Monoclonal antibody for specifically recognizing PZR protein and application thereof

Abstract

The invention relates to a monoclonal antibody capable of specifically recognizing a tumor-associated antigen PZR as well as a preparation method and application of the monoclonal antibody. The anti-PZR monoclonal antibody can specifically recognize natural PZR protein expressed by triple negative breast cancer cells, and can realize high-affinity binding with recombinant PZR extracellular domain protein. The compound also has the capability of remarkably inhibiting migration and metastasis of PZR positive triple negative breast cancer cells, and has the characteristic of repression function. The anti-PZR monoclonal antibody provided by the invention can be used for preventing and treating PZR positive triple negative breast cancer metastasis, and is suitable for developing antibody drugs for targeted therapy of PZR positive tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a monoclonal antibody targeting tumor-associated antigen PZR, its preparation technology and uses. Background Art

[0002] Breast cancer is the most common malignant tumor in women. The high incidence and mortality of breast cancer not only bring great pain to the patients themselves, but also impose a heavy economic burden on their families and society. Clinically, breast cancer is usually classified according to its receptor status, mainly divided into three subtypes: luminal type, HER2 overexpression type, and triple-negative breast cancer (TNBC). Luminal breast cancer expresses estrogen receptor (ER) and / or progesterone receptor (PR), and is the most common subtype of breast cancer, accounting for about 60%. HER2 overexpression breast cancer is negative for both ER and PR, and human epidermal growth factor receptor 2 (HER2) is positive, accounting for about 25% of breast cancer patients. Triple-negative breast cancer (TNBC) does not express ER, PR, and HER2, accounting for about 15% of breast cancer patients. Compared with luminal breast cancer, HER2 overexpression and TNBC breast cancers have lower survival rates in the first 4-5 years after onset. TNBC usually has highly malignant clinicopathological features, manifested as highly proliferative cells. Importantly, TNBC breast cancer patients are more likely to have early metastases, and the common metastatic sites are the lungs and liver, which is also the main reason for its high mortality.

[0003] In recent years, targeted therapies against ER and HER2 have made significant progress in the field of breast cancer treatment. For example, endocrine therapy drugs such as estrogen receptor antagonists (tamoxifen) and aromatase inhibitors (anastrozole and exemestane) have shown good efficacy in the treatment of luminal breast cancer. In addition, anti-HER2 therapy has also continuously made breakthroughs. For example, the combination of trastuzumab and the small molecule inhibitor lapatinib with standard chemotherapy has become the standard treatment regimen for metastatic HER2-positive breast cancer. However, the treatment of TNBC breast cancer faces great challenges. At present, the standard method for TNBC is still mainly chemotherapy, but chemotherapy has poor specificity, large side effects, and a short efficacy time, and is prone to recurrence and distant metastasis. This lack of specific therapies is also one of the important reasons for the high mortality of TNBC. Therefore, there is an urgent need to find new targets, develop more targeted and low-side-effect drugs, and provide new effective therapies for the treatment of refractory breast tumors.

[0004] PZR (Protein zero-related) is a type I transmembrane glycoprotein, and its encoding gene MPZL1(Myelinprotein zero like 1) is located on human chromosome 1q24.1-24.2. The PZR protein consists of a signal peptide, an extracellular region containing an immunoglobulin variable region (IgV)-like domain, a transmembrane region, and an intracellular region containing two immunoreceptor tyrosine-based inhibitory motifs (ITIM). PZR is widely expressed in human tissues, especially abundantly expressed in the placenta, kidney, and pancreas.

[0005] Studies have shown that PZR is closely related to the occurrence and development of various diseases, including schizophrenia, Noonan and Leopard syndrome. In addition, the PZR protein plays a key role in the metastasis process of various tumors. For example, in hepatocellular carcinoma, the expression level of PZR is positively correlated with the metastasis and malignancy of hepatocellular carcinoma. Src kinase promotes the migration and metastasis of hepatocellular carcinoma cells by mediating the phosphorylation of cortactin induced by PZR overexpression. In ovarian cancer, PZR may be a new pro-metastatic gene, promoting the proliferation and migration of tumor cells through Src-mediated phosphorylation of p130 and cortactin. In addition, in colorectal cancer (CRC), the expression of PZR not only increases in tumor tissues with a higher pathological stage, but also significantly rises in tumors with distant metastasis.

[0006] Cancer cell metastasis is a complex process involving multiple steps, including the migration of primary tumor cells, invasion of the stroma, entry into blood vessels, survival in the blood, attachment to vascular endothelial cells, crossing the vascular wall (extravasation), and colonization and expansion in distant tissues and organs. It is currently unclear whether PZR plays a role in the later stages of tumor cell metastasis (such as extravasation, colonization, and expansion in distant organs), but antibodies targeting PZR are expected to become potential drugs for the treatment of metastatic tumors.

[0007] Since the PZR protein was first reported in 1998, there has been no report on the research and development of monoclonal antibody drugs targeting the PZR protein. Given the key role of PZR in cancer cell migration, invasion, and metastasis, the development of humanized antibodies that specifically recognize PZR on the surface of cancer cells and inhibit cancer cell metastasis will help provide a new direction for the effective treatment of PZR-positive tumors. Summary of the Invention

[0008] Given the important role of PZR in cancer cell migration, invasion and metastasis, the present invention first used the eukaryotic cell-expressed PZR-ECD-127 recombinant protein (127 amino acids of the extracellular domain of PZR) mixed with an adjuvant to sensitize mice, prepared monoclonal antibodies that recognize the extracellular domain of PZR as an epitope, and detected the function of anti-PZR monoclonal antibodies in in vitro and in vivo models of TNBC. It was found that the anti-PZR monoclonal antibody (clone number: 12F6) specifically recognizes the native PZR protein expressed by TNBC cancer cells. Further, surface plasmon resonance analysis (SPR) was performed using a protein interaction instrument to measure the affinity of the anti-PZR monoclonal antibody for the recombinant PZR protein ( K D ) to be approximately 2.04 nM. The anti-PZR monoclonal antibody can significantly inhibit the migration of PZR-positive TNBC cells. Importantly, the anti-PZR monoclonal antibody can significantly inhibit the metastasis of PZR-positive TNBC cells to the lung tissue of mice. In addition, by sequence alignment, two human monoclonal antibody heavy and light chain variable regions with the highest homology to the variable region sequence of the parental mouse PZR monoclonal antibody were screened, and the CDRs of the human monoclonal antibody variable regions were replaced with the CDRs of the mouse PZR antibody, successfully preparing a humanized PZR (HuPZR) antibody. The HuPZR antibody can also recognize the native conformation PZR protein expressed by TNBC cells. The present invention will lay a solid foundation for the development of antibody drugs targeting PZR to overcome TNBC metastasis and has practical guiding significance for the targeted treatment of PZR-positive tumors.

[0009] Therefore, in order to solve the problem of developing monoclonal antibodies with inhibitory effects based on the target PZR, the inventors immunized animals with PZR-ECD-127 (127 amino acids of the extracellular region) as an antigen, coated an ELISA plate with the PZR-ECD-127 protein as an antigen for screening, and prepared a monoclonal antibody that specifically recognizes the recombinant PZR protein. This anti-PZR monoclonal antibody (12F6) specifically recognizes the native PZR protein expressed by tumor cells. And this monoclonal antibody has high affinity and strong specificity. Moreover, the anti-PZR monoclonal antibody can inhibit the in vivo metastasis of PZR-positive TNBC cells and has the functional characteristic of inhibition.

[0010] The present invention first provides an antigen, which is composed of 127 amino acids of the extracellular region of PZR.

[0011] Furthermore, it provides a monoclonal cell line that secretes a monoclonal antibody that specifically binds to the antigen as described in claim 1.

[0012] The present invention particularly provides an antibody or its antigen-binding fragment, which is prepared from the antigen and specifically binds to the antigen as described in claim 1.

[0013] Specifically, it is a monoclonal antibody.

[0014] Preferably, the amino acid sequences of VH-CDR1, VH-CDR2, and VH-CDR3 in the heavy chain variable region are shown as SEQ ID NO: 1; SEQ ID NO: 2; and SEQ ID NO: 3, respectively; and the amino acid sequences of VL-CDR1, VL-CDR2, and VL-CDR3 in the light chain variable region are shown as SEQ ID NO: 6; SEQ ID NO: 7; and SEQ ID NO: 8, respectively.

[0015] More preferably, the amino acid sequence of the heavy chain variable region is: SEQ ID NO: 4; and the amino acid sequence of its light chain variable region is: SEQ ID NO: 9.

[0016] The present invention further provides a coding nucleic acid for the antibody or its antigen-binding fragment as described above.

[0017] Preferably, the nucleic acid sequence of its heavy chain variable region is: SEQ ID NO: 5; and the nucleic acid sequence of its light chain variable region is: SEQ ID NO: 10.

[0018] The present invention also provides the use of the antibody or its antigen-binding fragment as described above in the preparation of a targeted therapeutic drug for preventing or treating PZR-positive tumors.

[0019] The beneficial effects of the present invention are as follows: The PZR protein is abnormally highly expressed in various tumors. The PZR protein plays an important role in promoting the metastasis of tumor cells. The present invention provides a monoclonal antibody targeting the tumor-associated antigen PZR, a preparation method, and uses thereof. The anti-PZR monoclonal antibody (12F6) specifically recognizes the native PZR protein expressed by tumor cells. The anti-PZR monoclonal antibody binds to the recombinant PZR protein with high affinity. The anti-PZR monoclonal antibody can inhibit the migration and metastasis of PZR-positive TNBC cells. In addition, the humanized PZR monoclonal antibody (HuPZR) can also recognize the native conformation PZR protein expressed by TNBC cells. The present invention lays a solid foundation for potential clinical translational applications in the targeted treatment of PZR-positive tumors.

[0020] The advantage of the antibody of the present invention is that a monoclonal antibody with the extracellular region of the PZR protein as the antigenic epitope is prepared. This antibody can specifically recognize the PZR protein on the surface of tumor cells and significantly inhibit the in vivo metastasis of TNBC cells, and has a clear inhibitory functional characteristic.

[0021] Murine antibodies have significant immunogenicity problems when applied in humans, manifested as immune rejection reactions caused by major histocompatibility antigens, which limits their clinical applications. Therefore, in this invention, the CDR transplantation method was used to humanize murine PZR monoclonal antibodies, and a novel recombinant humanized PZR monoclonal antibody (HuPZR) was successfully constructed. On the basis of retaining the high specificity of murine antibodies, this antibody can minimize immunogenicity, laying a foundation for subsequent clinical applications.

[0022] This invention shows significant innovation in aspects such as antigen target selection, antibody humanization, and the research and development of novel antibody drugs, and is expected to open up a new path for the precision treatment of breast cancer. Brief Description of the Drawings

[0023] Figure 1 Purification and identification of recombinant human PZR-ECD-127 protein. Coomassie brilliant blue staining and Western blotting were used to analyze the expression of recombinant human PZR protein.

[0024] Figure 2 Anti-PZR monoclonal antibody recognizes PZR protein on the surface of TNBC cells. Flow cytometry was used to detect the binding of anti-PZR monoclonal antibody to PZR protein on the surface of TNBC cells.

[0025] Figure 3 Anti-PZR monoclonal antibody recognizes PZR protein in the cytoplasm of TNBC cells. Immunoprecipitation was used to evaluate the recognition ability of anti-PZR monoclonal antibody to PZR protein expressed in TNBC cells.

[0026] Figure 4 Surface plasmon resonance analysis (SPR) was used to measure the affinity of 5 anti-PZR monoclonal antibodies. An SPR instrument was used to detect the affinity of the interaction between the antibody and the antigen.

[0027] Figure 5 Anti-PZR monoclonal antibody is effectively internalized by TNBC cells. (A) TNBC cells were stained on the cell surface, scale bar = 20 µm. (B) A laser confocal microscope system was used to detect the internalization efficiency of anti-PZR monoclonal antibody by TNBC cells, scale bar = 20 µm.

[0028] Figure 6A Graph showing the changes in bioluminescence signals in the lungs of mice in each experimental group.

[0029] Figure 6B Quantitative analysis graph of bioluminescence signals.

[0030] Figure 6C Histological examination results of the lung tissues of mice in each experimental group (scale bar in the figure = 200 µm).

[0031] Figure 7The humanized PZR antibody binds to the recombinant PZR protein with high affinity. The binding of the HuPZR antibody to the recombinant PZR protein was detected by ELISA.

[0032] Figure 8 The humanized PZR antibody recognizes the PZR protein on the surface of TNBC cells. The binding of the HuPZR antibody to the PZR protein on the cell surface was detected by flow cytometry.

[0033] Figure 9 The humanized PZR antibody recognizes the native PZR protein expressed in the cytoplasm of TNBC cells. The immunoprecipitation experiment was used to compare the efficiency of the HuPZR antibody and the murine monoclonal antibody 12F6 in precipitating the PZR protein. Detailed implementation mode

[0034] For the purpose, technical solutions and advantages of the present invention to be more clear, the following further detailed description of the present invention is made in conjunction with specific embodiments and with reference to the accompanying drawings.

[0035] Example 1: Selection and preparation of antigen I. Selection of antigen Since the PZR protein was first reported in 1998, there has been no report on the research and development of monoclonal antibody drugs targeting the PZR protein (the full-length PZR protein is 269 amino acids long, and its NCBI accession number is NP_003944.1).

[0036] The inventor of the present invention found in previous studies that PZR can significantly promote the migration and invasion of triple-negative breast cancer cells. Considering that the PZR protein is not only highly expressed in tumor cells, but also widely expressed in various cell types such as fibroblasts and vascular endothelial cells. And the study of the PZR crystal structure found that the PZR protein maintains its homodimer structure through the hydrogen bond interaction between Ser86 and Val145 sites between two PZR molecules. Therefore, the present invention attempts to further explore the mechanism by which the PZR protein promotes cancer cell metastasis, whether it is related to the interaction between PZR molecules, by preparing monoclonal antibodies that recognize the extracellular region of PZR. At the same time, from the aspect of antigen preparation, considering the proteins expressed on the surface of cancer cells, in order to be able to express through the eukaryotic cell expression system, the present invention finally determined to select all extracellular regions of the PZR protein as the antigen.

[0037] The present invention uses a recombinant PZR-ECD-127 protein (containing 127 amino acids of the extracellular domain of PZR) expressed by eukaryotic cells and mixed with an immune adjuvant to sensitize mice, and prepares monoclonal antibodies that recognize the extracellular domain of PZR as an antigenic epitope. Among them, the sequence containing 127 amino acids (SEQ ID No: 13) of the extracellular domain of PZR is: salevytpkeifvangtqgkltckfkststtggltsvswsfqpegadttvsffhysqgqvylgnyppfkdriswagdldkkdasinienmqfihngtyicdvknppdivvqpghirlyvvekenlpv.

[0038] II. Preparation, purification and identification of recombinant human PZR extracellular region protein To prepare the antigen, we extracted RNA from TNBC cells MDA-MB-231 and reverse transcribed it into cDNA. After PCR amplification, the extracellular region fragment of human PZR protein (PZR-ECD-127) was inserted into the pcDNA3.0 vector to construct a vector pcDNA3.0-PZR-ECD-127-6xHis expressing recombinant human PZR protein.

[0039] This expression vector (2.5 μg / mL) was mixed with polyethyleneimine (PEI, 7.5 μg / mL) and transiently transfected into CHO eukaryotic cell suspension culture (200 mL system). After six days, the supernatant was collected and the recombinant protein was purified with Ni-NTA beads, and 9 mg of protein was obtained. Coomassie brilliant blue staining experiment showed that the purity of the purified protein exceeded 90% ( Figure 1 ). We further used an anti-His-tag antibody for Western blot experiment to detect the expression of recombinant human PZR protein, and its molecular weight was about 15-25 kDa ( Figure 1 ).

[0040] Example 2: Animal immunization with recombinant human PZR extracellular region protein To prepare monoclonal antibodies specifically recognizing human PZR protein, we mixed recombinant PZR-ECD-127 protein (50 µg / mouse) with an immune adjuvant and intraperitoneally injected Balb / c mice (female, four weeks old, n = 3) three times. After immunizing the animals, orbital blood was collected from the mice, and the titer of mouse serum IgG (immunoglobulin G) was detected by enzyme-linked immunosorbent assay (ELISA). First, a 96-well enzyme-linked immunosorbent assay plate was coated with recombinant PZR-ECD-127 protein (4 µg / mL, 100 µL). After blocking with PBS-B (containing 3% bovine serum albumin) for 30 minutes, serums diluted in a serial dilution (a total of 8 gradients: 50-fold, 200-fold, 800-fold, 3200-fold, 12800-fold, 51200-fold, 204800-fold, 819200-fold) were incubated at room temperature for 2 hours (100 µL / well). The enzyme-linked immunosorbent assay plate was washed 5 times with PBS-T, and the anti-mouse IgG-HRP secondary antibody was incubated. After TMB color development, the absorbance value (OD value) was measured at a wavelength of 450 nm to determine whether there were specific antibodies recognizing recombinant PZR-ECD-127 protein. As shown in Table 1, the serum titers of Mouse No. 1 and Mouse No. 3 were higher than 51200-fold, reaching the available titer.

[0041] Table 1

[0042] Example 3: Screening of Monoclonal Hybridoma Cells Specifically Recognizing Recombinant Human PZR Protein We extracted lymphocytes from the spleen of Mouse No. 1 with a relatively high antibody titer. The spleen lymphocyte suspension was mixed with SP2 / 0 mouse myeloma cells at the logarithmic growth phase in a 5:1 ratio (the total cell amount was about 1.9×10 8 ), and the mixture was placed in a 50 mL centrifuge tube. After mixing, the cells were washed twice with DMEM medium. 1 mL of pre-warmed PEG (polyethylene glycol) solution was slowly added dropwise to the mixed cell precipitate in a 37°C water bath. The centrifuge tube was centrifuged at 1000 rpm for 90 seconds (18°C), and the supernatant was discarded. Then, 10.5 mL of pre-warmed DMEM medium was added drop by drop until it flowed, and the centrifuge tube was gently shaken throughout the process to mix, and the operation was completed within 5 minutes. The centrifuge tube was centrifuged at 1000 rpm for 5 minutes (37°C), and the supernatant was discarded. The cells were fully suspended in about 20 mL of DMEM medium containing 20% fetal bovine serum, 1% double antibody, and 2% HAT (hypoxanthine-aminopterin-thymine), and then plated in a 96-well plate and cultured in a 37°C cell culture incubator with 5% CO2.

[0043] Two weeks after cell fusion, the cell supernatant in the 96-well plate was taken for positive detection. The 96-well ELISA plate was coated with recombinant PZR-ECD-127 protein (4 μg / mL, 100 μL). Approximately 100 μL of the cell supernatant was added to the coated plate (diluted 3-fold, 9-fold, 27-fold, 81-fold, and 243-fold respectively), PBS was used as a blank control, and the serum from the immunized mouse orbit was used as a positive control, and incubated at room temperature for 2 hours; after washing 5 times with PBS-T (pH 7.4, 1% Tween), 100 μL of anti-mouse IgG-HRP secondary antibody was added and incubated at room temperature for 1 hour; after washing 5 times with PBS-T, 100 μL of TMB chromogenic solution was added, and after incubating for 10 minutes, 50 μL of 1 M sulfuric acid was added to terminate the reaction, and the absorbance value was measured at 450 nm using an ELISA reader. As shown in Table 2, the OD values of the cell supernatants in 15 cell culture wells were greater than 0.3, which were 5B5, 4D4, 12F6, 3A5, 18A12, 28A7, 6H12, 8D12, 22H12, 2E12, 4A8, 29A8, 6C9, 12E7, and 8F2 respectively.

[0044] Next, we plated the 10 clones (5B5, 4D4, 12F6, 3A5, 18A12, 28A7, 6H12, 8D12, 22H12, and 2E12) with OD values of the cell culture well supernatants greater than 0.35 onto 24-well cell culture plates and 6-well cell culture plates in sequence, and continuously amplified them in the medium containing HAT.

[0045] Table 2

[0046] Example 4: Preparation and Purification of Anti-PZR Monoclonal Antibody To purify the anti-PZR monoclonal antibody, we intraperitoneally injected Balb / c mice with pristane (500 μL / mouse). After 10 - 14 days, 10 hybridoma cells (5B5, 4D4, 12F6, 3A5, 18A12, 28A7, 6H12, 8D12, 22H12, and 2E12 respectively) (1x10 6 cells) were intraperitoneally injected into the mice to induce ascites production, and the anti-PZR monoclonal antibody was obtained by chromatography purification using protein-A beads. ELISA results showed that all these 10 anti-PZR monoclonal antibodies could specifically bind to the recombinant PZR protein, and the binding intensity showed a concentration gradient dependence (Table 3).

[0047] Table 3

[0048] Example 5: Anti-PZR Monoclonal Antibody Recognizes PZR Protein on the Surface of TNBC Cells To investigate whether anti-PZR monoclonal antibodies can recognize the PZR protein expressed by TNBC cells, we conducted experiments using flow cytometry. The results showed that the above-mentioned 7 anti-PZR monoclonal antibodies (6H12, 4D4, 8D12, 2E12, 5B5, 12F6, and 3A5) could recognize the PZR protein expressed on the surface of TNBC cell MDA-MB-468, while the other 3 antibodies (22H12, 18A12, and 28A7) could not recognize it ( Figure 2 ).

[0049] Example 6: Recognition of native PZR protein expressed in the cytoplasm of TNBC cells by anti-PZR monoclonal antibodies Furthermore, after lysing TNBC cells Hs578T, we added anti-PZR monoclonal antibodies and isotype control antibodies (mouse IgG1) respectively for immunoprecipitation (IP) experiments. The results showed that the above-mentioned 7 anti-PZR monoclonal antibodies (6H12, 4D4, 8D12, 2E12, 5B5, 12F6, and 3A5) could all effectively precipitate the endogenous PZR protein expressed in Hs578T cells, and among them, 5 antibodies (4D4, 8D12, 2E12, 5B5, and 12F6) had the highest immunoprecipitation efficiency. In contrast, 22H12, 18A12, and 28A7 could not precipitate the PZR protein ( Figure 3 ), and the isotype control antibody mouse IgG1 also failed to precipitate the PZR protein expressed by TNBC cells. These results indicate that the 7 anti-PZR monoclonal antibodies can recognize the PZR protein in the native conformation on the surface and in the cytoplasm of TNBC cells ( Figure 3 ).

[0050] Example 7: Detection of the affinity of 5 anti-PZR monoclonal antibodies To evaluate the affinity (equilibrium dissociation constant, K D ) of the interaction between 5 anti-PZR monoclonal antibodies (2E12, 4D4, 5B5, 8D12, and 12F6) and the antigen, we performed surface plasmon resonance (SPR) analysis using a protein interaction analyzer (Biacore X100). The results showed that the affinities ( K D ) of the 5 antibodies with the recombinant PZR protein were: 7.78 nM for 2E12, 4.54 nM for 4D4, 4.99 nM for 5B5, 0.6224 nM for 8D12, and 2.04 nM for 12F6 ( Figure 4 ). Among them, the anti-PZR monoclonal antibody 8D12 had the highest affinity, followed by 12F6. Generally, if the affinity of the interaction between an antibody and an antigen is less than 10 nM, it can be considered that the antibody has good affinity.

[0051] Example 8: Anti-PZR monoclonal antibody significantly inhibits the migration of PZR-positive TNBC cells Previous studies have shown that PZR plays an important role in the migration and invasion of cancer cells. To investigate the effect of anti-PZR monoclonal antibody on the migration of TNBC cells, we co-incubated five anti-PZR monoclonal antibodies and isotype control antibodies with PZR-positive TNBC cells MDA-MB-231 and Hs578T, respectively, and performed Trans-well assays. After 24 hours, the cell migration was analyzed by crystal violet staining assay. As shown in Table 4, compared with the isotype control antibody treatment group, anti-PZR monoclonal antibodies 5B5, 8D12, and 12F6 could significantly inhibit the migration of PZR-positive TNBC cells, and the inhibitory effect of anti-PZR monoclonal antibody 12F6 was the most significant.

[0052] Table 4

[0053] Example 9: Anti-PZR monoclonal antibody can be internalized by PZR-positive TNBC cells To confirm whether the anti-PZR monoclonal antibody (12F6 mAb) enters cells through receptor-mediated endocytosis on the cell surface, we first stained the cell surface with 12F6 mAb. The results showed obvious fluorescence signals on the cell membranes of MDA-MB-231 and Sum149PT ( Figure 5 A), while no fluorescence signals were detected in the isotype control group. These results indicate that 12F6 mAb can effectively recognize the native PZR protein on the surface of PZR-positive TNBC cells. Further, we treated MDA-MB-231 and Sum149PT cells with 12F6 mAb labeled with DyLight-488 fluorescence and incubated them at 37°C for different times. Analysis by a laser confocal microscopy system found that in MDA-MB-231 and Sum149PT cells with high PZR expression, the intracellular fluorescence signal gradually increased with the prolongation of the antibody treatment time. This phenomenon suggests that PZR on the cell surface may mediate the endocytosis of 12F6 mAb ( Figure 5 B).

[0054] Example 10: Anti-PZR monoclonal antibody significantly inhibits the in vivo metastasis of PZR-positive TNBC cells To detect whether 12F6 mAb could inhibit the in vivo metastasis of PZR-positive TNBC cells, we used luciferase-labeled MDA-MB-231-Luc cells and injected them into NOD / SCID mice via the tail vein, successfully establishing a mouse model of breast cancer metastasis (Figure 6). Subsequently, the metastasis of MDA-MB-231 cells in mice was monitored using IVIS. Three hours after cell injection, the bioluminescence signals in the lungs of mice in each group were similar ( Figure 6A ), indicating that the number of cells injected into each group of mice was basically the same. One day after cell injection, the mice were divided into three groups and injected with PBS, isotype control antibody (10 mg / kg), and 12F6 mAb (10 mg / kg) via the tail vein, respectively. During the experiment, antibody injection was performed twice a week, and the IVIS imaging system was used to observe the metastasis of MDA-MB-231 cells in the lung tissue of mice weekly. The results showed that over time, the bioluminescence signals in the lungs of mice in the PBS group and the isotype control group gradually increased, indicating that the metastasis of tumor cells in the lungs gradually worsened. However, compared with the isotype control group, the bioluminescence signals in the lungs of mice in the 12F6 mAb group were significantly reduced ( Figure 6A , B), indicating that 12F6 mAb could effectively inhibit the metastasis of tumor cells. On the 38th day of the experiment, the lungs of the mice were dissected and histological analysis was performed. The results showed that the number and size of metastatic foci in the lungs of mice in the 12F6 mAb group were significantly less or smaller than those in the PBS group or the isotype control antibody group ( Figure 6C ). The above data indicate that 12F6 mAb can significantly inhibit the in vivo metastasis of PZR-positive TNBC cells.

[0055] Example 11: Identification of the CDR sequences of the heavy and light chain variable regions of the anti-PZR monoclonal antibody To reduce the risk of immune responses induced by murine antibodies in humans, we humanized the murine PZR antibody 12F6 mAb. First, we extracted total RNA from 12F6 hybridoma cells and synthesized cDNA using reverse transcriptase. The Fab fragments of the heavy chain variable region (VH) and the light chain variable region (VL) were amplified by PCR technology. The primers are as follows: Heavy chain primers, IGG1: GGAAGATCTATAGACAGATGGGGGTGTCGTTTTGGC, 5'MH2: CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG; Light chain Kappa chain primers, 3'Kc:GGTGCATGCGGATACAGTTGGTGCAGCATC, 5'Mk: GGGAGCTCGAYATTGTGMTSACMCARWCTMCA.

[0056] Subsequently, the amplified Fab fragments were cloned into the pGEM-T Easy vector respectively, and through DNA sequencing analysis, the nucleic acid sequences of the heavy chain variable region (VH) and the light chain (VL) variable region of the 12F6 mAb were obtained. Based on the Kabat numbering method, we further analyzed and defined the amino acid sequences of the three CDRs (complementary determining regions) of the heavy chain variable region and the three CDRs of the light chain variable region.

[0057] Example 12: Humanization of the anti-PZR monoclonal antibody During the humanization process, the amino acid sequence and even the structure of the antibody will change. Therefore, it is crucial to maintain or basically maintain its affinity and specificity for the target antigen without affecting its drug developability, especially its druggability. The CDRs of monoclonal antibodies are the key regions for the interaction between antibodies and antigens. The classical humanization method is to transplant the CDR regions of murine antibodies onto the human antibody framework region (FR).

[0058] We input the amino acid sequences of the heavy chain variable region (m12F6D9-VH) and the light chain variable region (m12F6D9-VL) of the 12F6 mAb into the NCBI (National Center for Biotechnology Information) open-source database. Through IgBlast alignment, the human antibody heavy chain variable region and light chain variable region with the highest homology to the parental 12F6 mAb were screened out, which were IGHV2-70 and IGKV1-NL respectively. Subsequently, we transplanted the CDR regions of the parental 12F6 mAb onto the human antibody framework to obtain the sequences of the transplanted antibodies (which were defined as CDR grafting). By comparing the CDR grafting-VH and CDR grafting-VL after transplantation with the parental VH and VL sequences, we found all the different key residues in the transplanted and parental antibody framework sequences. According to the following principles, we gradually introduced one or more back mutations into the transplanted antibody sequences: (1) the key amino acids for the interaction of antibody loops; (2) the amino acids located at the interface between the heavy and light chains; (3) the key amino acids for the formation of the antibody core. After each back mutation, we performed modeling and structural analysis using the ROSETTA software. Finally, we screened out the candidate molecule Back mutation1 for humanization and defined it as the Fab fragment of the HuPZR antibody.

[0059] Example 13: The humanized PZR antibody recognizes the native PZR protein expressed by TNBC cells To verify whether the humanized PZR antibody can recognize the native PZR protein expressed by TNBC cells. First, we inserted the cDNA sequence of the variable region of the humanized PZR (HuPZR) antibody into the pcDNA3.4 expression vector and successfully constructed the vector pcDNA3.4-HuPZR capable of expressing recombinant HuPZR monoclonal antibody. Subsequently, we transiently transfected this expression vector into HEK-293F cells for suspension culture and collected the antibody from the culture supernatant, which was purified using protein A agarose beads. Then, we detected the binding ability of the HuPZR antibody to the recombinant PZR protein through ELISA assays. The results showed that the HuPZR antibody could specifically bind to the recombinant PZR protein, and this binding was concentration-dependent ( Figure 7 ), indicating that the HuPZR antibody has good antigen recognition ability.

[0060] To further evaluate the affinity of the HuPZR antibody, we compared it with the murine PZR antibody and detected the capture efficiency of the two antibodies for the PZR protein in immunoprecipitation experiments. In the experiment, we lysed MDA-MB-231 cells and added the HuPZR antibody and the murine PZR antibody respectively for immunoprecipitation. The results showed that the HuPZR antibody and the murine PZR antibody showed similar efficiency in precipitating the PZR protein in MDA-MB-231 cells ( Figure 9 ). This result preliminarily indicates that humanization did not weaken the recognition ability of the HuPZR antibody for the PZR protein. In addition, we also detected the recognition ability of the HuPZR antibody for the PZR protein on the surface of TNBC cells using flow cytometry. The results showed that the HuPZR antibody could recognize the PZR protein on the surface of TNBC cells ( Figure 8 ). The above results indicate that the HuPZR antibody can recognize the native PZR protein expressed on the surface and in the cytoplasm of TNBC cells.

Claims

1. An antigen, characterized in that It is composed of 127 amino acids in the extracellular region of PZR, and its amino acid sequence is shown in SEQ ID NO:

13.

2. A monoclonal cell line that secretes a protein that specifically binds to the antigen as claimed in claim 1.

3. An antibody or an antigen-binding fragment thereof, characterized in that: It is prepared from the antigen as claimed in claim 1 and specifically binds to the antigen as claimed in claim 1.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein: It is a monoclonal antibody.

5. The antibody or antigen-binding fragment thereof according to claim 4, wherein: The amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 in the heavy chain variable region are shown in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3, respectively; and the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region are shown in SEQ ID NO: 6; SEQ ID NO: 7; SEQ ID NO: 8, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein: The amino acid sequence of the heavy chain variable region is: SEQ ID NO: 4; The amino acid sequence of its light chain variable region is: SEQ ID NO:

9.

7. The antibody or antigen-binding fragment thereof according to claim 3, wherein: It is a humanized antibody; preferably, the amino acid sequence of its heavy chain variable region is: SEQ ID NO: 11; The amino acid sequence of its light chain variable region is: SEQ ID NO:

12.

8. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 3 to 7.

9. The encoding nucleic acid according to claim 8, characterized in that The nucleic acid sequence of the heavy chain variable region is: SEQ ID NO: 5; The nucleic acid sequence of its light chain variable region is: SEQ ID NO:

10.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 3 to 7 in the preparation of a targeted therapeutic drug for preventing or treating PZR-positive tumors.

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