another preferred embodiment, the antibody is a full-length antibody protein or an antigen-binding fragment.

[0058] In another preferred embodiment, the antibody is a bispecific antibody or a multispecific antibody.

[0059] In another preferred embodiment, the antibody is in the form of a drug conjugate.

[0060] In another preferred embodiment, the antibody has one or more functions selected from the group consisting of:

[0061] (a) Inhibits the release of Hedgehog signaling ligands from tumor cells;

[0062] (b) Inhibit the release of Hedgehog signaling ligand from tumor-peritum endothelial cells;

[0063] (c) Inhibit Hedgehog signaling ligand-mediated tumor cell growth;

[0064] (d) Inhibit Hedgehog signaling ligand-mediated tumor cell metastasis.

[0065] In another preferred embodiment, the Hedgehog signaling ligand is selected from the group consisting of SHH protein, IHH protein, and DHH protein.

[0066] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, melanoma, and lung cancer.

[0067] In another preferred embodiment, the tumor is breast cancer.

[0068] In another preferred embodiment, the transfer is selected from the group consisting of bone transfer.

[0069] In another preferred embodiment, the transfer is a bone transfer.

[0070] In a second aspect of the invention, a recombinant protein is provided, said recombinant protein comprising:

[0071] (i) the antibody as described in the first aspect of the invention; and

[0072] (ii) Optional tag sequences to assist in expression and / or purification.

[0073] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0074] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0075] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0076] In another preferred embodiment, the recombinant protein specifically binds to the SCUBE2 protein.

[0077] In another preferred embodiment, the recombinant protein specifically binds to the CUB functional region of the SCUBE2 protein.

[0078] In another preferred embodiment, the recombinant protein is a fusion protein.

[0079] In another preferred embodiment, the fusion protein is a monospecific antibody (i.e., a monospecific antibody against the SCUBE2 protein), a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0080] In another preferred embodiment, the bispecific or multispecific antibody not only targets the SCUBE2 protein but also specifically binds to additional target antigens (such as other tumor antigens, such as other antigens of breast cancer or other tumors).

[0081] In a third aspect of the invention, a chimeric antigen receptor (CAR) construct is provided, wherein the scFv segment of the antigen-binding region of the chimeric antigen receptor construct specifically binds to the SCUBE2 protein, and the scFv has a heavy chain variable region and a light chain variable region as described in the first aspect of the invention.

[0082] In a fourth aspect of the invention, a recombinant immune cell is provided, said immune cell expressing an exogenous CAR construct as described in the third aspect of the invention.

[0083] In another preferred embodiment, the immune cells are selected from the group consisting of NK cells and T cells.

[0084] In another preferred embodiment, the immune cells are derived from humans or non-human mammals (such as mice).

[0085] In a fifth aspect of the invention, an antibody conjugate is provided, the antibody conjugate comprising:

[0086] (a) An antibody portion, said antibody portion being selected from the group consisting of antibodies as described in the first aspect of the invention; and

[0087] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, or combinations thereof.

[0088] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0089] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0090] In a sixth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: antibodies as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, antibody-drug conjugates as described in the fifth aspect of the invention, or combinations thereof, wherein the active ingredient is used for:

[0091] (a) Preparation of diagnostic reagents, test plates or kits; and / or

[0092] (b) To prepare drugs for the prevention and / or treatment of diseases associated with abnormal expression or function of SCUBE2 protein.

[0093] In another preferred embodiment, the reagent is used to detect the SCUBE2 protein.

[0094] In another preferred embodiment, the reagent, detection plate, or kit is used to detect diseases associated with abnormal SCUBE2 protein expression or function.

[0095] In another preferred embodiment, the reagent, test plate, or kit is used to predict the risk of bone metastasis in breast cancer.

[0096] In another preferred embodiment, the agent is used to prevent and / or treat Hedgehog signaling ligand-mediated tumor cell metastasis.

[0097] In another preferred embodiment, the agent is used to prevent and / or treat the inhibition of bone metastases in breast cancer.

[0098] In a seventh aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0099] (i) an active ingredient selected from the group consisting of: antibodies as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, immune cells as described in the fourth aspect of the invention, antibody-drug conjugates as described in the fifth aspect of the invention, or combinations thereof; and

[0100] (ii) Pharmaceutically acceptable carriers.

[0101] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0102] In another preferred embodiment, the pharmaceutical composition is an injection.

[0103] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of an antibody as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, an immune cell as described in the fourth aspect of the invention, an antibody-drug conjugate as described in the fifth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0104] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of Hedgehog signaling ligand-mediated tumor cell metastasis.

[0105] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of inhibiting bone metastasis of breast cancer.

[0106] In an eighth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a polypeptide selected from the group consisting of:

[0107] (1) The antibody as described in the first aspect of the present invention;

[0108] (2) The recombinant protein as described in the second aspect of the present invention; or

[0109] (3) The chimeric antigen receptor (CAR) construct as described in the third aspect of the present invention.

[0110] In a ninth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the eighth aspect of the invention.

[0111] In another preferred embodiment, the vector includes: bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0112] In a tenth aspect of the invention, an engineered host cell is provided, the host cell containing a vector as described in the ninth aspect of the invention or a genome in which polynucleotides as described in the eighth aspect of the invention are integrated.

[0113] In an eleventh aspect of the present invention, a method for detecting SCUBE2 protein in a sample is provided, the method comprising the steps of:

[0114] (1) Contact the sample with the antibody as described in the first aspect of the present invention;

[0115] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of SCUBE2 protein in the sample.

[0116] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0117] In a twelfth aspect of the present invention, a composition for in vitro detection of SCUBE2 protein in a sample is provided, comprising an antibody as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, an antibody-drug conjugate as described in the fifth aspect of the present invention, an immune cell as described in the fourth aspect of the present invention, or a combination thereof as an active ingredient.

[0118] In a thirteenth aspect of the present invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing an antibody as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, an antibody-drug conjugate as described in the fifth aspect of the present invention, an immune cell as described in the fourth aspect of the present invention, or a combination thereof.

[0119] In a fourteenth aspect of the invention, a kit is provided, the kit comprising:

[0120] (1) A first container containing an antibody as described in the first aspect of the invention; and / or

[0121] (2) A second container containing a secondary antibody against the antibody as described in the first aspect of the present invention;

[0122] or,

[0123] The kit contains a detection plate as described in the thirteenth aspect of the present invention.

[0124] In a fifteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0125] (a) Culture the host cells as described in the tenth aspect of the present invention under suitable expression conditions;

[0126] (b) Isolating a recombinant polypeptide from a culture, said recombinant polypeptide being an antibody as described in the first aspect of the invention or a recombinant protein as described in the second aspect of the invention.

[0127] In a sixteenth aspect of the invention, a pharmaceutical composition is provided, comprising:

[0128] (i) A first active ingredient comprising an antibody as described in the first aspect of the present invention, or a recombinant protein as described in the second aspect of the present invention, or an immune cell as described in the fourth aspect of the present invention, or an antibody-drug conjugate as described in the fifth aspect of the present invention, or a pharmaceutical composition as described in the seventh aspect of the present invention, or a combination thereof;

[0129] (ii) A second active ingredient, which includes a Hedgehog signaling inhibitor, an anti-estrogenic drug, or a chemotherapeutic agent.

[0130] In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of SHH neutralizing antibodies or SMO inhibitors.

[0131] In another preferred embodiment, the anti-estrogenic drug is selected from the group consisting of tamoxifen, ovarian function inhibitors, aromatase inhibitors, or combinations thereof.

[0132] In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of anthracyclines (doxorubicin, epirubicin, daunorubicin, and aclarubicin), taxanes (paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, and docetaxel) or combinations thereof.

[0133] In a seventeenth aspect of the invention, the use of an antibody as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an antibody-drug conjugate as described in the fifth aspect of the invention, or an immune cell as described in the fourth aspect of the invention, and / or a pharmaceutical composition as described in the seventh aspect of the invention, in combination with a Hedgehog signaling inhibitor or a chemotherapeutic agent, in the preparation of a medicament for treating diseases associated with abnormal expression or function of the SCUBE2 protein.

[0134] In another preferred embodiment, the abnormal expression of SCUBE2 protein refers to the overexpression of SCUBE2 protein.

[0135] In another preferred embodiment, the overexpression refers to the ratio of the expression level (F1) of SCUBE2 protein to the expression level (F0) under physiological conditions (i.e., F1 / F0) being ≥2.0, preferably ≥3.0, and more preferably ≥5.0.

[0136] In another preferred embodiment, the drug is used to prevent and / or treat tumor occurrence, growth and / or metastasis.

[0137] In another preferred embodiment, the drug is used to prevent and / or treat bone metastases from breast cancer.

[0138] In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of SHH neutralizing antibodies or SMO inhibitors.

[0139] In an eighteenth aspect of the invention, a method for treating a disease associated with abnormal expression or function of the SCUBE2 protein is provided, comprising administering to a subject in need an effective amount of an antibody as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an antibody-drug conjugate as described in the fifth aspect of the invention, or an immune cell as described in the fourth aspect of the invention, or a pharmaceutical composition as described in the sixteenth aspect of the invention, or a combination thereof.

[0140] In another preferred embodiment, the diseases associated with abnormal expression or function of the SCUBE2 protein are cancer, central nervous system tuberculosis, and arteriovenous malformation of the brain.

[0141] In another preferred embodiment, the treatment includes prevention and / or treatment of tumor metastasis.

[0142] In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, melanoma, and lung cancer.

[0143] In another preferred embodiment, the tumor metastasis is a Hedgehog signaling-mediated tumor metastasis.

[0144] In another preferred embodiment, the tumor metastasis is a bone metastasis.

[0145] In another preferred embodiment, the method further includes applying a safe and effective amount of a second active ingredient to the subject before, during, and / or after the application of the first active ingredient.

[0146] In another preferred embodiment, the second active ingredient includes a Hedgehog signaling inhibitor, an anti-estrogenic drug, or a chemotherapeutic agent.

[0147] In another preferred embodiment, the Hedgehog signaling inhibitor is selected from the group consisting of SHH neutralizing antibodies or SMO inhibitors.

[0148] In another preferred embodiment, the anti-estrogenic drug is selected from the group consisting of tamoxifen, ovarian function inhibitors, aromatase inhibitors, or combinations thereof.

[0149] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0150] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0151] Figure 1 The purification and identification of the SCUBE2 monoclonal antibody are shown. Figure 1A) Ascites fluid from mice after injection of hybridoma cells, and Coomassie brilliant blue staining of purified antibodies in the ascites fluid. Figure 1 B) ELISA kits were used to identify SCUBE2 monoclonal antibody subtypes. Figure 1 C) SCUBE2 expression was detected using a monoclonal antibody in the human breast cancer cell line MDA-MB-231, which overexpresses SCUBE2.

[0152] Figure 2 The in vitro inhibition of the SCUBE2 protein by the SCUBE2 monoclonal antibody was demonstrated. Figure 2 A) Activation of the GLI reporter system induced by MCF7 cell supernatant after treatment with IgG or SCUBE2 monoclonal antibody. Figure 2 B) Osteoblast differentiation induced by MCF7 cell supernatant after treatment with IgG or SCUBE2 monoclonal antibody. Figure 2 C) Expression of SHH protein in MCF7 cell supernatant after treatment with different concentrations of IgG or SCUBE2 monoclonal antibody.

[0153] Figure 3 The in vivo efficacy of the SCUBE2 monoclonal antibody was demonstrated. Figure 3 AB) Schematic diagram of whole-body bone metastasis in mice treated with IgG or SCUBE2 monoclonal antibody after left ventricular injection of human breast cancer cell line. Figure 3 A) and the optical signal and schematic diagram of in vitro bone tissue transfer ( Figure 3 B). Figure 3 Schematic diagram of whole-body bone metastasis in mice after left ventricular injection of mouse breast cancer cell lines into the control group or the SCUBE2 monoclonal antibody treatment group (CD) Figure 3 C) and the optical signals and schematic diagram of in vitro bone tissue transfer ( Figure 3 D). Detailed Implementation

[0154] Through extensive and in-depth research, the inventors unexpectedly obtained a class of neutralizing monoclonal antibodies against SCUBE2 for the first time. These neutralizing antibodies competitively bind to the SCUBE2 protein and exhibit high affinity. Surprisingly, these antibodies not only inhibit the ability of SCUBE2 protein to cleave and release SHH in vitro, but also inhibit breast cancer cell-mediated SHH release and the resulting bone metastasis process in vivo. Therefore, these antibodies show promise for treating breast cancer bone metastases. Based on this, the present invention was completed.

[0155] Experiments show that the SCUBE2 neutralizing antibody of the present invention can bind to the CUB functional region of the SCUBE2 protein, competitively blocking the binding of Hedgehog signaling ligands (such as SHH protein) to the CUB domain of the SCUBE2 protein, thereby preventing Hedgehog signaling-mediated bone metastasis of breast cancer.

[0156] the term

[0157] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0159] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0160] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent to a patient, comprising an antibody or combination thereof against a respiratory syncytial virus fusion protein (preferably pre-fused F protein) of the present invention, wherein the patient has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves one or more disease symptoms (therapeutic effective amount).

[0161] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0162] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0163] SCUBE2

[0164] SCUBE2 is an evolutionarily conserved protein containing nine EGF-like repeat sequences, three cysteine-rich repeat components, and a CUB region. SCUBE2 is a crucial protein in Hedgehog signaling, responsible for cleaving and releasing Hedgehog signaling ligands anchored to the cell membrane, such as SHH. This allows free SHH to bind to the Hedgehog receptor, initiating downstream Hedgehog signaling. Studies have found that the CUB region of SCUBE2 plays a key role in the cleavage and release of SHH protein; truncated proteins lacking the CUB region cannot bind to SHH protein and cannot release it from the cell membrane. Previous research has shown that SCUBE2 plays an important role in breast cancer bone metastasis; SCUBE2 helps cancer cells colonize the bone microenvironment by promoting the release of SHH from breast cancer cells.

[0165] Antibody

[0166] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0167] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0168] Vertebrate antibodies (immunoglobulins) can be classified into two distinct classes (denoted as κ and λ) based on the amino acid sequence of their constant region. Immunoglobulins can be further classified into different types based on the amino acid sequence of their heavy chain constant region. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are respectively called α, δ, ε, γ, and μ. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0169] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies in this population are identical, except for a few possible naturally occurring mutations. Monoclonal antibodies target a single antigenic site with high specificity. Moreover, unlike conventional polyclonal antibody formulations (which typically contain different antibodies targeting different determinants), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized through hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the antibody's properties and that it is obtained from a largely homogeneous population of antibodies; this should not be interpreted as requiring any special methods to produce the antibody.

[0170] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable regions of the heavy and light chains, called variable regions (CDRs). These regions are divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0171] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bridges on the lower chain region; (iii) Fd fragments consisting of VH and CH1 domains; and (iv) Fv fragments consisting of the VH and VL domains of a single arm of the antibody. Fv antibodies contain variable regions of the antibody heavy chain and light chain, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.

[0172] This invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0173] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional spatial site on the antigen, recognized by the antibody or antigen-binding fragment of the present invention.

[0174] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10 M or lower affinity (KD) binding.

[0175] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0176] In this invention, antibodies include mouse, chimeric, humanized, or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, can be obtained using standard DNA recombination techniques and are all useful antibodies. A chimeric antibody is a molecule in which different portions are derived from different animal species, such as a chimeric antibody having a variable region derived from a mouse monoclonal antibody and a constant region derived from a human immunoglobulin (see, for example, U.S. Patents 4,816,567 and 4,816,397, which are incorporated herein by reference in their entirety). A humanized antibody is an antibody molecule derived from a non-human species, having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent 5,585,089, which is incorporated herein by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombination techniques well known in the art.

[0177] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0178] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0179] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0180] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242.

[0181] In a preferred embodiment of the present invention, the light chain of the antibody includes the aforementioned light chain variable region and light chain constant region, wherein the light chain constant region may be mouse-derived or human-derived.

[0182] In this invention, the antibody also includes its conserved variants, which are polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0183] Table A

[0184] Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0185] SCUBE2 antibody

[0186] As used herein, the terms "antibody of the present invention", "SCUBE2 antibody of the present invention" and "SCUBE2 neutralizing antibody of the present invention" are used interchangeably and all refer to the antibody that binds to the SCUBE2 protein as described in the first aspect of the present invention.

[0187] The function of the antibody of this invention is determined by the specific gene sequences of the variable regions of its light and heavy chains. The antibody of this invention can competitively bind to the SCUBE2 protein with high affinity, competitively blocking the binding of Hedgehog signaling ligands (such as SHH protein) to the CUB domain of the SCUBE2 protein, thereby further inhibiting Hedgehog signaling-mediated bone metastasis in breast cancer. Using the variable region gene or complementarity-determining region (CDR) gene of the antibody of this invention, different forms of genetically engineered antibodies can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0188] In a first aspect of the invention, an antibody is provided that binds to the SCUBE2 protein, the antibody specifically binding to the CUB domain of the SCUBE2 protein, and the antibody competitively blocking the binding of the Hedgehog signal ligand to the SCUBE2 protein, wherein the amino acid sequence of the CUB domain is shown in SEQ ID NO:2.

[0189] In a preferred embodiment of the present invention, the antibody comprises a heavy chain and a light chain.

[0190] The variable region of the heavy chain has a complementary determinant region (CDR) selected from the following group:

[0191] VH-CDR1 as shown in SEQ ID NO:9, 15 or 20,

[0192] VH-CDR2 shown in SEQ ID NO:10, 16 or 21, and

[0193] VH-CDR3 as shown in SEQ ID NO: 11, 17 or 22;

[0194] Furthermore, the variable region of the light chain has a complementary determinant region (CDR) selected from the following group:

[0195] VL-CDR1 shown in SEQ ID NO:12, 18 or 23,

[0196] VL-CDR2 shown in SEQ ID NO:13 or 24, and

[0197] VL-CDR3 as shown in SEQ ID NO:14, 19 or 25;

[0198] Furthermore, any amino acid sequence in the aforementioned CDR sequence may also include a derivative sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid, such that the derivative antibody composed of the heavy and light chains containing the derived CDR sequence can retain SCUBE2 binding affinity.

[0199] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:3; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:4.

[0200] In another preferred embodiment of the present invention, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:5; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:6.

[0201] In another preferred embodiment of the present invention, the heavy chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:7; and the light chain variable region of the antibody contains the amino acid sequence shown in SEQ ID NO:8.

[0202] Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence, preferably with at least 80% homology or sequence identity, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence.

[0203] Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0204] Preferably, the antibody described herein is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0205] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0206] The antibody of the present invention can be a double-chain or single-chain antibody, and can be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies, and even more preferably fully humanized antibodies.

[0207] The antibody derivatives described in this invention may be single-chain antibodies and / or antibody fragments, such as Fab, Fab', (Fab')2 or other known antibody derivatives in the field, as well as any one or more of IgA, IgD, IgE, IgG and IgM antibodies or other subtypes of antibodies.

[0208] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0209] The animal is preferably a mammal, such as a mouse.

[0210] The antibodies of this invention may be chimeric antibodies targeting SCUBE2 (e.g., human SCUBE2), humanized antibodies, CDR-grafted and / or modified antibodies.

[0211] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0212] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0213] The SCUBE2 antibody of this invention can interrupt the source of SHH release from tumor cells. At the same time, it can be used in combination with Hedgehog signaling inhibitors, such as SHH neutralizing antibodies or SMO inhibitors, to inhibit Hedgehog signaling and thus inhibit tumor progression.

[0214] Recombinant protein

[0215] The present invention also provides a recombinant protein comprising one or more of the heavy chain CDR1 (VH-CDR1), heavy chain CDR2 (VH-CDR2), and heavy chain CDR3 (VH-CDR3) of the antibody of the present invention, and / or one or more of the light chain CDR1 (VL-CDR1), light chain CDR2 (VL-CDR2), and light chain CDR3 (VL-CDR3) of SCUBE2 of the present invention.

[0216] Preferably, the recombinant protein further includes an antibody heavy chain constant region and / or an antibody light chain constant region. The antibody heavy chain constant region is conventional in the art, preferably a rat-derived antibody heavy chain constant region or a human-derived antibody heavy chain constant region, and more preferably a human-derived antibody heavy chain constant region. The antibody light chain constant region is conventional in the art, preferably a rat-derived antibody light chain constant region or a human-derived antibody light chain constant region, and more preferably a human-derived antibody light chain constant region.

[0217] In another preferred embodiment, the recombinant protein comprises the antibody of the present invention.

[0218] The recombinant protein is a conventional protein in the art. Preferably, it is one or more of the following: full-length antibody protein, antigen-antibody binding domain protein fragment, bispecific antibody, multispecific antibody, single-chain antibody fragment (scFv), single-domain antibody (sdAb), and single-domain antibody, as well as monoclonal or polyclonal antibodies prepared from the above antibodies. The monoclonal antibody can be developed using various methods and techniques, including hybridoma technology, phage display technology, and single-lymphocyte gene cloning technology. The mainstream method is to prepare monoclonal antibodies from wild-type or transgenic mice using hybridoma technology.

[0219] The full-length antibody protein is a conventional full-length antibody protein in the art, comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region. The heavy chain variable region and light chain variable region of the protein, together with the human heavy chain constant region and the human light chain constant region, constitute a fully human full-length antibody protein. Preferably, the full-length antibody protein is IgG1, IgG2, IgG3, or IgG4.

[0220] The single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region, a light chain variable region, and a short peptide of 15 to 20 amino acids.

[0221] The antigen-antibody binding domain protein fragment is a conventional antigen-antibody binding domain protein fragment in the art, comprising an Fd segment of a light chain variable region, a light chain constant region, and a heavy chain constant region. Preferably, the antigen-antibody binding domain protein fragment is Fab and F(ab').

[0222] The single-domain antibody is a conventional single-domain antibody in the art, which includes a heavy chain variable region and a heavy chain constant region.

[0223] The single-region antibody described is a conventional single-region antibody in the art, which includes only the heavy chain variable region.

[0224] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from a recombinant expression transformant or obtaining it through artificial synthesis of the protein sequence. The preferred method for isolating the protein from the recombinant expression transformant is as follows: cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0225] Nucleic acid

[0226] The present invention also provides a nucleic acid encoding the above-described antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0227] The method for preparing the nucleic acid is a conventional method in the art, and preferably includes the following steps: obtaining a nucleic acid molecule encoding the above-mentioned protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above-mentioned protein by artificial full-sequence synthesis.

[0228] Those skilled in the art will understand that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homologue. The polynucleotide homologue of this invention can be prepared by substituting, deleting, or adding one or more bases of the gene encoding the protein sequence, while maintaining antibody activity.

[0229] carrier

[0230] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0231] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0232] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0233] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any common host cells in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells (expressing single-chain antibodies or Fab antibodies), or HEK293 or CHO cells (expressing full-length IgG antibodies). Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably chemical transformation, heat shock, or electroporation.

[0234] Antibody preparation

[0235] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0236] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0237] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0238] Currently, the DNA sequence encoding the antibody (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0239] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0240] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0241] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, which are well known to those skilled in the art.

[0242] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0243] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0244] Antibody-drug conjugates (ADCs)

[0245] The present invention also provides antibody-drug conjugates (ADCs) based on the antibodies of the present invention.

[0246] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0247] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0248] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0249] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0250] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0251] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly linked to the linker, the drug has a reactive group before being linked to the antibody.

[0252] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines) and vinca alkaloids.

[0253] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0254] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0255] The present invention also provides a method for preparing an ADC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (ADC).

[0256] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0257] application

[0258] The present invention also provides the use of the antibodies, antibody-drug conjugates (ADCs), recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0259] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal SCUBE2 expression or function.

[0260] In this invention, the diseases associated with abnormal SCUBE2 expression or function are conventional diseases associated with abnormal SCUBE2 expression or function in the art. Preferably, the diseases associated with abnormal SCUBE2 expression or function are cancer, central nervous system tuberculosis, and cerebral arteriovenous malformations.

[0261] In this invention, the diseases associated with abnormal SCUBE2 expression or function also include cancer metastasis, preferably bone metastasis, such as breast cancer bone metastasis.

[0262] In this invention, the cancer is a conventional cancer in the art, preferably breast cancer, melanoma, or lung cancer.

[0263] The antibodies of this invention can effectively prevent and / or alleviate excessive SHH release caused by abnormal SCUBE2 expression or function, thereby preventing and / or treating cancer bone metastases. In cancers where excessive SHH release has been observed, the antibodies of this invention can be co-administered with SHH neutralizing antibodies, SMO inhibitors, or any other therapeutic agents with Hedgehog signaling inhibition to enhance the inhibitory effect on cancer bone metastases.

[0264] Detection uses and kits

[0265] The antibody or its ADC of the present invention can be used in detection applications, such as for testing samples, thereby providing diagnostic information.

[0266] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0267] The samples used in this invention include fixed or preserved cell or tissue samples.

[0268] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the antibody of the present invention can be immobilized on a detection plate.

[0269] Pharmaceutical Composition

[0270] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0271] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0272] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0273] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal SCUBE2 expression or function.

[0274] The pharmaceutical composition of the present invention can be directly used to bind to the SCUBE2 protein molecule and block the binding of SCUBE2 to SHH, thereby inhibiting the release of SHH, and thus can be used to prevent and treat diseases such as tumors and tumor metastases.

[0275] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0276] In a preferred embodiment of the invention, the polypeptide of the invention can be used in combination with therapeutic agents for the treatment and / or prevention of cancer and / or cancer metastasis, such as Hedgehog signaling inhibitors, anti-estrogen drugs, or chemotherapeutic agents. The Hedgehog signaling inhibitor may be an SHH neutralizing antibody or an SMO inhibitor; the anti-estrogen drug may be tamoxifen, an ovarian function inhibitor, an aromatase inhibitor, or a combination thereof. The chemotherapeutic agent may be anthracyclines (doxorubicin, epirubicin, daunorubicin, and aclarubicin), taxanes (paclitaxel, paclitaxel liposomes, albumin-bound paclitaxel, and docetaxel), or a combination thereof.

[0277] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0278] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0279] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0280] This invention provides the use of the above-described pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of diseases associated with abnormal SCUBE2 expression or function. Preferably, the diseases associated with abnormal SCUBE2 expression or function are cancer, central nervous system tuberculosis, and cerebral arteriovenous malformations. More preferably, the diseases associated with abnormal SCUBE2 expression or function are breast cancer or breast cancer bone metastases.

[0281] Methods and compositions for detecting SCUBE2 protein in samples

[0282] The present invention also provides a method for detecting SCUBE2 protein in a sample (e.g., detecting SCUBE2 overexpressing cells), comprising the following steps: contacting the antibody with the sample to be tested in vitro, and detecting whether the antibody and the sample to be tested bind to form an antigen-antibody complex.

[0283] The term "overexpression" is conventional in the art and refers to the overexpression of SCUBE2 protein in RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and changes in protein degradation), as well as local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (e.g., in the case of increased enzymatic hydrolysis of the substrate).

[0284] In this invention, the detection method for whether or not an antigen-antibody complex is formed is a conventional detection method in the art, and preferably a flow cytometry (FACS) assay.

[0285] This invention provides a composition for detecting SCUBE2 protein in a sample, comprising the above-described antibody, recombinant protein, antibody-drug conjugate, immune cells, or combinations thereof as active ingredients. Preferably, it further comprises a compound composed of functional fragments of the above-described antibody as an active ingredient.

[0286] The main advantages of this invention include:

[0287] 1) The antibody of the present invention can competitively bind to the SCUBE2 protein, preventing the SCUBE2 protein from binding to SHH. In vitro, it can inhibit the ability of the SCUBE2 protein to release SHH through cleavage. In vivo, it can significantly inhibit the release of SHH mediated by breast cancer cells and the bone metastasis process caused therefrom, and has the prospect of treating breast cancer bone metastasis.

[0288] 2) The SCUBE2 neutralizing antibody of the present invention also has therapeutic potential for other tumors that highly express SCUBE2 and initiate downstream Hedgehog signaling, except for breast cancer bone metastases, and has corresponding application prospects.

[0289] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0290] Example 1: Screening of SCUBE2 monoclonal antibodies

[0291] In this embodiment, mouse monoclonal neutralizing antibodies against human SCUBE2 protein were screened.

[0292] In this embodiment, the full-length sequence of SCUBE2 (SEQ ID NO: 1) was cloned from human breast cancer cells MCF7. The CUB functional region (809-918aa SEQ ID NO: 2) of the full-length SCUBE2 sequence annotated in NCBI was selected and constructed into the pGEX4T-AB1 vector, and the protein was expressed and purified in a prokaryotic system.

[0293] Full-length SCUBE2 sequence:

[0294]

[0295] CUB structural domain (SEQ ID NO:1, 809-918aa)

[0296] CGGELGDFTGYIESPNYPGNYPANTECTWTINPPPKRRILIVVPEIFLPIEDDCGDYLVMRKTSSSNSVTTYETCQTYERPIAFTSRSKKLWIQFKSNEGNSARGFQVPY(SEQ ID NO:2)

[0297] Purified recombinant CUB domain protein was used as an antigen to immunize Balb / C mice. After three immunizations, the binding ability of mouse serum to the antigen was detected by ELISA, and mice with anti-SCUBE2 immunoglobulin titers were obtained. Simultaneously, human recombinant SHH protein was used as a competitive binding protein for SCUBE2 function detection, and competitive ELISA was performed on serum along with the antigen protein. The two mice with the best detection results were selected, sacrificed, and their spleens were fused with SP2 / 0 myeloma cells.

[0298] The fused hybridoma cells were screened for antibodies, and a competitive ELISA was performed on the hybridoma cell supernatant using recombinant human SHH protein. Positive monoclonal antibodies were selected for two subsequent subcloning processes, and three positive mouse monoclonal antibodies were finally obtained, named LTMA1F1, LTMA16D5, and LTMA24C10, respectively.

[0299] Example 2: Determination of the binding ability of SCUBE2 monoclonal antibody to CUB domain

[0300] In this embodiment, the ability of the SCUBE2 monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 screened in Example 1 to bind to the CUB domain (SCUBE2 protein 809-918aa) was tested.

[0301] First, a 96-well ELISA plate was coated with CUB domain antigen protein. After adding hybridoma cell supernatant, it was incubated at room temperature for 2 hours. After washing three times, goat secondary antibody bound to mouse antibody was added, and the plate was incubated at room temperature for 1 hour. After washing three times, the reaction substrate was added, and the reaction was stopped after 5-10 minutes. The absorbance signal at 450 nM was immediately detected using an ELISA reader.

[0302] Results: As shown in Table 1, compared with the negative control and blank control group, the three SCUBE2 mouse monoclonal antibodies screened in this invention all showed strong binding ability to the CUB domain in the SCUBE2 protein.

[0303] Table 1: Binding ability of SCUBE2 monoclonal antibody to CUB domain

[0304] LTMA1F1 1.268 LTMA16D5 1.279 LTMA24C10 0.695 negative control 0.067 Blank control 0.061

[0305] Example 3: SCUBE2 mouse monoclonal antibody blocks SCUBE2 protein function

[0306] In this embodiment, it was tested whether the three SCUBE2 mouse monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 screened in Example 1 could block the protein function of SCUBE2.

[0307] Because the CUB domain of the SCUBE2 protein binds to the SHH protein, it can cleave and release SHH. If the antibody screened in Example 1 can competitively block the binding of the CUB domain to the SHH protein, it can inhibit the ability of the SCUBE2 protein to cleave and release SHH.

[0308] In this embodiment, a competitive ELISA experiment was first used for verification. A 96-well ELISA plate was coated with SCUBE2 antigen protein, and hybridoma cell supernatant and different concentrations of the competitive protein SHH were added and incubated at room temperature for 2 hours. The negative control was the competitive binding result of adding commercially available SCUBE2 antibody (which recognizes the non-CUB region). After washing three times, goat secondary antibody bound to mouse antibody was added, and the plate was incubated at room temperature for 1 hour. After washing three times, the reaction substrate was added, and the reaction was stopped after 5-10 minutes. The absorbance signal at 450 nM was immediately detected using an ELISA reader.

[0309] Results: Table 2 shows the competitive binding of different antibodies to SCUBE2 protein with SHH protein.

[0310] Table 2: Competitive binding of SCUBE2 monoclonal antibody and SHH protein to SCUBE2 protein

[0311]

[0312] SHH protein specifically binds to the CUB region of SCUBE2 protein. Table 2 shows that as the concentration of the competitive protein SHH added to the system increases, the binding of the negative control antibody, whose antibody recognition site is not in the CUB region, to SCUBE2 does not change significantly, while the binding of the three SCUBE2 mouse monoclonal antibodies screened in this invention to the CUB domain gradually weakens.

[0313] This suggests that the binding sites of these three SCUBE2 mouse monoclonal antibodies to SCUBE2 are the same as the binding sites of SHH protein and SCUBE2 protein, thus possessing the ability to competitively block the binding of SHH protein and SCUBE2 protein.

[0314] Example 4: SCUBE2 Monoclonal Antibody

[0315] In this embodiment, the positive hybridoma cells that produced the above three antibodies were subjected to next-generation sequencing, and the amino acid sequences corresponding to the antibody variable region sequences were obtained as shown in Table 3.

[0316] Table 3. Variable region sequence of the antibody of the present invention

[0317]

[0318] Note: The sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italicized parts of the sequence are FR sequences, and the underlined parts are CDR sequences.

[0319] The amino acid sequences corresponding to the CDR regions of the antibody heavy and light chains are shown in Table 4.

[0320] Table 4. CDR region sequences of each heavy chain and light chain

[0321]

[0322] Example 5: Purification and Isotype Detection of SCUBE2 Monoclonal Antibody

[0323] In this embodiment, antibody collection and purification were performed. First, 2 million hybridoma cells were injected into the peritoneal cavity of pre-sensitized Balb / C mice. After 7-14 days of observation, the mice were sacrificed, and ascites fluid was collected. High concentrations of SCUBE2 monoclonal antibody were obtained through purification using a Protein G antibody affinity column. The purity of the obtained antibody was then detected using Coomassie Brilliant Blue staining. Differences in the position and size of the heavy and light chain bands were observed in the Coomassie Brilliant Blue staining results. Antibody subtypes were detected using an antibody subtype identification ELISA kit.

[0324] Results: After purification, clear heavy and light chain bands of the antibody appeared in the protein lanes, with no impurities, indicating that the obtained antibody had high purity. Figure 1 A). Antibodies LTMA1F1 and LTMA16D5 were detected as belonging to the IgG1 type, and antibody LTMA24C10 belonged to the IgM subtype. Figure 1 B).

[0325] Example 6: Detection of binding specificity of SCUBE2 monoclonal antibody

[0326] To detect the binding specificity of the monoclonal antibodies screened in this invention, this embodiment uses Western blot to detect the recognition of the full-length SCUBE2 protein in breast cancer cells by the three SCUBE2 mouse monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 in this invention.

[0327] Results: In the protein lanes overexpressing SCUBE2, the SCUBE2 monoclonal antibodies all exhibited accurate and specific recognition performance. Figure 1 C). This demonstrates that the SCUBE2 monoclonal antibody obtained in this invention has high recognition specificity for the full-length human SCUBE2 protein.

[0328] Example 7: In vitro neutralization effect of SCUBE2 monoclonal antibody

[0329] In this embodiment, the in vitro neutralizing effect of the SCUBE2 monoclonal antibody on the CUB region was tested.

[0330] SCUBE2 monoclonal antibody treatment inhibited MCF7 cell supernatant-induced GLI reporter system activation.

[0331] SCUBE2 neutralizing antibodies inhibit the binding of SHH to SCUBE2 by competitively binding to the CUB functional domain of SCUBE2, thereby blocking the ability of SCUBE2 to release SHH through cleavage. GLI reporter systems are commonly used in the art to detect cellular SHH release levels. Therefore, this embodiment employs an in vitro GLI reporter system initiation experiment to detect the effect of SCUBE2 antibodies on SHH release from tumor cells.

[0332] After co-incubating the SCUBE2 monoclonal antibody with breast cancer cells MCF7 for 24 hours, the supernatant suspension of the cancer cells was collected and used to treat target cells MC3T3 that had been transferred into the GLI reporter system.

[0333] Results: Compared with the blank group (DMEM), the breast cancer supernatant in the control IgG treatment group significantly initiated the GLI reporter system, while the ability of MCF7 cell supernatant to induce GLI initiation was significantly inhibited after the addition of SCUBE2 monoclonal antibody. Figure 2 A). This indicates that the SCUBE2 monoclonal antibody inhibits the release of SHH into the extracellular space from breast cancer cells.

[0334] SCUBE2 monoclonal antibody treatment inhibited osteoblast differentiation induced by MCF7 cell supernatant.

[0335] SHH significantly promotes osteoblast differentiation, manifested by upregulation of ALP activity. In this embodiment, the effect of SCUBE2 monoclonal antibody on SHH release from tumor cells was detected by examining ALP activity. Supernatants from MCF7 cells treated with control IgG and SCUBE2 antibody were collected separately for in vitro osteoblast differentiation induction experiments.

[0336] Results: The supernatant from MCF7 in the control IgG treatment group significantly promoted ALP activity in osteoblasts, indicating that breast cancer cells release high levels of SHH, which can initiate osteoblast differentiation. In contrast, the SCUBE2 monoclonal antibody treatment groups all inhibited the degree of osteoblast differentiation induced by breast cancer cells. Figure 2 B) This indicates that all three neutralizing antibodies in this invention can inhibit the release of SHH from breast cancer cells, thereby inhibiting tumor cell-induced osteogenic differentiation.

[0337] Expression of SHH in the supernatant of breast cancer cells after treatment with different concentrations of SCUBE2 monoclonal antibody

[0338] This embodiment also detected the expression of SHH in breast cancer cell supernatant after treatment with different concentrations of SCUBE2 monoclonal antibody.

[0339] Results: The results showed that as the concentration of SCUBE2 monoclonal antibody increased, the amount of SHH released extracellularly by breast cancer cells decreased. Figure 2 C).

[0340] The results of this embodiment demonstrate that the three SCUBE2 monoclonal antibodies LTMA1F1, LTMA16D5 and LTMA24C10 in this invention can inhibit the cleavage and release of SCUBE2 protein from SHH in vitro, thereby neutralizing the function of SCUBE2 protein.

[0341] Example 8: Inhibition of Bone Metastasis in Breast Cancer by SCUBE2 Monoclonal Antibody

[0342] This embodiment tested whether the SCUBE2 monoclonal antibody had the effect of inhibiting bone metastasis of breast cancer in vivo.

[0343] After injecting luciferase-labeled MCF7 human breast cancer cells into immunodeficient mice, luciferase substrate was injected, and the light signal corresponding to the metastatic load was observed. Results showed that tail vein injection of SCUBE2 monoclonal antibody significantly inhibited bone metastasis of breast cancer in mice. Figure 3 A, B).

[0344] 4T1.2 mouse breast cancer cells labeled with luciferase were injected into the left ventricle of Balb / C mice. Results showed that intraperitoneal injection of the SCUBE2 monoclonal antibody also significantly inhibited bone metastasis signaling in immunocompetent mice. Figure 3 C, D).

[0345] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences <120> SCUBE2 neutralizing antibodies and their pharmaceutical uses <130> P2021-2822 <160> 25 <170> PatentIn version 3.5 <210> 1 <211> 999 <212> PRT <213> Homo sapiens <400> 1 Met Gly Val Ala Gly Arg Asn Arg Pro Gly Ala Ala Trp Ala Val Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Pro Pro Leu Leu Leu Leu Ala Gly Ala Val 20 25 30 Pro Pro Gly Arg Gly Arg Ala Ala Gly Pro Gln Glu Asp Val Asp Glu 35 40 45 Cys Ala Gln Gly Leu Asp Asp Cys His Ala Asp Ala Leu Cys Gln Asn 50 55 60 Thr Pro Thr Ser Tyr Lys Cys Ser Cys Lys Pro Gly Tyr Gln Gly Glu 65 70 75 80 Gly Arg Gln Cys Glu Asp Ile Asp Glu Cys Gly Asn Glu Leu Asn Gly 85 90 95 Gly Cys Val His Asp Cys Leu Asn Ile Pro Gly Asn Tyr Arg Cys Thr 100 105 110 Cys Phe Asp Gly Phe Met Leu Ala His Asp Gly His Asn Cys Leu Asp 115 120 125 Val Asp Glu Cys Leu Glu Asn Asn Gly Gly Cys Gln His Thr Cys Val 130 135 140 Asn Val Met Gly Ser Tyr Glu Cys Cys Cys Lys Glu Gly Phe Phe Leu 145 150 155 160 Ser Asp Asn Gln His Thr Cys Ile His Arg Ser Glu Glu Gly Leu Ser 165 170 175 Cys Met Asn Lys Asp His Gly Cys Ser His Ile Cys Lys Glu Ala Pro 180 185 190 Arg Gly Ser Val Ala Cys Glu Cys Arg Pro Gly Phe Glu Leu Ala Lys 195 200 205 Asn Gln Arg Asp Cys Ile Leu Thr Cys Asn His Gly Asn Gly Gly Cys 210 215 220 Gln His Ser Cys Asp Asp Thr Ala Asp Gly Pro Glu Cys Ser Cys His 225 230 235 240 Pro Gln Tyr Lys Met His Thr Asp Gly Arg Ser Cys Leu Glu Arg Glu 245 250 255 Asp Thr Val Leu Glu Val Thr Glu Ser Asn Thr Thr Ser Val Val Asp 260 265 270 Gly Asp Lys Arg Val Lys Arg Arg Leu Leu Met Glu Thr Cys Ala Val 275 280 285 Asn Asn Gly Gly Cys Asp Arg Thr Cys Lys Asp Thr Ser Thr Gly Val 290 295 300 His Cys Ser Cys Pro Val Gly Phe Thr Leu Gln Leu Asp Gly Lys Thr 305 310 315 320 Cys Lys Asp Ile Asp Glu Cys Gln Thr Arg Asn Gly Gly Cys Asp His 325 330 335 Phe Cys Lys Asn Ile Val Gly Ser Phe Asp Cys Gly Cys Lys Lys Gly 340 345 350 Phe Lys Leu Leu Thr Asp Glu Lys Ser Cys Gln Asp Val Asp Glu Cys 355 360 365 Ser Leu Asp Arg Thr Cys Asp His Ser Cys Ile Asn His Pro Gly Thr 370 375 380 Phe Ala Cys Ala Cys Asn Arg Gly Tyr Thr Leu Tyr Gly Phe Thr His 385 390 395 400 Cys Gly Asp Thr Asn Glu Cys Ser Ile Asn Asn Gly Gly Cys Gln Gln 405 410 415 Val Cys Val Asn Thr Val Gly Ser Tyr Glu Cys Gln Cys His Pro Gly 420 425 430 Tyr Lys Leu His Trp Asn Lys Lys Asp Cys Val Glu Val Lys Gly Leu 435 440 445 Leu Pro Thr Ser Val Ser Pro Arg Val Ser Leu His Cys Gly Lys Ser 450 455 460 Gly Gly Gly Asp Gly Cys Phe Leu Arg Cys His Ser Gly Ile His Leu 465 470 475 480 Ser Ser Asp Val Thr Thr Ile Arg Thr Ser Val Thr Phe Lys Leu Asn 485 490 495 Glu Gly Lys Cys Ser Leu Lys Asn Ala Glu Leu Phe Pro Glu Gly Leu 500 505 510 Arg Pro Ala Leu Pro Glu Lys His Ser Ser Val Lys Glu Ser Phe Arg 515 520 525 Tyr Val Asn Leu Thr Cys Ser Ser Gly Lys Gln Val Pro Gly Ala Pro 530 535 540 Gly Arg Pro Ser Thr Pro Lys Glu Met Phe Ile Thr Val Glu Phe Glu 545 550 555 560 Leu Glu Thr Asn Gln Lys Glu Val Thr Ala Ser Cys Asp Leu Ser Cys 565 570 575 Ile Val Lys Arg Thr Glu Lys Arg Leu Arg Lys Ala Ile Arg Thr Leu 580 585 590 Arg Lys Ala Val His Arg Glu Gln Phe His Leu Gln Leu Ser Gly Met 595 600 605 Asn Leu Asp Val Ala Lys Lys Pro Pro Arg Thr Ser Glu Arg Gln Ala 610 615 620 Glu Ser Cys Gly Val Gly Gln Gly His Ala Glu Asn Gln Cys Val Ser 625 630 635 640 Cys Arg Ala Gly Thr Tyr Tyr Asp Gly Ala Arg Glu Arg Cys Ile Leu 645 650 655 Cys Pro Asn Gly Thr Phe Gln Asn Glu Glu Gly Gln Met Thr Cys Glu 660 665 670 Pro Cys Pro Arg Pro Gly Asn Ser Gly Ala Leu Lys Thr Pro Glu Ala 675 680 685 Trp Asn Met Ser Glu Cys Gly Gly Leu Cys Gln Pro Gly Glu Tyr Ser 690 695 700 Ala Asp Gly Phe Ala Pro Cys Gln Leu Cys Ala Leu Gly Thr Phe Gln 705 710 715 720 Pro Glu Ala Gly Arg Thr Ser Cys Phe Pro Cys Gly Gly Gly Leu Ala 725 730 735 Thr Lys His Gln Gly Ala Thr Ser Phe Gln Asp Cys Glu Thr Arg Val 740 745 750 Gln Cys Ser Pro Gly His Phe Tyr Asn Thr Thr Thr His Arg Cys Ile 755 760 765 Arg Cys Pro Val Gly Thr Tyr Gln Pro Glu Phe Gly Lys Asn Asn Cys 770 775 780 Val Ser Cys Pro Gly Asn Thr Thr Thr Asp Phe Asp Gly Ser Thr Asn 785 790 795 800 Ile Thr Gln Cys Lys Asn Arg Arg Cys Gly Gly Glu Leu Gly Asp Phe 805 810 815 Thr Gly Tyr Ile Glu Ser Pro Asn Tyr Pro Gly Asn Tyr Pro Ala Asn 820 825 830 Thr Glu Cys Thr Trp Thr Ile Asn Pro Pro Pro Lys Arg Arg Ile Leu 835 840 845 Ile Val Val Pro Glu Ile Phe Leu Pro Ile Glu Asp Asp Cys Gly Asp 850 855 860 Tyr Leu Val Met Arg Lys Thr Ser Ser Ser Asn Ser Val Thr Thr Tyr 865 870 875 880 Glu Thr Cys Gln Thr Tyr Glu Arg Pro Ile Ala Phe Thr Ser Arg Ser 885 890 895 Lys Lys Leu Trp Ile Gln Phe Lys Ser Asn Glu Gly Asn Ser Ala Arg 900 905 910 Gly Phe Gln Val Pro Tyr Val Thr Tyr Asp Glu Asp Tyr Gln Glu Leu 915 920 925 Ile Glu Asp Ile Val Arg Asp Gly Arg Leu Tyr Ala Ser Glu Asn His 930 935 940 Gln Glu Ile Leu Lys Asp Lys Lys Leu Ile Lys Ala Leu Phe Asp Val 945 950 955 960 Leu Ala His Pro Gln Asn Tyr Phe Lys Tyr Thr Ala Gln Glu Ser Arg 965 970 975 Glu Met Phe Pro Arg Ser Phe Ile Arg Leu Leu Arg Ser Lys Val Ser 980 985 990 Arg Phe Leu Arg Pro Tyr Lys 995 <210> 2 <211> 110 <212> PRT <213> Homo sapiens <400> 2 Cys Gly Gly Glu Leu Gly Asp Phe Thr Gly Tyr Ile Glu Ser Pro Asn 1 5 10 15 Tyr Pro Gly Asn Tyr Pro Ala Asn Thr Glu Cys Thr Trp Thr Ile Asn 20 25 30 Pro Pro Pro Lys Arg Arg Ile Leu Ile Val Val Pro Glu Ile Phe Leu 35 40 45 Pro Ile Glu Asp Asp Cys Gly Asp Tyr Leu Val Met Arg Lys Thr Ser 50 55 60 Ser Ser Asn Ser Val Thr Thr Tyr Glu Thr Cys Gln Thr Tyr Glu Arg 65 70 75 80 Pro Ile Ala Phe Thr Ser Arg Ser Lys Lys Leu Trp Ile Gln Phe Lys 85 90 95 Ser Asn Glu Gly Asn Ser Ala Arg Gly Phe Gln Val Pro Tyr 100 105 110 <210> 3 <211> 116 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ile Tyr 20 25 30 Asn Ile His Trp Val Lys Leu Thr Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ser Ile Tyr Pro Gly Asp Gly His Thr Ser Tyr Asn Pro Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Phe Cys 85 90 95 Ala Arg Leu Glu Lys Tyr Thr Asp Tyr Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115 <210> 4 <211> 112 <212> PRT <213> Artificial Sequence <400> 4 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Ala Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Leu Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <400> 5 Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Ile Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 亮氨酸-谷氨酰胺-异亮氨酸-天冬酰胺-天冬酰胺-亮氨酸-赖氨酸-天冬氨酸-苏氨酸-丙氨酸-苏氨酸-酪氨酸-苯丙氨酸-半胱氨酸 85 90 95 甘氨酸-精氨酸-苯丙氨酸-苏氨酸-脯氨酸-缬氨酸-缬氨酸-谷氨酸-天冬氨酸-酪氨酸-色氨酸-甘氨酸-谷氨酰胺-甘氨酸-丝氨酸-苏氨酸 100 105 110 亮氨酸-苏氨酸-缬氨酸-丝氨酸-丝氨酸 115 <210> 6 <211> 112 <212> PRT <213> Artificial Sequence <400> 6 天冬氨酸-缬氨酸-缬氨酸-甲硫氨酸-苏氨酸-谷氨酰胺-苏氨酸-脯氨酸-亮氨酸-苏氨酸-亮氨酸-丝氨酸-缬氨酸-苏氨酸-异亮氨酸-甘氨酸 1 5 10 15 谷氨酰胺-脯氨酸-丙氨酸-丝氨酸-甲硫氨酸-丝氨酸-半胱氨酸-赖氨酸-丝氨酸-丝氨酸-谷氨酰胺-丝氨酸-亮氨酸-亮氨酸-酪氨酸-丝氨酸 20 25 30 天冬酰胺-甘氨酸-赖氨酸-苏氨酸-酪氨酸-亮氨酸-天冬酰胺-色氨酸-亮氨酸-亮氨酸-谷氨酰胺-精氨酸-脯氨酸-甘氨酸-谷氨酰胺-丝氨酸 35 40 45 脯氨酸-赖氨酸-精氨酸-亮氨酸-异亮氨酸-酪氨酸-亮氨酸-缬氨酸-丝氨酸-赖氨酸-亮氨酸-天冬氨酸-丝氨酸-甘氨酸-缬氨酸-脯氨酸 50 55 60 天冬氨酸-精氨酸-苯丙氨酸-苏氨酸-甘氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-苏氨酸-天冬氨酸-苯丙氨酸-苏氨酸-亮氨酸-赖氨酸-异亮氨酸 65 70 75 80 丝氨酸-精氨酸-缬氨酸-谷氨酸-丙氨酸-谷氨酸-天冬氨酸-亮氨酸-甘氨酸-缬氨酸-酪氨酸-酪氨酸-半胱氨酸-缬氨酸-谷氨酰胺-丙氨酸 85 90 95 Thr His Phe Pro His Thr Phe Gly Ser Gly Thr Glu Leu Glu Ile Lys 100 105 110 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Tyr Gly Ser Ser Ser Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 8 <211> 111 <212> PRT <213> Artificial Sequence <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ile Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 9 <211> 8 <212> PRT <213> Artificial Sequence <400> 9 Gly Tyr Thr Phe Thr Ile Tyr Asn 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial Sequence <400> 10 Ile Tyr Pro Gly Asp Gly His Thr 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <400> 11 Ala Arg Leu Glu Lys Tyr Thr Asp Tyr 1 5 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <400> 12 Gln Ser Leu Leu Ala Ser Asp Gly Lys Thr Tyr 1 5 10 <210> 13 <211> 3 <212> PRT <213> Artificial Sequence <400> 13 Leu Val Ser 1 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <400> 14 Trp Gln Gly Thr His Phe Pro Phe Thr 1 5 <210> 15 <211> 8 <212> PRT <213> Artificial Sequence <400> 15 Gly Tyr Thr Phe Thr Asp Tyr Ser 1 5 <210> 16 <211> 8 <212> PRT <213> Artificial Sequence <400> 16 Ile Asn Thr Glu Thr Gly Glu Pro 1 5 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <400> 17 Gly Arg Phe Thr Pro Val Val Glu Asp Tyr 1 5 10 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <400> 18 Gln Ser Leu Leu Tyr Ser Asn Gly Lys Thr Tyr 1 5 10 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <400> 19 Val Gln Ala Thr His Phe Pro His Thr 1 5 <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <400> 20 Gly Tyr Ser Phe Thr Gly Tyr Thr 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial Sequence <400> twenty one Ile Asn Pro Tyr Asn Gly Gly Thr 1 5 <210> twenty two <211> 13 <212> PRT <213> Artificial Sequence <400> twenty two Ala Arg Arg Asp Tyr Tyr Gly Ser Ser Ser Phe Asp Tyr 1 5 10 <210> twenty three <211> 10 <212> PRT <213> Artificial Sequence <400> twenty three Gln Ser Val Ser Thr Ser Ser Tyr Ser Tyr 1 5 10 <210> twenty four <211> 3 <212> PRT <213> Artificial Sequence <400> twenty four Tyr Ala Ser 1 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <400> 25 Gln His Ser Trp Glu Ile Pro Trp Thr 1 5

Claims

1. An antibody that binds to the SCUBE2 protein, characterized in that, The antibody specifically binds to the CUB domain of the SCUBE2 protein, and it competitively blocks the binding of the Hedgehog signaling ligand to the SCUBE2 protein. The amino acid sequence of the CUB domain is shown in SEQ ID NO: 2; The antibody has three complementary determinant HCDRs selected from the heavy chain variable region and three complementary determinant liquid regions LCDRs selected from the following: (A) VH-CDR1: The amino acid sequence is shown in SEQ ID NO:15; VH-CDR2: The amino acid sequence is shown in SEQ ID NO:16; VH-CDR3: The amino acid sequence is shown in SEQ ID NO:17; VL-CDR1: The amino acid sequence is shown in SEQ ID NO:18; VL-CDR2: The amino acid sequence is shown in SEQ ID NO:13; and VL-CDR3: The amino acid sequence is shown in SEQ ID NO:19; or (B) VH-CDR1: The amino acid sequence is shown in SEQ ID NO:9; VH-CDR2: The amino acid sequence is shown in SEQ ID NO:10; VH-CDR3: The amino acid sequence is shown in SEQ ID NO:11; VL-CDR1: The amino acid sequence is shown in SEQ ID NO:12; VL-CDR2: The amino acid sequence is shown in SEQ ID NO:13; and VL-CDR3: The amino acid sequence is shown in SEQ ID NO:

14.

2. The antibody as described in claim 1, characterized in that, The heavy chain variable region and light chain variable region of the antibody are selected from the following group: (a) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:5, and the light chain variable region with the amino acid sequence shown in SEQ ID NO:6; or (b) The heavy chain variable region with the amino acid sequence shown in SEQ ID NO:3 and the light chain variable region with the amino acid sequence shown in SEQ ID NO:

4.

3. A recombinant protein, characterized in that, The recombinant protein consists of the following: (i) the antibody as described in claim 1; and (ii) Tag sequences that assist in expression and / or purification.

4. An antibody conjugate, characterized in that, The antibody conjugate contains: (a) The antibody portion, wherein the antibody portion is selected from the group consisting of: The antibody as described in claim 1; and (b) A conjugation portion coupled to the antibody portion, wherein the conjugation portion is a detectable marker.

5. Use of an active ingredient, said active ingredient being selected from the group consisting of: the antibody of claim 1, the recombinant protein of claim 3, the antibody-drug conjugate of claim 4, or combinations thereof, wherein said active ingredient is used for: (a) Prepare a detection plate or kit for detecting diseases associated with abnormal SCUBE2 protein expression; and / or (b) To develop drugs for the prevention and / or treatment of diseases associated with abnormal SCUBE2 protein expression; The disease associated with abnormal SCUBE2 protein expression is breast cancer bone metastasis.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) an active ingredient, said active ingredient being selected from the group consisting of: The antibody as claimed in claim 1, the recombinant protein as claimed in claim 3, the antibody conjugate as claimed in claim 4, or a combination thereof; and (ii) Pharmaceutically acceptable carriers.

7. A polynucleotide, characterized in that, The polynucleotide encodes a polypeptide selected from the following group: (1) The antibody as described in claim 1; or (2) The recombinant protein as described in claim 3.

8. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 7.

9. An engineered host cell, characterized in that, The host cell contains the vector as described in claim 8 or the genome is integrated with the polynucleotide as described in claim 7.

10. A method for detecting SCUBE2 protein in a sample for non-diagnostic or therapeutic purposes, characterized in that, The method includes the following steps: (1) Contact the sample with the antibody as described in claim 1; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of SCUBE2 protein in the sample.

11. A detection plate, characterized in that, The detection plate comprises a substrate and a test strip, wherein the test strip contains the antibody as described in claim 1, the recombinant protein as described in claim 3, the antibody-drug conjugate as described in claim 4, or a combination thereof.

12. A reagent kit, characterized in that, The kit includes: (1) A first container containing the antibody as described in claim 1; and / or (2) A second container containing a secondary antibody against the antibody as described in claim 1; or, The kit contains the detection plate as described in claim 11.

13. A method for preparing an antibody or recombinant protein, characterized in that, The method includes: (a) Culturing the host cells as described in claim 9 under suitable expression conditions; (b) Isolate the antibody as described in claim 1 or the recombinant protein as described in claim 3 from the culture.

14. A drug combination comprising: (i) A first active ingredient, comprising the antibody as claimed in claim 1, the recombinant protein as claimed in claim 3, the antibody-drug conjugate as claimed in claim 4, or the pharmaceutical composition as claimed in claim 6, or a combination thereof; (ii) A second active ingredient, which includes a Hedgehog signaling inhibitor, an anti-estrogenic drug, or a chemotherapeutic agent.

15. Use of the antibody of claim 1, the recombinant protein of claim 3, the antibody-drug conjugate of claim 4, and / or the pharmaceutical composition of claim 6 in combination with a Hedgehog signaling inhibitor or chemotherapeutic agent in the preparation of a medicament for treating diseases associated with abnormal SCUBE2 protein expression, wherein the abnormal SCUBE2 protein expression is associated with breast cancer bone metastasis.

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