Biomarker for triple negative breast cancer metastasis, antibody targeting same and application of biomarker

Abstract

The invention discloses a biomarker for triple negative breast cancer metastasis, an antibody targeting the biomarker and application of the biomarker. The biomarker is PEAR1 (PEAR1) or phosphorylated PEAR1 (PEAR1). PEAR1 is used as a biomarker, LOXL2 / PEAR1 / CD44 signal channels can be adjusted, and the PEAR1 has wide application prospects in treatment of triple negative breast cancer such as metastatic triple negative breast cancer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to a biomarker for metastasis of triple-negative breast cancer, an antibody targeting the same and application thereof, in particular to application of PEAR1 as a biomarker and an antibody targeting the same in treatment of metastatic triple-negative breast cancer. BACKGROUND

[0002] Breast cancer is the most common malignant tumor in women worldwide, and its incidence is gradually increasing, and it shows a trend of younger age. It occurs in the terminal ductal lobular unit of breast collecting duct, and is prone to metastasis and recurrence, which is the main cause of death in breast cancer patients. Breast cancer is a highly heterogeneous disease with rich intrinsic subtypes, among which triple-negative breast cancer has the highest pathogenicity and the worst prognosis, with characteristics of small age of onset, large potential of recurrence and metastasis, poor prognosis, short overall survival, etc. Due to the lack of expression of hormone receptors, it is not sensitive to endocrine therapy, and the lack of or low level of HER2 expression makes it not sensitive to HER2 targeted therapy, and the effect of chemotherapy is not good, and there is a lack of effective targeted therapy drugs, which is the most challenging subtype of breast cancer. Compared with traditional treatment methods, new adjuvant chemotherapy, platinum drug chemotherapy, new targeted therapy and immunotherapy programs have emerged in recent years, which have been improved, but the overall benefit is limited.

[0003] In the face of the current situation of triple-negative breast cancer with high malignancy, high risk of invasion and metastasis, poor prognosis and limited clinical treatment methods, the field urgently needs to find new targets for TNBC treatment and develop a drug that can improve metastasis and recurrence of triple-negative breast cancer. SUMMARY

[0004] To solve the defect that there is a lack of a drug that can improve metastasis and recurrence of triple-negative breast cancer in the prior art, the present application provides a target point related to treatment of metastatic triple-negative breast cancer, namely PEAR1 and application thereof, and in particular provides an antibody targeting PEAR1 for anti-triple-negative breast cancer metastasis and application thereof.

[0005] The present inventors have conducted extensive and in-depth research, and for the first time systematically proved that PEAR1 is an important potential target for TNBC metastasis, PEAR1 is highly expressed in TNBC and is related to poor prognosis of TNBC patients, and it is clear that PEAR1 S891 phosphorylation is a decisive phosphorylation site for mediating PEAR1 to play a role in promoting TNBC metastasis.

[0006] The present inventors identified a new endogenous ligand LOXL2 of PEAR1, which can induce phosphorylation of PEAR1 after binding to the PEAR1-EMI domain. Phosphorylated PEAR1 is a new chaperone protein of CD44, and the interaction between the two can maintain the stable expression of CD44 on the cell membrane, so that it is protected from endocytosis-mediated lysosomal degradation.

[0007] Based on the above research, the inventors independently developed a high-specificity monoclonal antibody targeting the PEAR1-EMI domain, and to avoid dimerization activation of the antibody, the single arm of the antibody was selected and coupled with human HSA to prolong its half-life in the body. The PEAR1 fab-HSA monoclonal antibody can competitively block the binding of LOXL2 and PEAR1, and can significantly inhibit TNBC metastasis.

[0008] In TNBC patients, the expression levels of PEAR1, PEAR1 Ser891 phosphorylation, LOXL2 and CD44 are all significantly up-regulated, and are positively correlated, but are negatively correlated with overall survival, wherein the PEAR1 Ser891 phosphorylation level is considered to be the best independent factor for the prognosis of TNBC patients, and the combined prognostic effect of PEAR1 Ser891 phosphorylation and CD44 expression levels is better.

[0009] On this basis, the present application is completed.

[0010] The present application solves the above technical problems through the following technical solutions.

[0011] The first aspect of the present application provides a use of a reagent for detecting a biomarker or a combination of biomarkers in the preparation of a product for diagnosing or assisting in the diagnosis of triple-negative breast cancer;

[0012] The biomarker is PEAR1 or phosphorylated PEAR1;

[0013] The combination of biomarkers comprises at least two of the following:

[0014] (1) PEAR1;

[0015] (2) phosphorylated PEAR1;

[0016] (3) LOXL2;

[0017] (4) CD44;

[0018] The reagent is used to detect the protein expression level of the biomarker or the combination of biomarkers.

[0019] In some embodiments, the triple-negative breast cancer is metastatic triple-negative breast cancer.

[0020] In some embodiments, the phosphorylated PEAR1 is serine-phosphorylated PEAR1, for example, PEAR1 phosphorylated at one or more of Ser795, Ser891, Ser953, Ser976 and Ser1029.

[0021] In some embodiments, the phosphorylated PEAR1 is Ser891 phosphorylated PEAR1.

[0022] In some embodiments, the phosphorylated PEAR1 is Ser795, Ser891, Ser953, Ser976 and Ser1029 phosphorylated PEAR1.

[0023] In some embodiments, the product comprises a kit, a chip, a test paper and a high-throughput sequencing platform.

[0024] The second aspect of the present application provides an antibody targeting PEAR1, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, respectively; the light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are according to the definition of Kabat.

[0025] In some embodiments, the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 14 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 14.

[0026] In some embodiments, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 19 or having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.2%, at least 98.5%, at least 98.8%, at least 99%, at least 99.2%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID NO: 19.

[0027] In some embodiments, the antibody is a full-length antibody, a Fab, a Fab’-SH, a Fv or a (Fab’)2; the Fv is, for example, a scFv.

[0028] The third aspect of the present application provides a fusion protein comprising the antibody of the second aspect, and a protein fragment; the protein fragment is capable of promoting the antibody to remain stable and prolonging the half-life of the antibody.

[0029] In the present application, the way to prolong the half-life is not limited to the form of the fusion protein mentioned in the present application, but can also be other modifications, such as PEGylation, fatty acid coupling, etc.

[0030] In some embodiments, the protein fragment is human serum albumin, an antibody or fragment thereof that binds human serum albumin, an immunoglobulin constant region or a fragment thereof.

[0031] In some embodiments, the human serum albumin is linked to the N-terminus of the antibody.

[0032] In some embodiments, the human serum albumin is linked to the C-terminus of the antibody.

[0033] In some embodiments, the antibody further comprises a heavy chain constant region and a light chain constant region.

[0034] In some preferred embodiments, the human serum albumin is linked to the C-terminus of the heavy chain constant region.

[0035] In some embodiments, the human serum albumin has an amino acid sequence as shown in SEQ ID NO: 22.

[0036] In some specific embodiments, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 21.

[0037] In some specific embodiments, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 23.

[0038] In some specific embodiments, the fusion protein comprises a first polypeptide having an amino acid sequence as shown in SEQ ID NO: 15 and a second polypeptide having an amino acid sequence as shown in SEQ ID NO: 20.

[0039] The fourth aspect of the present application provides a polypeptide having an amino acid sequence as shown in SEQ ID NO: 24.

[0040] In some embodiments, the polypeptide specifically binds to the antibody or variant thereof as described in the second aspect, or the fusion protein as described in the third aspect;

[0041] The variant has at least 80% identity with the antibody and retains the function of the antibody.

[0042] The fifth aspect of the present application provides a polynucleotide encoding the antibody as described in the second aspect, the fusion protein as described in the third aspect, or the polypeptide as described in the fourth aspect.

[0043] A sixth aspect of the present application provides an expression vector comprising the polynucleotide according to the fifth aspect.

[0044] A seventh aspect of the present application provides a transformant comprising the polynucleotide according to the fifth aspect or the expression vector according to the sixth aspect.

[0045] An eighth aspect of the present application provides a method for preparing a polypeptide, an antibody or a fusion protein, comprising culturing the transformant according to the seventh aspect, and obtaining the polypeptide, the antibody or the fusion protein from the culture.

[0046] A ninth aspect of the present application provides a pharmaceutical composition comprising the antibody according to the second aspect or the fusion protein according to the third aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0047] A tenth aspect of the present application provides use of a PEAR1 inhibitor in the preparation of a medicament for diagnosing or treating cancer.

[0048] In some embodiments, the PEAR1 inhibitor reduces or blocks the binding of PEAR1 to LOXL2, inhibits PEAR1 serine phosphorylation and / or inhibits the expression of CD44.

[0049] In some preferred embodiments, the PEAR1 inhibitor is an agent that mutates one or more of Ser795, Ser891, Ser953, Ser976 and Ser1029 of PEAR1 to Ala.

[0050] In some preferred embodiments, the PEAR1 inhibitor is an agent that mutates Ser891 of PEAR1 to Ala.

[0051] In some preferred embodiments, the PEAR1 inhibitor is an agent that mutates Ser795, Ser891, Ser953, Ser976 and Ser1029 of PEAR1 to Ala.

[0052] In some embodiments, the cancer is selected from breast cancer, blood cancer, brain cancer, colon cancer and ovarian cancer.

[0053] In some embodiments, the breast cancer is triple negative breast cancer.

[0054] In some embodiments, the triple negative breast cancer is metastatic triple negative breast cancer.

[0055] In some embodiments, the PEAR1 inhibitor is the antibody according to the second aspect, the fusion protein according to the third aspect, or the pharmaceutical composition according to the ninth aspect.

[0056] The eleventh aspect of the present application provides use of the antibody according to the second aspect, the fusion protein according to the third aspect, the polynucleotide according to the fifth aspect, the expression vector according to the sixth aspect or the pharmaceutical composition according to the ninth aspect in the preparation of a medicament for treating cancer.

[0057] In some embodiments, the cancer is selected from breast cancer, blood cancer, brain cancer, colon cancer and ovarian cancer.

[0058] In some embodiments, the breast cancer is triple negative breast cancer, for example metastatic triple negative breast cancer.

[0059] The twelfth aspect of the present application provides a method for detecting a biomarker or a combination of biomarkers of triple negative breast cancer in a sample, the biomarker or the combination of biomarkers being according to the first aspect.

[0060] The method comprises the step of contacting the sample with the antibody according to the second aspect, or the fusion protein according to the third aspect.

[0061] In some embodiments, the method is not for therapeutic or diagnostic purposes.

[0062] In the present application, firstly, the important application value of PEAR1 (Platelet endothelial aggregation receptor 1) as a new target for anti-triple negative breast cancer metastasis is proved through a series of in vitro cell experiments and in vivo mouse tumor-bearing experiments. Secondly, it is proved that LOXL2 (Lysyl oxidase like protein 2) binds to the EMI domain of PEAR1, and is a new type of ligand for inducing the phosphorylation of PEAR1 Ser891. Then, the inventors proved that Ser891 phosphorylation is the key active form of PEAR1 to promote triple negative breast cancer metastasis. Ser891 phosphorylated PEAR1 acts as an important chaperone protein of CD44 (Cluster of differentiation 44), which can maintain the stable expression of CD44 on the cell membrane to normally exert its function. In addition, the inventors have also developed a high-specificity monoclonal antibody targeting the EMI domain of PEAR1. In order to avoid the dimerization and activation of the antibody, the single arm of the antibody is selected and coupled with human serum albumin (HSA) to prolong its half-life in the body. PEAR1 fab-HSA monoclonal antibody mainly binds to PEAR1-EMI recombinant protein, but does not bind to PEAR1-EMI polypeptide, indicating that the interaction occurs in the spatial epitope.

[0063] The inventors proved by experiments that PEAR1 fab-HSA monoclonal antibody competes with LOXL2 to bind to PEAR1, that is, PEAR1 fab-HSA monoclonal antibody can block the binding of LOXL2 to PEAR1. PEAR1 fab-HSA monoclonal antibody can inhibit the PEAR1 serine phosphorylation induced by LOXL2 and the stable expression of downstream CD44. Through a series of in vitro cell experiments and in vivo mouse tumor-bearing experiments, it is proved that PEAR1 fab-HSA monoclonal antibody can effectively inhibit the invasion and migration of MDA-MB-231 cells, and inhibit the lung metastasis and liver metastasis of MDA-MB-231 cells by hematogenous metastasis.

[0064] It is known in the art that CD44 is a molecular marker of various tumor stem cells and is closely related to the metastasis of pan-cancer. Based on the analysis of TIMER2.0 database, the inventors found that the expression of PEAR1 in Her2 + In various cancers such as breast cancer, blood cancer, brain cancer, colon cancer, ovarian cancer, etc., the expression of PEAR1 and CD44 is positively correlated, and the expression amount of PEAR1 is negatively correlated with prognosis. The above results show that PEAR1 regulates the expression of CD44 and PEAR1-CD44 pathway inhibitors have pan-cancer universality.

[0065] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, that is, to obtain each preferred example of the present application.

[0066] The reagents and raw materials used in the present application are commercially available.

[0067] The positive progress effect of the present application is that:

[0068] PEAR1 of the present application has the potential as a target for anti-triple negative breast cancer metastasis and a biomarker for triple negative breast cancer metastasis, and regulates the signal pathway; anti-PEAR1 antibody can specifically bind to the spatial epitope, thereby inhibiting the binding of PEAR1 and LOXL2, and the anti-metastasis effect is verified in in vivo experiments. BRIEF DESCRIPTION OF DRAWINGS

[0069] Figure 1 shows that PEAR1 is a potential target for triple negative breast cancer. A, the expression level of PEAR1 in human cancer adjacent and breast cancer tissues is detected by immunohistochemical staining, and the staining score of PEAR1 is detected. B, Log-rank test analyzes the relationship between PEAR1 expression and overall survival rate of breast cancer patients. C, Log-rank test analyzes the relationship between PEAR1 expression and overall survival rate of triple negative breast cancer patients. D, the expression of PEAR1 mRNA in various human breast cancer cell lines (data from HPA database).

[0070] Figure 2 is a schematic diagram of construction of PEAR1 knockdown and PEAR1 overexpression triple negative breast cancer cell lines. A, qPCR detection of the relative expression level of PEAR1 mRNA in PEAR1 knockdown and overexpression MDA-MB-231 cell lines. B, qPCR detection of the relative expression level of PEAR1 mRNA in PEAR1 knockdown and overexpression SUM159 cell lines. C, qPCR detection of the relative expression level of PEAR1 mRNA in PEAR1 overexpression MDA-MB-468 cell lines. D, Western blot detection of the expression level of PEAR1 in PEAR1 knockdown and overexpression MDA-MB-231 cell lines. E, Western blot detection of the expression level of PEAR1 in PEAR1 knockdown and overexpression SUM159 cell lines. F, Western blot detection of the expression level of PEAR1 in PEAR1 overexpression MDA-MB-468 cell lines.

[0071] Figure 3 shows that PEAR1 promotes triple negative breast cancer cell invasion and migration. A, transwell experiment, scratch experiment, CCK8 experiment were used to detect the invasion, migration and proliferation of PEAR1 knockdown and overexpression MDA-MB-231 cells, respectively. B, transwell experiment, scratch experiment, CCK8 experiment were used to detect the invasion, migration and proliferation of PEAR1 knockdown and overexpression SUM159 cells, respectively. C, transwell experiment, scratch experiment, CCK8 experiment were used to detect the invasion, migration and proliferation of PEAR1 overexpression MDA-MB-468 cells, respectively. D, MDA-MB-231 cells were injected into the tail vein of nude mice, and the effect of PEAR1 knockdown on lung metastasis and liver metastasis was detected.

[0072] Figure 4 shows that PEAR1 is a novel chaperone protein of CD44. A, Western blot detection of the expression level of PEAR1 and CD44 in each different human breast cancer cell line. B, co-immunoprecipitation detection of the binding of PEAR1 and CD44 in MDA-MB-231 cells. C, immunofluorescence staining detection of the co-localization of PEAR1 and CD44 in MDA-MB-231 cells. D, immunofluorescence staining detection of the co-localization of PEAR1 and CD44 in triple negative breast cancer tissues.

[0073] Figure 5 shows that PEAR1 protects CD44 from endocytic degradation. A, Western blotting was used to detect the expression level of CD44 protein in MDA-MB-231 cells with PEAR1 knockdown and overexpression. B, qPCR was used to detect the relative expression level of CD44 mRNA in MDA-MB-231 cells with PEAR1 knockdown and overexpression. C, Western blotting was used to detect the expression level of CD44 protein in MDA-MB-231 cells with PEAR1 knockdown and intervention with different inhibitors. Dynasore: dynamin inhibitor, SGC-AAK1-1: AP2-associated kinase 1 inhibitor, Pitstop 2: clathrin inhibitor, MG132: proteasome inhibitor. D, Immunofluorescence staining was used to detect the colocalization of CD44 and LAMP1 in MDA-MB-231 cells with PEAR1 knockdown and intervention with different inhibitors.

[0074] Figure 6 shows that S891A mutation inhibits CD44 function in MDA-MB-231 cells. A, Western blotting was used to detect the phosphorylation of PEAR1 serine / threonine in MDA-MB-231 cells with overexpression of PEAR1 SA full-point mutation and PEAR1 S891A single-point mutation. B, Western blotting was used to detect the binding of PEAR1 and CD44 in MDA-MB-231 cells with overexpression of PEAR1 SA full-point mutation and PEAR1 S891A single-point mutation. C, Western blotting was used to detect the expression level of CD44 protein in MDA-MB-231 cells with overexpression of PEAR1 SA full-point mutation and PEAR1 S891A single-point mutation. D, qPCR was used to detect the relative expression level of CD44 mRNA in MDA-MB-231 cells with overexpression of PEAR1 SA full-point mutation and PEAR1 S891A single-point mutation. E, Western blotting was used to detect the expression level of CD44 protein in MDA-MB-231 cells with overexpression of PEAR1 S891A single-point mutation and intervention with different endocytic inhibitors or proteasome inhibitors. F, Immunofluorescence staining was used to detect the colocalization of CD44 and LAMP1 in MDA-MB-231 cells with overexpression of PEAR1 S891A single-point mutation and intervention with different endocytic inhibitors or proteasome inhibitors.

[0075] Figure 7 shows that PEAR1 Ser891 phosphorylation is a decisive factor for PEAR1 promoting TNBC metastasis. A, transwell assay was used to detect the invasion of MDA-MB-231 cells overexpressing PEAR1 SA full point mutation and PEAR1 S891A single point mutation. B, scratch assay was used to detect the migration of MDA-MB-231 cells overexpressing PEAR1 SA full point mutation and PEAR1 S891A single point mutation. C, CCK8 assay was used to detect the proliferation of MDA-MB-231 cells overexpressing PEAR1 SA full point mutation and PEAR1 S891A single point mutation. D, MDA-MB-231 cells were injected into the tail vein of nude mice to detect the effect of overexpression of PEAR1 SA full point mutation and PEAR1 S891A single point mutation on lung metastasis and liver metastasis.

[0076] Figure 8 shows that LOXL2 activates PEAR1 phosphorylation and promotes TNBC cell invasion and migration. A, pull-down and immunoblotting were used to detect the binding of his-tagged PEAR1 extracellular segment protein to LOXL2 protein. B, co-immunoprecipitation and immunoblotting were used to detect the phosphorylation of PEAR1 serine / threonine and the expression level of CD44 protein in MDA-MB-231 and SUM159 cells stimulated with different concentrations of LOXL2 protein. C, transwell assay was used to detect the invasion of MDA-MB-231 cells stimulated with different concentrations of LOXL2 protein. D, scratch assay was used to detect the migration of MDA-MB-231 cells stimulated with different concentrations of LOXL2 protein. E, CCK8 assay was used to detect the proliferation of MDA-MB-231 cells stimulated with different concentrations of LOXL2 protein.

[0077] Figure 9 shows that LOXL2 binds to the EMI domain of PEAR1. A, ELISA was used to detect the polypeptide binding of LOXL2 to each PEAR1 extracellular segment domain. B, co-immunoprecipitation and immunoblotting were used to detect the phosphorylation of PEAR1 serine / threonine and the expression level of CD44 protein in MDA-MB-231 cells stimulated by using different concentrations of PEAR1-EMI recombinant protein to compete for binding to LOXL2. C, transwell assay was used to detect the invasion of MDA-MB-231 cells stimulated by using different concentrations of PEAR1-EMI recombinant protein to compete for binding to LOXL2. D, scratch assay was used to detect the migration of MDA-MB-231 cells stimulated by using different concentrations of PEAR1-EMI recombinant protein to compete for binding to LOXL2. E, CCK8 assay was used to detect the proliferation of MDA-MB-231 cells stimulated by using different concentrations of PEAR1-EMI recombinant protein to compete for binding to LOXL2.

[0078] Figure 10 shows that PEAR1 fab-HSA mAb competitively blocks LOXL2 binding to PEAR1 to inhibit metastasis of TBC cells. A, ELISA was used to detect the binding of PEAR1 fab-HSA mAb to PEAR1-EMI recombinant protein and polypeptides of each PEAR1 ectodomain domain. B, ELISA was used to detect the competitive blocking of PEAR1 fab-HSA mAb on the binding of LOXL2 to PEAR1 ectodomain recombinant protein. C, Co-immunoprecipitation and immunoblotting were used to detect the PEAR1 serine / threonine phosphorylation and CD44 protein expression levels in MDA-MB-231 cells after competitive inhibition of LOXL2 stimulation by different concentrations of PEAR1 fab-HSA mAb. D, Transwell assay was used to detect the invasion of MDA-MB-231 cells after competitive inhibition of LOXL2 stimulation by different concentrations of PEAR1 fab-HSA mAb. E, Scratch assay was used to detect the migration of MDA-MB-231 cells after competitive inhibition of LOXL2 stimulation by different concentrations of PEAR1 fab-HSA mAb. F, CCK8 assay was used to detect the proliferation of MDA-MB-231 cells after competitive inhibition of LOXL2 stimulation by different concentrations of PEAR1 fab-HSA mAb. G, MDA-MB-231 cells were injected into the tail vein of nude mice, and the effect of PEAR1 fab-HSA mAb treatment on lung metastasis and liver metastasis was detected.

[0079] Figure 11 is a schematic diagram showing the preparation of phospho-PEAR1 Ser891 detection antibody and verification of its specificity. A, ELISA was used to detect the titer of PEAR1 S891 phosphorylation antibody and its corresponding non-phosphorylation antibody. B, Dot blotting was used to detect the specificity of PEAR1 S891 phosphorylation antibody. C, Immunoblotting was used to detect the expression levels of non-phosphorylated PEAR1 and PEAR1 S891 phosphorylation in MDA-MB-231 cells overexpressing PEAR1 SA full point mutation and PEAR1 S891A single point mutation.

[0080] Figure 12 shows that PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 are highly expressed in TNBC and are associated with poor prognosis of patients. A, Immunohistochemical staining was used to detect the expression levels of PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 in human paracancer and breast cancer tissues and their staining scores. B, Log-rank test was used to analyze the relationship between the expression of PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 and the overall survival rate of triple-negative breast cancer patients.

[0081] Figure 13 shows that the expression levels of PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 are positively correlated in TNBC, in which PEAR1 S891 phosphorylation is the best independent prognostic factor for TNBC patients, and the prognostic effect is better when combined with CD44. A, Pearson analysis of the correlation between the expression of PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 in TNBC samples, and plotted as a heat map (B). C, the prognostic effect of different single factors and two two-combination schemes was analyzed using time-dependent ROC curve, and the best prognostic scheme was determined by comparing the area under the curve (AUC). DETAILED DESCRIPTION

[0082] TERMS

[0083] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 Da molecular mass that is composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains differs depending on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. At one end of each light chain is a variable region (VL) followed by a constant region; the constant region of the light chain is aligned with the first constant region of the heavy chain, and the variable region of the light chain is aligned with the variable region of the heavy chain. Particular amino acid residues at the interface between the variable regions of the light and heavy chains form an interface. The amino acid composition and order of the immunoglobulin heavy chain constant regions differ, and thus their antigenicities differ. Accordingly, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, whose respective heavy chains are μ, δ, γ, α, and ε. The same class of Ig can be further divided into subclasses or isotypes, e.g., IgG can be divided into IgG1, IgG2, IgG3, and IgG4, depending on the amino acid composition and the number and location of the disulfide bonds in the hinge region of the heavy chain. The light chains are differentiated by the constant region, either κ or λ. Each of the five classes of Ig can have either κ or λ light chains.

[0084] The sequences of about 110 amino acids at the N-terminus of both the heavy and light chains are highly conserved, forming the variable (V) region of the antibody; the remainder of the chain is the constant region (C), which is relatively invariant. The variable region includes three hypervariable regions (HVRs) and four relatively conserved framework regions (FWRs). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity-determining region (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) is composed of three CDR regions and four FWR regions, arranged in the order of FWR1, CDR1, FWR2, CDR2, FWR3, CDR3, FWR4 from the amino-terminus to the carboxy-terminus. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

[0085] "Identity" means the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of identity between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions in the shorter of the two sequences, multiplied by 100. For example, if 6 of 10 positions in two sequences are matched or homologous, then the two sequences are 60% identical. In general, the comparison is made when the two sequences are aligned for maximum correspondence.

[0086] As used herein, the term "Fab'" comprises one light chain and a portion of one heavy chain comprising a VH domain and a CH1 domain and the region between CH1 and CH2 domains, whereby an interchain disulfide bond can form between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule. "F(ab')2" comprises two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, whereby an interchain disulfide bond forms between the two heavy chains. Thus a F(ab')2 fragment is composed of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0087] As used herein, the term "Fv" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, but lacking the constant region.

[0088] In the present application, the scFv (single chain antibody fragment) can be a conventional single chain antibody in the art, which includes a heavy chain variable region, a light chain variable region, and a short peptide of 15-20 amino acids. Among them, the VL and VH domains are paired through a linker that enables them to be produced as a single polypeptide chain to form a monovalent molecule [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0089] As used herein, the term "Fc" (fragment crystallizable) consists of the constant region CH2 and CH3 domains and hinge region of an immunoglobulin, e.g., IgG.

[0090] As used herein, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term can include vectors primarily used for the insertion of DNA or RNA into a cell, vectors primarily used for the replication of DNA or RNA, and expression vectors for the transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.

[0091] As used herein, the term "host cell" for making a transformant generally includes a single cell, a cell line, or a cell culture that can be or has been the recipient of a plasmid or vector of the subject disclosure, which contains a polynucleotide of the subject disclosure, or expresses a protein heterodimer (e.g., a heterodimeric protein) of the subject disclosure. The host cell can include progeny of the single host cell. The progeny can not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The host cell can include a cell transfected in vitro with a vector of the subject disclosure. The host cell can be a bacterial cell (e.g., E. coli), a yeast cell, or other eukaryotic cell, such as a HEK293 cell, a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, or a myeloma cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the mammalian cell is a CHO cell.

[0092] As used herein, the terms "treatment," "treat," and "treating" can be used interchangeably. The term "treatment" includes controlling the progression of a disease, disorder, condition, and associated symptoms, preferably reducing the impact of the disease, disorder, condition, or one or more symptoms of the disease, disorder, condition. This term includes curing the disease or completely eliminating the symptoms. This term includes remission of symptoms. This term also includes, but is not limited to, non-cure palliative treatment. The term "treatment" includes administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an antibody or fusion protein of the present application to prevent or delay, reduce or alleviate the progression of a disease, disorder, condition, or the impact of one or more symptoms of the disease, disorder, condition.

[0093] The present application is further illustrated by the following examples, which in no way should be construed as limiting the scope of the application. The experimental methods in the following examples, where no specific conditions are indicated, were carried out according to routine methods and conditions, or according to the instructions of the commercial suppliers.

[0094] I. Experimental materials and methods

[0095] (I) Experimental animals and cells

[0096] 6-week-old female BALB / c mice, BALB / c-nu / nu mice, NOD-SCID mice and wild-type C57BL / 6 mice were purchased from Shanghai Sino-British Biotech Co., Ltd. Human breast cancer cell lines and mouse breast cancer cell lines were purchased from the ATCC cell library.

[0097] (II) Immunohistochemistry

[0098] Immunohistochemistry is a technique that uses the principle of highly specific binding between antigen and antibody and chemical coloration. The antigen in the tissue or cell is first combined with the primary antibody, and then the primary antibody is reacted with the secondary antibody labeled with biotin, etc. The biotin is then combined with horseradish peroxidase (HRP) and other anti-biotin (such as streptavidin, etc.), and finally the antigen-antibody reaction product in the tissue or cell is indicated by coloration. This technique has the functions of localization, qualification and quantification, and is an important detection means in clinical pathological diagnosis.

[0099] The specific steps are as follows:

[0100] 1. Baking slices: Paraffin sections (purchased from Shanghai Xincuo Biotech Co., Ltd.) were baked at 60°C overnight, and the paraffin on the glass slide melted into tear-drop shape.

[0101] 2. Dewaxing to water: Tissue sections were deparaffinated by soaking in xylene I -> xylene II -> xylene III, each for 10 min. Then rehydrated by soaking in alcohol stepwise, 100% alcohol I -> 100% alcohol II -> 95% alcohol I -> 95% alcohol II -> 85% alcohol -> 75% alcohol -> ddH2O, each for 5 min.

[0102] 3. Endogenous peroxidase removal: After 3 rinses in lx PBS, incubate with 3% hydrogen peroxide (methanol dilution) for 30 min at room temperature in the dark.

[0103] 4. Membrane permeabilization: After 3 rinses in lx PBS, treat with 0.5% Triton-X 100 (lx PBS dilution) for 15 min at room temperature.

[0104] 5. Antigen retrieval: After 3 rinses in lx PBS, perform antigen retrieval with sodium citrate antigen retrieval solution, heat in a microwave at high for 3 min to boiling, 95°C water bath for 30 min, and cool to room temperature naturally. Antigen retrieval can destroy cross-linking between molecules, expose blocked or hidden antigenic determinants, and restore the original spatial conformation.

[0105] 6. Blocking: After 3 rinses in lx PBS, block with 10% goat serum (lx PBS dilution) for 2 h at room temperature in a humidified chamber.

[0106] 7. Discard the goat serum and incubate the primary antibody (lx PBS dilution) overnight at 4°C in a humidified chamber.

[0107] 8. After 3 rinses in lx PBS, add the biotin-labeled secondary antibody (lx PBS dilution), avoid light, 37°C for 30 min.

[0108] 9. After 3 rinses in lx PBS, add the tertiary antibody (VECTASTAIN Elite ABC-Peroxidase kit), avoid light, 37°C for 30 min.

[0109] 10. After 3 rinses in lx PBS, develop with DAB (DAB: diluent: ddH2O = 1:50:50), mix well, and observe under a microscope. Stop when the experimental group is moderately colored and the control group is not colored. Strictly control the developing time.

[0110] 11. Place the slide in ddH2O for 5 min to stop the color development.

[0111] 12. Hematoxylin stain the nucleus: After staining with hematoxylin stain for 3-5 min, observe under a microscope to ensure moderate coloration, and place the slide in water for rinsing.

[0112] 13. Decolorization: The slide is decolorized with 1% hydrochloric acid alcohol for several seconds, and the cytoplasm is observed to disappear non-specifically under a microscope.

[0113] 14. Blueing: The slide is soaked in distilled water for 5 min, and the hematoxylin color is gradually changed to blue.

[0114] 15. Decolorization: The slide is soaked in graded alcohol, 75% alcohol→85% alcohol→95% alcohol I→95% alcohol II→100% alcohol I→100% alcohol II, each for 5 min.

[0115] 16. Transparency: The slide is soaked in xylene, xylene I→xylene II→xylene III, each for 10 min.

[0116] 17. Mounting: Neutral gum is dropped on the slide, and a cover glass is covered to avoid bubbles, and is dried in a fume hood.

[0117] 18. Observation and imaging are performed using a Nikon microscope. The slide after the experiment is stored at -20°C.

[0118] Table 1: Immunohistochemical antibodies and dilution ratios

[0119]

[0120] (III) Immunofluorescence

[0121] Immunofluorescence is a technique for locating, identifying and quantifying antigens or hapten substances in tissues or cells based on antigen-antibody reactions and using fluorescent substances to label antibodies. At present, the method of tracing or checking corresponding antigens using fluorescent antibodies is called the fluorescent antibody method, and the method of tracing or checking corresponding antibodies using known fluorescent antigen markers is called the fluorescent antigen method, and the former is more commonly used in actual work. Immunofluorescence technology has the advantages of strong specificity and high sensitivity, but is often interfered by non-specific staining.

[0122] The specific steps are as follows:

[0123] 1. A new clean round slide is placed in a 24-well plate, and the human breast cancer cell density is adjusted and plated, and is cultured in a 37°C, 5% CO2 cell incubator. After adhering for 12-24 h, the cell density and state are observed under a microscope.

[0124] 2. The culture medium is discarded, and the cells are fixed with 4% paraformaldehyde at room temperature for 20 min, and then at 4°C for 2 h.

[0125] 3. After being washed with 1×PBS for 3 times, the cells are treated with 0.5% Triton-X 100 (diluted with 1×PBS) at room temperature for 10 min.

[0126] 4. After 3 rinses with 1 x PBS, block with 2% BSA (diluted with 1 x PBS) for 1 h at room temperature.

[0127] 5. Add primary antibody (diluted with 2% BSA), incubate overnight at 4°C.

[0128] 6. After 3 rinses with 1 x PBS, add secondary antibody (diluted with 2% BSA), incubate for 1 h at room temperature in the dark.

[0129] 7. After 3 rinses with 1 x PBS, add DAPI staining solution (diluted with 1 x PBS, final concentration 5 pg / mL), stain for 10 min at room temperature in the dark.

[0130] 8. After 3 rinses with 1 x PBS, mount with anti-fluorescence mounting medium, fix the slide with nail polish, and air dry at room temperature in the dark.

[0131] 9. Observe and image using a fluorescence microscope. Store the slides at -20°C after the experiment.

[0132] Table 2: Immunofluorescence antibodies and dilution ratios

[0133]

[0134] (iv) Western blotting

[0135] After sample preparation, perform BCA quantification, electrophoresis, gel running, membrane transfer, blocking, incubation of primary antibody, washing, incubation of secondary antibody, washing, and development.

[0136] The specific steps are as follows:

[0137] 1. Preparation of protein samples

[0138] 1) Discard the culture medium, wash the cells twice with pre-cooled 1 x PBS, add 500 pL of RIPA lysis buffer (with added protease inhibitors and phosphatase inhibitors, final concentration 1 x) to each 10 cm cell culture dish, scrape the cells with a cell scraper and transfer them to a 1.5 mL EP tube, lyse on ice for 20 min, ultrasonic at 100 w, start for 5 s, pause for 3 s, 8 cycles, and ultrasonic until the cell lysate is clear and not viscous.

[0139] 2) Centrifuge at 4°C, 12000 rpm, 20 min, and transfer the supernatant to a new 1.5 mL EP tube.

[0140] 3) Perform protein quantification using a BCA quantification kit (refer to the kit instructions), add 5 x SDS loading buffer, mix well, lyse on ice for 15 min, denature at 100°C for 10 min, and centrifuge, and store at -80°C after preparation for electrophoresis.

[0141] 2. BCA quantification

[0142] BCA quantification is one of the most commonly used methods for protein concentration determination. Under alkaline conditions, proteins will convert Cu... 2+ Reduced to Cu + Cu + It can bind with bispyridinecarboxylic acid (BCA) to form a purple complex with a strong absorption peak at 562 nm. The amount of this complex is directly proportional to the protein concentration, and the protein concentration in the sample can be calculated based on the standard curve.

[0143] 1) Preparation of BSA standard system: The concentration of the BSA standard stock solution is 2 mg / mL. BSA standards with concentrations of 1, 0.5, 0.25, 0.0125, 0.0625, 0.03125 and 0 mg / mL are prepared by serial dilution with sterile ddH2O.

[0144] 2) Dilute the protein sample to be tested 10 times, i.e., take 6 μL of the sample to be tested and mix it with 54 μL of sterile ddH2O.

[0145] 3) Prepare BSA working solution: Mix 50 times the volume of reagent A with 1 times the volume of reagent B, and use immediately after preparation.

[0146] 4) Add the diluted BSA standard and the protein sample to be tested to a 96-well plate, 25 μL / well, with 2 replicates. Then add the BSA working solution, 200 μL / well. Gently shake to mix and place the 96-well plate in a 37°C incubator for 30 min.

[0147] 5) Use an ELISA reader to read the plate. The absorbance (optical density, OD) is 562 nm. Plot a standard curve based on the concentration of BSA standard and the corresponding absorbance, and then calculate the protein concentration of the sample to be tested based on this curve.

[0148] 3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0149] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PADE) is an electrophoretic separation technique that separates proteins in a gel medium based on their different migration rates caused by differences in molecular weight and charge.

[0150] 1) Preparation of SDS-polyacrylamide gel: Clean and dry the gel plate (Tanon, 1.5 mm size), install the mold, and check for leaks with ddH2O. Prepare 8% to 12% separating gel according to the protein molecular weight, mix well, and pour into the mold. Add 1 mL of isopropanol to press the gel flat. After the separating gel solidifies, discard the isopropanol, rinse with ddH2O, and invert to dry. Prepare 5% concentrated gel, mix well, and pour into the mold. Carefully insert the comb vertically to avoid air bubbles. After the concentrated gel solidifies, it is ready for use. The SDS-polyacrylamide gel can be stored at 4°C and used as soon as possible.

[0151] Table 3: SDS-polyacrylamide gel formula (total volume is 10 mL)

[0152]

[0153] 1) Loading: Assemble the SDS-polyacrylamide gel into the electrophoresis tank, add 1x Running buffer to the tank, fill the inner tank, and half fill the outer tank. Carefully remove the comb, clean the loading wells, and load the protein samples and protein marker. Fill the remaining loading wells with an equal volume of 1x SDS loading buffer. Typically, the loading amount of protein is 20-50 μg.

[0154] 2) Electrophoresis: Constant voltage, 85 V, until the protein samples in each lane are pressed into a straight line and enter the separating gel. Adjust the voltage to 120 V and continue constant voltage electrophoresis. Stop the electrophoresis when the bromophenol blue runs out of the separating gel.

[0155] 3) Membrane transfer: First, activate the PVDF membrane (0.45 μm) with methanol for about 30 s. Then, soak the PVDF membrane, membrane transfer clamp, sponge, and filter paper in 1x Transfer buffer. Assemble the "sandwich" structure according to the order of "membrane transfer clamp blackboard (negative) - sponge - filter paper - gel - PVDF membrane - filter paper - sponge - membrane transfer clamp whiteboard (positive)". After removing the air bubbles, place it in the membrane transfer tank, fill it with pre-cooled 1x Transfer buffer, and perform membrane transfer in an ice bath at a constant voltage of 100 V for 100 min.

[0156] 4) Blocking: Place the PVDF membrane protein side up in 1x TBST and rinse once. Then, block with 5% skim milk at room temperature on a shaker at 40-50 rpm for 1 h.

[0157] 5) Incubation of primary antibody: Discard the blocking solution and wash the PVDF membrane with 1x TBST at room temperature on a shaker at 90-100 rpm for 3 times, 10 min each time. Then, add the primary antibody (diluted with antibody diluent), and incubate at 4°C on a shaker at 40-50 rpm overnight.

[0158] 6) Incubate secondary antibody: The next day, the primary antibody was recovered, the PVDF membrane was washed with 1x TBST, 90-100 rpm on a shaker at room temperature, for 3 times, 10 min each time. Then, the HRP-labeled secondary antibody was added (diluted with 5% skim milk), incubated for 1 h at room temperature, 40-50 rpm on a shaker.

[0159] 7) Development: The secondary antibody was discarded, the PVDF membrane was washed with 1x TBST, 90-100 rpm on a shaker at room temperature, for 3 times, 10 min each time. The ECL chemiluminescence kit (A solution: B solution = 1:1, prepared immediately before use) was used, and the membrane was exposed to imaging in a Tanon automatic chemiluminescence imaging system.

[0160] Table 4: Western blot antibodies and dilution ratios

[0161]

[0162]

[0163]

[0164] (V) Co-immunoprecipitation

[0165] Co-immunoprecipitation (Co-IP) is one of the classic methods for studying the physiological interaction of proteins in intact cells. When cells are lysed under non-denaturing conditions, the interactions between many proteins present in the cells are preserved, and if protein A is immunoprecipitated with an antibody against A, protein B bound to A will also be precipitated.

[0166] The specific steps are as follows:

[0167] 1. Preparation of protein samples

[0168] 1) Discard the culture medium, wash the cells with pre-cooled 1x PBS for 2 times, 500 μL of Western and IP cell lysis buffer (containing protease inhibitors and phosphatase inhibitors, final concentration 1x) was added to each 10 cm cell culture dish, the cells were scraped with a cell scraper and transferred to a 1.5 mL EP tube, lysed on ice for 20 min, and then subjected to mild ultrasonic treatment, 80 w, 5 s on and 5 s off, 6 cycles, until the cell lysis buffer was clear.

[0169] 2) Centrifugation at 4°C, 12000 rpm, 20 min, and transfer the supernatant to a new 1.5 mL EP tube.

[0170] 3) BCA protein quantification, take the total protein sample (Input) and add 5x SDS loading buffer, mix well, ice bath lysis for 20 min, 100°C denaturation for 10 min, instant separation, ice bath for WB detection or stored at -80°C.

[0171] 2. Remaining samples for IP

[0172] 1) After mixing Protein A / G PLUS-Agarose beads, take 20 μL beads and add to the protein sample (1 mg / mL protein / tube), mix well, rotate enrichment at 4°C for 2 h, and pre-pull to remove non-specific proteins.

[0173] 2) Centrifuge at 4°C, 12000 rpm, 1 min, transfer the supernatant to a new 1.5 mL EP tube, add IP antibody, and rotate enrichment at 4°C overnight.

[0174] 3) After mixing Protein A / G PLUS-Agarose beads, take 50 μL beads and add to the protein sample, mix well, rotate enrichment at 4°C for 4 h.

[0175] 4) Centrifuge at 4°C, 12000 rpm, 1 min, carefully discard the supernatant, wash the beads with 1x TBS, 1 mL / tube, repeat 3 times.

[0176] 5) After carefully discarding the supernatant, add 50 μL 2x SDS loading buffer, mix well, ice bath lysis for 20 min, 100°C denaturation for 10 min, centrifuge at 4°C, 12000 rpm, 2 min, transfer the supernatant to a new 1.5 mL EP tube, ice bath for WB detection or stored at -80°C.

[0177] Table 5: Immunoprecipitation antibodies and usage

[0178]

[0179]

[0180] (VI) Extraction of RNA

[0181] Trizol is a common and widely used total RNA extraction reagent, containing phenol, guanidine isothiocyanate and other substances, which can quickly break cells and inhibit the release of nucleases from cells, so it is of great value for extracting high-purity and high-stability RNA. When extracting RNA, chloroform can effectively and quickly separate the organic and inorganic phases. After centrifugation, the sample is layered from top to bottom, with the water sample layer (RNA exists), the middle layer (DNA and protein exist), and the organic layer. RNA degradation should be avoided throughout the experiment.

[0182] The specific steps are as follows:

[0183] 1) Discard the culture medium, wash the cells twice with pre-cooled 1×PBS, add 1mL Trizol to each 10cm cell culture dish, blow off and disperse the cells, transfer to 1.5mL EP tubes, and lyse on ice for 15min.

[0184] 2) Add 200 μL of chloroform, vortex rapidly for 15 seconds until a milky white lysate appears, lyse in an ice bath for 3 minutes, then centrifuge at 4°C, 12000 rpm for 20 minutes.

[0185] 3) Transfer the supernatant to a new 1.5 mL EP tube, add an equal volume of isopropanol, gently invert to mix, incubate on ice for 20 min to allow the RNA to precipitate fully, then centrifuge at 4°C, 12000 rpm for 20 min.

[0186] 4) Discard the supernatant, wash the precipitate with 1 mL of 75% ethanol (prepared with DEPC water), centrifuge at 4℃, 12000 rpm, for 5 min.

[0187] 5) Discard the supernatant, invert the 1.5mL EP tube onto a piece of clean paper, and place it in a fume hood to air dry until the precipitate begins to become transparent.

[0188] Add 40 μL of DEPC water, incubate on ice for 20 min, and after the RNA has completely dissolved and precipitated, gently mix. Use Nanodrop to determine the RNA concentration and A260 / A280 ratio, label, and store at -80℃.

[0189] (vii) Reverse transcription-polymerase chain reaction (quantitative real-time PCR, qPCR)

[0190] By using reverse transcriptase to synthesize cDNA complementary to RNA as a template, and then using the cDNA strand as a template for PCR amplification, a large number of copies can be obtained, which greatly improves the sensitivity of RNA detection.

[0191] Table 6: qPCR primer information

[0192]

[0193] (viii) Construction of stable cell lines

[0194] The plasmids were synthesized by Shanghai Saiheng Biotechnology Co., Ltd. and Shanghai Jikai Gene Medical Technology Co., Ltd., and used for transfection, lentiviral packaging and infection of target cells, drug screening or flow cytometry sorting.

[0195] (ix) CCK8 Cell Counting Kit (Dojindo)

[0196] For detecting cell proliferation or drug toxicity experiment, cell counting is performed. The kit uses a new type of water-soluble tetrazolium salt WST-8 formazan dye as a chromogenic substrate, which has high sensitivity and low toxicity. Because WST-8 has high water solubility, it exists outside the cell and does not penetrate the living cell membrane. It is reduced by 1-methoxy PMS to receive the coenzyme NADH of lactate dehydrogenase, generating water-soluble WST-8 formazan dye. By measuring the absorbance (OD=450nm) of formazan dye, the number of living cells can be measured, and the number of formazan generated is proportional to the number of living cells.

[0197] (X) Scratch test

[0198] Scratch test refers to artificially creating a blank area on a confluent monolayer of cells, and the cells on both sides will grow towards the blank area, gradually healing the "scratch", also known as "wound healing experiment". Scratch test utilizes the perception and migration ability of cells to external space factors when cultured in monolayer, and is the most commonly used method for measuring cell migration and repair ability.

[0199] The specific steps are as follows:

[0200] 1. Draw a line every 0.5 cm on the bottom of a 6-well plate, crossing the well (at least 5 lines per well).

[0201] 2. Collect breast cancer cells in the logarithmic growth phase, count the cells, adjust the cell density, and inoculate 2x10 6 cells / 2mL / well in a 6-well plate, and incubate in a 37°C, 5% CO2 incubator overnight.

[0202] 3. After adhering, replace the medium with DMEM basic medium and starve for 24 hours to minimize the impact of cell proliferation on the experiment.

[0203] 4. Use a 200μL gun head vertically against a ruler, and make sure it is perpendicular to the marked line. Draw 3 parallel lines in each well, and try to ensure that each scratch is of uniform width.

[0204] 5. Discard the supernatant, wash 3 times with sterile 1xPBS, and continue to culture in DMEM basic medium at 37°C, 5% CO2 in a constant temperature cell incubator. Observe and image under a microscope at 0h, 12h, 24h, 36h, and 48h (make sure the observation and imaging positions are the same throughout the process, and the intersection of the scratch and the line can be used as a fixed point).

[0205] Use Image J software to calculate the area of each field scratch, calculate the relative wound healing area, formula=(initial area-some time point same position area) / initial area, and analyze the migration ability of cells.

[0206] (xi) Transwell assay

[0207] Transwell assay is a common laboratory technique for measuring the migration or invasive movement of cells across a porous membrane, which well mimics the physiological conditions of cells crossing tissues in vivo. This experiment is usually used to study the chemotactic response of cells to various exogenous stimuli (such as chemotactic factors, growth factors or extracellular matrix components, etc.) and to reflect the migration and invasion behavior of cells. If no Matrigel is added in the chamber, it is often used to measure the ability of cells to migrate in response to inducers, i.e. cells migrate from the upper chamber to the lower chamber without the need to degrade or invade the membrane. If Matrigel is added in the chamber, it is used to measure the invasion ability of cells, in which cells not only migrate through the porous membrane, but also need to degrade the extracellular matrix layer coated on the surface of the upper chamber membrane to achieve invasion, which is usually used to study the invasion behavior of tumor cells.

[0208] The specific steps are as follows:

[0209] 1. Matrigel pretreatment: After thawing Matrigel in an ice bath at 4°C, operate on ice, and divide into 250 μL / tube. Note that the sterile EP tube and gun head need to be pre-cooled at -20°C, and strict precautions should be taken to avoid repeated freezing and thawing of Matrigel.

[0210] 2. Hydrate the PET membrane of the insert cell culture chamber with sterile 1x PBS to remove the surface tension of the PET membrane.

[0211] 3. Dilute Matrigel with 3 times the volume of DMEM basal medium, mix well, and then take 80 μL and add to the upper surface of the PET membrane of the insert cell culture chamber (pore size 8 μm), and let it stand at 37°C for 3 h.

[0212] 4. Collect breast cancer cells in the logarithmic growth phase, wash them 3 times with sterile 1x PBS, resuspend the cell pellet with DMEM basal medium, count the cells, adjust the cell density, and inoculate 1x10 5 cells / 200 μL in the Matrigel-coated insert cell culture upper chamber, and add 700 μL of DMEM complete medium containing 20% FBS to the lower chamber 24-well plate. Set up 3 replicate wells, and avoid air bubbles between the upper and lower chambers.

[0213] 5. Incubate at 37°C, 5% CO2 in a constant temperature cell incubator for 48 h, remove the insert cell culture chamber, discard the culture medium in the chamber, and gently wipe off the residual gel and uninvaded cells on the PET membrane with a cotton swab, then wash 2 times with 1x PBS.

[0214] 6. Fix the lower surface of the PET membrane of the chamber with methanol at room temperature for 30 min, wash 2 times with 1x PBS, and then dry naturally.

[0215] 7. Stain with 0.1% crystal violet, avoid light, 37°C, 30 min, wash with 1x PBS for 2 times, and dry naturally.

[0216] 8. Use microscope to observe 5 random fields, image and count the number of cells with invasion, and analyze the invasion ability of cells.

[0217] (ix) Enzyme-linked immunosorbent assay

[0218] Enzyme-linked immunosorbent assay (ELISA) is a comprehensive technology based on antigen-antibody immune reaction and efficient catalysis of enzymes, and is widely used for quantitative and qualitative detection of antibodies (antigens). The antigen or antibody is fixed on a solid carrier to maintain its immune reaction activity and enzyme catalytic activity; then the test substance is added to combine with the solidified substance to form an "antigen-antibody" complex; then the enzyme-labeled antibody or antigen is added to further combine to form an enzyme-labeled complex; finally, the substrate is added to occur enzyme catalysis of the substrate color reaction, and qualitative or quantitative analysis is performed according to the color depth. Mainly including double antibody sandwich method, indirect method, competition method, double antigen sandwich method, neutralization method, etc.

[0219] The specific steps are as follows:

[0220] 1. Coating: dilute recombinant Streptavidin to 10 μg / mL with coating solution, add to ELISA special 96-well plate, 100 μL / well, 37°C for 1.5 h.

[0221] 2. Wash the plate: PBST, 300 μL / well, wash 3 times, and dry on the blotting paper.

[0222] 3. Blocking: 2% BSA (diluted with 1x PBS), 150 μL / well, 37°C for 1 h.

[0223] 4. Wash the plate: PBST, 300 μL / well, wash 3 times, and dry on the blotting paper.

[0224] 5. Add biotin-coupled polypeptide (10 μg / mL, diluted with 1x PBS) and PEAR1-EMI recombinant protein (5 μg / mL, diluted with 1x PBS), 100 μL / well, 4°C for overnight.

[0225] 6. Wash the plate: PBST, 300 μL / well, wash 3 times, and dry on the blotting paper.

[0226] 7. Blocking: 2% BSA (diluted with 1x PBS), 150 μL / well, 37°C for 1 h.

[0227] 8. Wash plates: PBST, 300 μL / well, wash 3 times, tap dry on absorbent paper.

[0228] 9. Add primary antibody (LOXL2-his recombinant protein or PEAR1 fab-HSA-his antibody, 2 μg / mL, diluted with 1% BSA), 100 μL / well, incubate at 37°C for 1 h.

[0229] 10. Wash plates: PBST, 300 μL / well, wash 3 times, tap dry on absorbent paper.

[0230] 11. Add HRP-labeled secondary antibody (Mouse monoclonal anti-His-tag HRP-conjugated, 1:10000, diluted with 1% BSA), 100 μL / well, incubate at 37°C for 30 min.

[0231] 12. Wash plates: PBST, 300 μL / well, wash 6 times, tap dry on absorbent paper.

[0232] 13. Color development: add substrate Tetramethylbenzidine (TMB), 100 μL / well, positive wells turn blue, control time no more than 15 min.

[0233] 14. Stop: add 2M H2SO4, 50 μL / well, positive wells turn yellow.

[0234] Read plates: immediately read plates using a microplate reader (OD = 450 nm).

[0235] (Thirteen) Pulldown experiment

[0236] Pulldown experiment is an in vitro experimental technique for detecting the interaction between two proteins. The protein to be purified is expressed in the form of a fusion protein using genetic engineering methods, i.e. the "bait" protein is fused to a ligand protein that is easy to purify, and the fusion protein is then fixed on the affinity resin corresponding to the tag. When the solution containing the "bait" protein passes through the chromatography column, the "bait" protein will be adsorbed by the column, and if the target protein interacts with the "bait" protein, it will also be adsorbed when passing through the column, thereby achieving the separation and identification of proteins. Pulldown experiment can identify unknown proteins interacting with known fusion proteins, and can also identify whether there is interaction between two known proteins.

[0237] The specific steps are as follows:

[0238] 1. Collect 20 mL supernatant of normal cultured MDA-MB-231 cells, centrifuge at 2000 rpm for 15 min at 4°C, take the supernatant and concentrate to 2 mL using 10 kD concentrator tube, and place the concentrated solution in an ice bath for standby.

[0239] 2. Mix the Dynabeads His-Tag well by vortexing, take 10 μL into a 1.5 mL EP tube, and attract using a magnet for 2 min, and discard the supernatant.

[0240] 3. Add 500 μL of 1x binding / washing buffer and 20 μg of PEAR1-ECD-his recombinant protein, mix well, and enrich at 4°C for 1 h, with no PEAR1-ECD-his recombinant protein as a blank control.

[0241] 4. After attracting using a magnet for 2 min and discarding the supernatant, resuspend the Dynabeads His-Tag by adding 500 μL of 1x binding / washing buffer, wash at 4°C for 10 min, and repeat for 3 times.

[0242] 5. Add 500 μL of the concentrated solution prepared in "step 1", add an equal volume of 2x pull-down buffer, mix well, and enrich at 4°C for 2 h.

[0243] 6. After attracting using a magnet for 2 min and discarding the supernatant, resuspend the Dynabeads His-Tag by adding 1 mL of 1x binding / washing buffer, wash at 4°C for 10 min, and repeat for 3 times.

[0244] 7. Add 50 μL of His elution solution, mix once for 2 min in an ice bath, and repeat for a total of 10 min.

[0245] 8. Add 50 μL of 2x SDS loading buffer, mix once for 2 min, lyse in an ice bath for 15 min, and denature at 100°C for 10 min.

[0246] Attract using a magnet for 2 min, and transfer the supernatant containing PEAR1-ECD-his recombinant protein and its interacting protein (the ligand of PEAR1) to a new 1.5 mL EP tube for Western blotting.

[0247] (Twelfth) Intravenous injection of mice

[0248] Mice have 3 tail veins, including 1 on the back and 1 on each side, because the back vein is deep and thin, generally choose the 2 sides of the tail vein injection. Select 1 mL specification syringe, after sucking in the cell suspension or drug, empty the air to prevent air into the vein causing the mouse to die. The mouse is loaded into a special tail vein injection fixer, the tail is exposed, and the device is turned on to illuminate, making it easy to observe and operate. Use 75% alcohol cotton ball to wipe the mouse's tail vein to make the blood vessels full, the needle insertion site should be close to the mouse tail end (distal end), the needle tip is about 30° angle with the blood vessel, and the needle tip is kept inclined upward, the needle head is immediately parallel to the blood vessel after piercing the skin, and the needle head is ensured in the blood vessel to push the injection. Under normal circumstances, there is no obvious resistance during the injection. If there is a large resistance or the blood vessel is swollen, the needle should be immediately pulled out and the opposite proximal end should be selected for needle insertion. After injection, the needle is left in the blood vessel for 3 s to prevent the inoculated cell suspension or drug from leaking and losing due to too fast needle removal, dry cotton ball is used to stop bleeding, tail vein injection fixer is disassembled, and the mouse is put back into the cage.

[0249] II. Experimental results

[0250] Figure 1 shows that PEAR1 can be used as a potential target for triple-negative breast cancer, wherein in order to reveal the clinical correlation between PEAR1 expression level and breast cancer, IHC staining is performed on breast tumor tissues and their corresponding paracancer tissues, and the results show that the expression level of PEAR1 in breast tumor tissues is significantly higher than that in paracancer tissues. In-depth analysis combined with the background information of the tissue chip shows that high expression of PEAR1 is related to poor overall survival of breast cancer patients, and high expression of PEAR1 is significantly related to poor prognosis of TNBC breast cancer patients. The HPA database (Human Protein Atlas) collects the expression of PEAR1 mRNA in various human breast cancer cell lines, and the results show that PEAR1 is mainly significantly highly expressed in TNBC cells.

[0251] Figure 2 shows that PEAR1 overexpression and knockdown cell lines are successfully constructed, wherein PEAR1 knockdown and overexpression MDA-MB-231 cell lines and SUM159 cell lines and PEAR1 overexpression MDA-MB-468 cell lines are constructed by slow virus infection method, qPCR is used to detect the relative expression level of PEAR1 mRNA, and WB is used to detect the expression level of PEAR1 protein.

[0252] Figure 3 shows that PEAR1 promotes the invasion and migration of triple-negative breast cancer cells, wherein the transwell experiment results show that PEAR1 can significantly promote the invasion of MDA-MB-231, SUM159 and MDA-MB-468 cells; the scratch experiment results show that PEAR1 can significantly promote the migration of MDA-MB-231, SUM159 and MDA-MB-468 cells; and the CCK8 experiment results show that PEAR1 has no significant effect on the proliferation of MDA-MB-231, SUM159 and MDA-MB-468 cells. Further, MDA-MB-231 cells with PEAR1 knocked down were injected into nude mice through the tail vein, and the results showed that knocking down PEAR1 can significantly inhibit the lung metastasis and liver metastasis of MDA-MB-231 cells by blood.

[0253] Figure 4 shows that PEAR1 can be a novel chaperone protein of CD44, wherein WB detects the expression levels of PEAR1 and CD44 in different subtypes of breast cancer cells, and the results show that PEAR1 is significantly highly expressed in MDA-MB-231 cells, while CD44 is only expressed in TNBC cells, suggesting that the potential biological functions of the two may be most common in TNBC cells. The combination between PEAR1 and CD44 in MDA-MB-231 cells was verified by endogenous co-IP and IF experiments. In addition, IF experiments further proved that PEAR1 and CD44 significantly co-localize in human TNBC tissues, mainly in tumor cell nests and individual interstitial cells.

[0254] Figure 5 shows that PEAR1 can protect CD44 from endocytic degradation, wherein in MDA-MB-231 cells, the function of CD44 is regulated by PEAR1, it has been observed that PEAR1 can directly regulate the expression level of CD44 protein, but it is found that the CD44 mRNA level is not consistent with its protein level, so it is further speculated that PEAR1 may regulate the degradation pathway of CD44. WB detection results show that knocking down PEAR1 can significantly inhibit the expression of CD44 in MDA-MB-231 cells, and after intervention with a series of endocytosis inhibitors, the stable expression of CD44 can be effectively restored, but intervention with proteasome inhibitors is ineffective. In addition, the IF staining results directly show that after knocking down PEAR1, CD44 cannot be stably expressed on the cell membrane, but enters the lysosome for degradation, and the morphology of the cells also appears an epithelioid change, and after intervention with endocytosis inhibitors, the endocytic degradation of CD44 is significantly inhibited, while proteasome inhibitors cannot effectively inhibit the degradation of CD44. Therefore, PEAR1 is an important chaperone protein of CD44, which can protect it from endocytosis-mediated lysosomal degradation, and is not related to ubiquitin-proteasome degradation.

[0255] Figure 6 shows that S891A mutation can inhibit CD44 function in MDA-MB-231 cells, wherein, in order to evaluate the biological function of PEAR1 Ser891, MDA-MB-231 cell lines overexpressing PEAR1 S891A single point mutation and PEAR1 All SA full point mutation were first constructed, and the results of co-IP experiment showed that S891A single point mutation and All SA full point mutation can completely inhibit the serine phosphorylation of PEAR1 in MDA-MB-231 cells, and at this time the combination of PEAR1 and CD44 is also completely destroyed. In addition, S891A single point mutation and All SA full point mutation significantly inhibit the expression of CD44 protein in MDA-MB-231 cells, but the CD44 mRNA level is not affected, and it is speculated that the point mutation also affects the degradation of CD44 protein, and the results of WB detection and IF staining prove that CD44 in MDA-MB-231 cells after PEAR1 S891A mutation is endocytosed and degraded.

[0256] Figure 7 shows that PEAR1 Ser891 phosphorylation is a decisive factor for PEAR1 to promote TNBC metastasis, wherein, further in vitro cell experiments and in vivo animal experiments were carried out to verify the biological function of PEAR1 Ser891 phosphorylation. The results of Transwell experiment and scratch experiment respectively showed that after PEAR1 S891A single point mutation and PEAR1 All SA full point mutation, the enhancing effect of PEAR1 on the invasion and migration of MDA-MB-231 cells also disappeared. The results of CCK8 showed that PEAR1 S891A single point mutation and PEAR1 All SA full point mutation had no obvious effect on the proliferation ability of MDA-MB-231 cells. In addition, the tail vein of nude mice was injected with MDA-MB-231 cells with PEAR1 S891A single point mutation and PEAR1 All SA full point mutation and their control groups, and the results showed that the lung metastasis and liver metastasis of MDA-MB-231 cells with PEAR1 S891A single point mutation and PEAR1 All SA full point mutation were significantly inhibited. The above results show that the function of PEAR1 to promote TNBC metastasis depends on the Ser891 phosphorylation of PEAR1, which indicates that Ser891 phosphorylation is an important functional activated form of PEAR1.

[0257] Figure 8 shows that LOXL2 activates PEAR1 phosphorylation and promotes TNBC cell invasion and migration. The inventors sought novel ligands for PEAR1, capturing PEAR1 ligands in the supernatant of MDA-MB-231 cells using the PEAR1-ECD-his recombinant protein and enriching and pulling them using his magnetic beads. After elution, the PEAR1-ECD-his recombinant protein and its interacting proteins were subjected to gel electrophoresis and silver staining. Mass spectrometry was used to identify the differentially expressed bands, ultimately identifying the LOXL2 protein. Stimulation of MDA-MB-231 and SUM159 cells with different concentrations of LOXL2 protein showed that LOXL2 could induce PEAR1 serine / threonine phosphorylation in a dose-dependent manner and increase CD44 expression in both MDA-MB-231 and SUM159 cells. In addition, cell function experiments showed that exogenous LOXL2 could promote the invasion and migration of MDA-MB-231 cells in a significant dose-dependent manner, while LOXL2 had no significant effect on the proliferation of MDA-MB-231 cells.

[0258] Figure 9 shows the binding of LOXL2 to the EMI domain of PEAR1. PEAR1 consists of an extracellular EMI domain, a NIM domain, 15 epidermal growth factor-like repeat units, a transmembrane region, and an intracellular NPXY motif, two ITAM motifs, and five C-terminal proline-rich domains. ELISA results showed that LOXL2, as an endogenous ligand of PEAR1, mainly binds to the EMI domain of PEAR1. Pretreatment of MDA-MB-231 cells with different concentrations of PEAR1-EMI recombinant protein, followed by stimulation with 10 ng / mL LOXL2 protein, showed that PEAR1-EMI recombinant protein effectively inhibited LOXL2-induced PEAR1 phosphorylation in MDA-MB-231 cells, inhibiting downstream CD44 signaling, i.e., inhibiting CD44 expression in MDA-MB-231 cells. Cell function experiments showed that PEAR1-EMI recombinant protein significantly inhibited cell invasion and migration in a dose-dependent manner, while having no significant effect on cell proliferation.

[0259] Figure 10 shows that PEAR1 fab-HSA monoclonal antibody competitively blocks the binding of LOXL2 to PEAR1 to inhibit the metastasis of TBC cells, wherein the inventors independently developed a high-specificity monoclonal antibody targeting the PEAR1-EMI domain based on existing monoclonal antibody preparation technology, and to avoid the dimerization and activation of the antibody, the inventors selected the single arm of the antibody and coupled it with human serum albumin (HSA) to prolong its half-life in the body. ELISA results showed that PEAR1 fab-HSA monoclonal antibody mainly binds to PEAR1-EMI recombinant protein, but not to PEAR1-EMI polypeptide, indicating that the interaction occurs in the spatial epitope. In addition, the ELISA results also directly prove that PEAR1 fab-HSA monoclonal antibody has a competitive effect on the binding of LOXL2 to PEAR1, i.e. PEAR1 fab-HSA monoclonal antibody can block the binding of LOXL2 to PEAR1. WB detection results showed that PEAR1 fab-HSA monoclonal antibody can significantly inhibit the LOXL2-induced PEAR1 serine phosphorylation and downstream CD44 stable expression. Cell function experiment results showed that PEAR1 fab-HSA monoclonal antibody can significantly inhibit the invasion and migration of MDA-MB-231 cells caused by LOXL2 stimulation, and shows obvious dose-dependent effect, but has no obvious effect on cell proliferation. In addition, in the NOD-SCID mouse in vivo metastasis model, tail vein injection of MDA-MB-231 cells and drug treatment, the results showed that PEAR1 fab-HSA monoclonal antibody can effectively inhibit the lung and liver metastasis of MDA-MB-231 cells by hematogenous spread.

[0260] The amino acid sequence of the high-specificity monoclonal antibody targeting the PEAR1-EMI domain after sequencing is shown in the following table:

[0261] Table 7: Amino acid sequence of monoclonal antibody and coupled polypeptide

[0262]

[0263]

[0264] In FIG. 11, phospho-PEAR1 Ser891 detection antibody was prepared by Shanghai Jier Biochemical Co., Ltd. First, phospho-peptide (Cys-RGSSRLDRSY(pS)YSYSNGP) (SEQ ID NO: 25) and control non-phospho-peptide (Cys-RGSSRLDRSYSYSYSNGP) (SEQ ID NO: 26) were synthesized to ensure purity greater than 90%; then the two were coupled with keyhole limpet hemocyanin (KLH) and immunized into two New Zealand rabbits, respectively, for a total of 8 times, and after the 6th immunization, a small sample of antisera was taken to test the serum titer; the rabbit antisera were collected, and all the rabbit sera were subjected to polypeptide affinity purification, i.e., first, the non-phospho-peptide corresponding antigen affinity purification column was used to purify the antisera to obtain non-phospho-antibody, and after the non-phospho-antibody was removed completely (until the peak of non-phospho-antibody was at a low level), the phospho-peptide corresponding antigen affinity purification column was used to purify the antisera to obtain phospho-antibody; finally, ELISA and dot blot (DB) detection were performed, ELISA detection ensured that the antibody titer was greater than 1:32000, and DB detection ensured that there was no cross reaction between the phospho-antibody and the non-modified polypeptide. In addition, the inventors verified the specificity of phospho-PEAR1 Ser891 antibody and the corresponding non-phospho-antibody in MDA-MB-231 cells with PEAR1 S891A single-point mutation and PEAR1 All SA full-point mutation and their corresponding control cells.

[0265] FIG. 12 shows that PEAR1, PEAR1 S891 phosphorylation, LOXL2 and CD44 are all highly expressed in TNBC and are associated with poor prognosis of patients, wherein in order to reveal the clinical correlation of PEAR1, PEAR1 Ser891 phosphorylation, LOXL2, CD44 expression with TNBC, IHC staining was performed on TNBC tissues and their corresponding para-carcinoma tissues, and the results showed that the expression levels of LOXL2, PEAR1, PEAR1 Ser891 phosphorylation and CD44 in TNBC tissues were all significantly increased compared with para-carcinoma tissues. Combined with the analysis of background information of tissue chips, the results showed that the expression levels of PEAR1, PEAR1 Ser891 phosphorylation, LOXL2 and CD44 in TNBC were all negatively correlated with the overall survival rate of patients.

[0266] Figure 13 shows that the expression levels of EAR1, PEAR1 Ser891 phosphorylation, LOXL2 and CD44 in TNBC are positively correlated, wherein the pearson correlation analysis results show that there is a significant pairwise positive correlation between the expression levels of PEAR1, PEAR1 Ser891 phosphorylation, LOXL2 and CD44. In order to further compare the effects of the risk scores of PEAR1, PEAR1 Ser891 phosphorylation, LOXL2, CD44 and their pairwise combination schemes on the prognosis of TNBC patients, the best prognosis scheme is determined by using time-dependent receiver operating characteristic (ROC) curve analysis, and the results show that PEAR1 Ser891 phosphorylation is considered to be the best independent prognostic factor for TNBC patients, and the combination scheme thereof with CD44 shows a better prognostic effect.

Claims

1. Use of a reagent for detecting a biomarker or a combination of biomarkers in the preparation of a product for diagnosing or aiding in the diagnosis of triple negative breast cancer; the biomarker is PEAR1 or phosphorylated PEAR1; the combination of biomarkers comprises at least two of the following: (1) PEAR1; (2) phosphorylated PEAR1; (3) LOXL2; (4) CD44; the reagent is used to detect the protein expression level of the biomarker or the combination of biomarkers.

2. Use according to claim 1, wherein the triple negative breast cancer is metastatic triple negative breast cancer; and / or, the phosphorylated PEAR1 is serine-phosphorylated PEAR1, for example, PEAR1 phosphorylated at one or more of Ser795, Ser891, Ser953, Ser976 and Ser1029; and / or, the product comprises a kit, a chip, a test paper and a high-throughput sequencing platform.

3. An antibody targeting PEAR1, characterized in that, the antibody comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NOs: 11, 12 and 13, respectively, and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined according to Kabat.

4. The antibody of claim 3, wherein the heavy chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 14 or having at least 90% sequence identity to SEQ ID NO: 14; and / or, the light chain variable region comprises an amino acid sequence set forth in SEQ ID NO: 19 or having at least 90% sequence identity to SEQ ID NO: 19; preferably, the antibody is a full-length antibody, Fab, Fab'-SH, Fv or (Fab')2; the Fv is, for example, a scFv.

5. A fusion protein, characterized in that, the fusion protein comprises the antibody of claim 3 or 4, and a protein fragment; the protein fragment is capable of promoting the antibody to remain stable and prolong the half-life of the antibody; preferably, the protein fragment is human serum albumin, an antibody or fragment thereof that binds human serum albumin, an immunoglobulin constant region or a fragment thereof; more preferably, the human serum albumin is connected to the N-terminus or C-terminus of the antibody; and / or, the human serum albumin has an amino acid sequence set forth in SEQ ID NO: 22; and / or, the antibody further comprises a heavy chain constant region and a light chain constant region; more preferably, the human serum albumin is connected to the C-terminus of the heavy chain constant region; and / or, the heavy chain constant region has an amino acid sequence set forth in SEQ ID NO: 21; and / or, the light chain constant region has an amino acid sequence set forth in SEQ ID NO: 23; for example, the fusion protein comprises a first polypeptide having an amino acid sequence set forth in SEQ ID NO: 15 and a second polypeptide having an amino acid sequence set forth in SEQ ID NO:

20.

6. A polypeptide, characterized in that, The polypeptide has an amino acid sequence as set forth in SEQ ID NO:

24.

7. The polypeptide of claim 6, wherein, The polypeptide specifically binds to the antibody or variant thereof as claimed in claim 3 or 4, or the fusion protein as claimed in claim 5; The variant has at least 80% identity to the antibody and retains the function of the antibody.

8. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-7. The polynucleotide encodes the antibody as claimed in claim 3 or 4, the fusion protein as claimed in claim 5, or the polypeptide as claimed in claim 6 or 7.

9. An expression vector, characterized by, The expression vector comprises the polynucleotide as claimed in claim 8.

10. A transformant characterized in that, The transformant comprises the polynucleotide as claimed in claim 8 or the expression vector as claimed in claim 9.

11. A method of producing a polypeptide, antibody or fusion protein, characterized in that, The method comprises culturing the transformant as claimed in claim 10, and obtaining the polypeptide, antibody or fusion protein from the culture.

12. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the antibody as claimed in claim 3 or 4, or the fusion protein as claimed in claim 5, and a pharmaceutically acceptable carrier and / or excipient.

13. Use of a PEAR1 inhibitor in the preparation of a medicament for the diagnosis or treatment of cancer. Preferably, the cancer is selected from breast cancer, blood cancer, brain cancer, colon cancer and ovarian cancer. More preferably, the cancer is triple negative breast cancer, for example metastatic triple negative breast cancer.

14. Use according to claim 13, wherein the compound is ###0006### The PEAR1 inhibitor reduces or blocks the binding of PEAR1 to LOXL2, inhibits the serine phosphorylation of PEAR1 and / or inhibits the expression of CD44. and / or, the triple negative breast cancer is metastatic triple negative breast cancer; and / or, the PEAR1 inhibitor is an agent that mutates one or more of Ser795, Ser891, Ser953, Ser976 and Ser1029 of PEAR1 to Ala; and / or, the PEAR1 inhibitor is the antibody as claimed in claim 3 or 4, the fusion protein as claimed in claim 5, or the pharmaceutical composition as claimed in claim 12.

15. Use of the antibody as claimed in claim 3 or 4, the fusion protein as claimed in claim 5, the polynucleotide as claimed in claim 8, the expression vector as claimed in claim 9, or the pharmaceutical composition as claimed in claim 12 in the preparation of a medicament for the treatment of cancer. Preferably, the cancer is selected from breast cancer, blood cancer, brain cancer, colon cancer and ovarian cancer. More preferably, the breast cancer is triple negative breast cancer, for example metastatic triple negative breast cancer.

16. A method of detecting a biomarker or a combination of biomarkers of triple negative breast cancer in a sample, characterized in that, The biomarker or combination of biomarkers is as claimed in claim 1. The method comprises the step of contacting the sample with the antibody as claimed in claim 3 or 4, or the fusion protein as claimed in claim 5. Preferably, the method is for non-therapeutic or diagnostic purposes.

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