CBLN1 monoclonal antibody, humanized antibody, and use

Abstract

Provided are a CBLN1 monoclonal antibody, a humanized antibody, and a use. The CBLN1 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the light chain variable region comprises three complementarity-determining regions (CDRs), i.e., CDR1, CDR2, and CDR3, the amino acid sequences of CDR1 and CDR3 are as shown in SEQ ID NO. 1 and SEQ ID NO. 2, and the amino acid sequence of CDR2 is KVS; and the amino acid sequences of three CDRs, i.e., CDR1, CDR2, and CDR3 of the heavy chain variable region are as shown in SEQ ID NOs. 3-5, respectively. Further provided is a use of the monoclonal antibody in the preparation of a drug for treating breast cancer and prostate cancer, especially castration-resistant prostate cancer and androgen receptor-dependent prostate cancer. The CBLN1 monoclonal antibody is further humanized, and the humanized CBLN1 monoclonal antibody can inhibit the metastasis of prostate cancer.

Description

A CBLN1 monoclonal antibody and a humanized antibody and their uses Technical Field

[0001] This invention relates to the field of monoclonal antibody technology in genetic engineering, and more specifically, to a CBLN1 monoclonal antibody and a humanized CBLN1 antibody and their uses. Background Technology

[0002] For patients with early-stage prostate cancer, active surveillance, radical resection, and adjuvant radiotherapy are commonly used clinically. However, for intermediate-stage and metastatic prostate cancer, due to its complex and diverse pathogenesis, there is still no optimal treatment plan. Androgen deprivation therapy targeting the AR signaling pathway is the first-line treatment for prostate cancer, but the vast majority of patients who receive this treatment will relapse and develop castration-resistant prostate cancer (CRPC). Furthermore, the immunosuppressive tumor microenvironment of prostate cancer prevents immune checkpoint therapy from achieving significant efficacy in prostate cancer patients.

[0003] For breast cancer, most clinical anticancer drugs are still targeted therapies against the tumor cells themselves, but many patients still eventually relapse and develop metastatic breast cancer. Immunotherapy has been used in clinical practice for breast cancer patients since 1998, and these drugs have improved the overall survival rate of patients with early or late-stage breast cancer. However, tumor tissue often develops intrinsic or acquired resistance mechanisms after treatment, eventually leading to disease progression, and there are no effective subsequent treatment options. Therefore, the development of novel and effective targeted therapies for prostate and breast cancer is urgently needed.

[0004] Studies have shown that the peripheral autonomic nervous system plays a crucial role in tissue growth, homeostasis, and organ function. Since tumor tissue also requires key regulatory signals during organ development to maintain growth and survival, research suggests that targeting the intratumoral nervous system can effectively control tumor progression. Both the breast and prostate are organs richly infiltrated by nerves, and the nervous system plays a promoting role in breast cancer tumor progression; blocking sympathetic nerve signals can effectively inhibit tumor development. The autonomic nervous system also plays an important role in the development and progression of prostate cancer. Of particular note is the positive correlation between the density of infiltrating nerves in tumor tissue and the degree of PSA expression in tumor tissue, indicating a potential role of the nervous system in the progression of androgen receptor-dependent CRPC prostate cancer and castration-resistant CRPC. Neurochemical signal transduction is the main way the peripheral nervous system exerts its regulatory effects. Besides classical neurotransmitters, there are currently no studies exploring the potential roles of neurosecreted proteins in prostate and breast cancer and related targeted therapies.

[0005] CBLN1 is a highly conserved neurosecretory protein expressed and secreted by cerebellar granule cells. The loss of expression of this protein directly leads to defects in neural network construction under physiological conditions. Most commercially available CBLN1 antibodies are polyclonal recognition antibodies, capable only of recognizing the CBLN1 protein and lacking the ability to target and block the CBLN1 protein in vivo or in vitro.

[0006] Studies have shown that neural tissue plays a crucial role in tumor initiation, proliferation, and invasion / metastasis, and surgical resection or pharmacological blocking of nerves can significantly inhibit tumor progression. Currently, there are few neuropharmaceuticals used in tumor treatment, and most target classical neurotransmitter signaling axes, leading to numerous side effects in early clinical trials. Neuropeptides, however, possess advantages such as diversity and good tissue specificity, making them a promising option for targeted tumor therapy. Currently, there are no studies exploring the potential role of neuropeptides in immunotherapy tolerance and tumor metastasis.

[0007] CBLN1, a neurocerebellar peptide secreted by cerebellar granule cells, is a highly homologous secreted protein found in both humans and mice. It plays a crucial role in synapse formation, functional maintenance, and neural network construction by forming a supercomplex with proteins such as neural adhesion molecules. We found a positive correlation between intratumoral innervation and the inhibitory nature of the immune microenvironment, and CBLN1 protein was significantly enriched in patients unresponsive to immunotherapy. Furthermore, the objective response rate (ORR) of immune checkpoint therapy was low in patients with metastatic castration-resistant prostate cancer (mCRPC).

[0008] Currently, there are no studies on CBLN1 in the context of immunotherapy resistance and metastatic malignant tumors, and there are no CBLN1 humanized antibodies on the market with functional blocking effects. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a CBLN1 monoclonal antibody and a humanized CBLN1 antibody, along with their uses. Specifically, it provides the use of a CBLN1 antibody in the preparation of drugs for treating breast and prostate cancer. Based on clinical needs in tumor treatment, it has been found that CBLN1 is significantly enriched in patients resistant to immunotherapy and has a role in promoting tumor metastasis, making it a potential target for improving the response rate of immune checkpoint therapy and treating tumor metastasis. Using genetic engineering technology to obtain a humanized CBLN1 antibody, and through mouse prostate cancer xenograft treatment experiments, the present invention provides a novel use of the humanized CBLN1 antibody ZH003 in improving the response rate of tumor immune checkpoint therapy and treating tumor metastasis (taking prostate cancer as an example).

[0010] The objective of this invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a CBLN1 monoclonal antibody ZH-DT7792, comprising a heavy chain variable region and a light chain variable region.

[0012] The three complementary determinants of the variable region in the ZH-DT7792 light chain are:

[0013] CDR1: QSLVHSNGNTY(SEQ ID NO.1),

[0014] CDR2: KVS

[0015] CDR3: SQNTHVPWT(SEQ ID NO.2);

[0016] The three complementary determinants of the variable region of the ZH-DT7792 heavy chain are:

[0017] CDR1: GFNIKDYF(SEQ ID NO.3),

[0018] CDR2: IDPANGNT(SEQ ID NO.4),

[0019] CDR3: NAFHYGHYGRGYAVDY (SEQ ID NO. 5).

[0020] As some specific embodiments of the present invention, the amino acid sequence of the light chain variable region of the CBLN1 monoclonal antibody ZH-DT7792 is shown in SEQ ID NO.6;

[0021] And / or, the amino acid sequence of the heavy chain variable region of the CBLN1 monoclonal antibody ZH-DT7792 is shown in SEQ ID NO.7.

[0022] Secondly, the present invention provides a method for preparing a CBLN1 monoclonal antibody ZH-DT7792, wherein the CBLN1 monoclonal antibody is obtained by immunization with a recombinant CBLN1 protein as an immunogen; the recombinant CBLN1 protein is obtained by adding a signal peptide and a C-His tag to the full-length human CBLN1 protein sequence, using HEK293 / CHO as the expression host cell, and through codon optimization and gene synthesis, expression and purification.

[0023] As some specific embodiments of the present invention, the amino acid sequence of the human CBLN1 protein is shown in SEQ ID NO.8, the amino acid sequence of the signal peptide is shown in SEQ ID NO.9, and the amino acid sequence of the C-His tag is shown in SEQ ID NO.10.

[0024] As some specific embodiments of the present invention, the amino acid sequence of the CBLN1 recombinant protein is shown in SEQ ID NO.11; the gene sequence of the CBLN1 recombinant protein after codon optimization is shown in SEQ ID NO.12.

[0025] As some specific embodiments of the present invention, the preparation method of the CBLN1 monoclonal antibody ZH-DT7792 specifically includes the following steps:

[0026] Step 1: Select Balb / c mice as immunization subjects, use CBLN1 recombinant protein as immunogen, and after multiple rounds of immunization, collect a small amount of blood from the mice to determine the serum titer by indirect ELISA; when the serum titer reaches the B cell differentiation standard, take a certain amount of immunogen to perform a final shock immunization on the mice.

[0027] Step 2: Collect peripheral blood from target mice to sort memory B cells, obtain antibody heavy and light chain gene fragments through single-cell gene amplification technology and construct expression plasmids. After transient expression, collect the supernatant and detect positive cell supernatant using indirect ELISA.

[0028] Step 3: Sequencing the heavy and light chain plasmids of the antibodies corresponding to the supernatant of positive cells, constructing the expression plasmid and transiently expressing it, collecting and purifying the plasmids, and obtaining the CBLN1 recombinant protein-specific monoclonal antibody ZH-DT7792.

[0029] Thirdly, the present invention provides the application of the neurocerebellar peptide CBLN1 monoclonal antibody ZH-DT7792 in the preparation of a drug for treating breast cancer.

[0030] The inventors discovered that the 4T1 breast cancer cell line can induce tumor-associated neutrophils to express the CBLN1 receptor NRXN1. By injecting 4T1 breast cancer-bearing mice with intraperitoneal injection of CBLN1 monoclonal antibody, the size and mass of the tumors were analyzed to clarify the possible use of CBLN1 monoclonal antibody in the treatment of breast cancer.

[0031] Fourthly, the present invention provides the application of the neurocerebellar peptide CBLN1-specific monoclonal antibody ZH-DT7792 in the preparation of a drug for treating prostate cancer.

[0032] As some specific embodiments of the present invention, the prostate cancer includes castration-resistant prostate cancer. By intraperitoneally injecting Probasin-cre;Trp53f / f;Ptenf / f castration-resistant prostate cancer-bearing mice with CBLN1 monoclonal antibody, the tumor size and mass were analyzed to clarify the potential use of CBLN1 monoclonal antibody in the treatment of castration-resistant prostate cancer.

[0033] As some specific embodiments of the present invention, the prostate cancer includes androgen receptor-dependent prostate cancer. By intraperitoneally injecting Probasin-cre; Ptenf / f; Mychi androgen receptor-dependent prostate cancer-bearing mice with tumors, including CBLN1 monoclonal antibody, the tumor size and mass were analyzed to clarify the potential use of CBLN1 monoclonal antibody in the treatment of androgen receptor-dependent prostate cancer.

[0034] This invention provides the application of the CBLN1 monoclonal antibody ZH-DT7792 in alleviating the inhibitory effect of the immune microenvironment in prostate cancer. By intraperitoneally injecting the CBLN1 monoclonal antibody into Probasin-cre; Ptenf / f; Mychi orthotopic prostate cancer-bearing mice, the composition of major immune cells in the immune microenvironment was analyzed to clarify the potential use of the CBLN1 monoclonal antibody in alleviating the inhibitory effect of the immune microenvironment in prostate cancer.

[0035] Fifthly, the present invention provides a humanized CBLN1 antibody, comprising a heavy chain and a light chain;

[0036] The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.17;

[0037] The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.25.

[0038] As some specific embodiments of the present invention, the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.22.

[0039] As some specific embodiments of the present invention, the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.30.

[0040] As some specific embodiments of the present invention, the heavy chain further includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.23.

[0041] Furthermore, the nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.24.

[0042] As some specific embodiments of the present invention, the light chain further includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.31.

[0043] Furthermore, the nucleotide sequence of the light chain constant region is shown in SEQ ID NO.32.

[0044] In a sixth aspect, the present invention provides the use of the CBLN1 humanized antibody as described in any of the preceding claims in the preparation of a medicament for inhibiting prostate cancer metastasis.

[0045] In a seventh aspect, the present invention provides an antibody composition comprising the humanized CBLN1 antibody as described in any one of the preceding claims, and a PD1 antibody.

[0046] Eighthly, the present invention provides the use of the antibody composition described above in the preparation of a medicament for treating prostate cancer.

[0047] This invention involves intraperitoneal injection of the CBLN1 monoclonal antibody ZH003 into humanized tumor-bearing mice, followed by analysis of tumor size and mass to clarify the potential use of the humanized CBLN1 antibody ZH003 in enhancing the response rate of tumor immune checkpoint therapy. Furthermore, humanized mice are injected with a prostate cancer cell line into their hearts, followed by intraperitoneal injection of the humanized CBLN1 antibody ZH003. In vivo imaging of small animals is used to analyze the metastatic lesions, clarifying the potential use of the humanized CBLN1 antibody ZH003 in treating tumor metastases.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1) The recombinant CBLN1 protein of the present invention can be used as an immunogen to successfully immunize Balb / c mice and obtain the CBLN1 monoclonal antibody ZH-DT7792, which has the effect of specifically recognizing the neurocerebellar peptide CBLN1.

[0050] 2) The CBLN1 monoclonal antibody ZH-DT7792 of the present invention can be used to prepare drugs for treating breast cancer;

[0051] 3) The CBLN1-specific monoclonal antibody ZH-DT7792 of the present invention has the effect of functionally blocking CBLN1 in prostate cancer, and has the effect of alleviating the immunosuppressive effect of the tumor microenvironment in prostate cancer; and can be used to prepare drugs for treating castration-resistant prostate cancer and androgen receptor-dependent prostate cancer.

[0052] 4) The humanized antibody ZH003 for neurocerebellar peptide CBLN1 of the present invention has the effect of specifically recognizing neurocerebellar peptide CBLN1 and has a strong affinity for neurocerebellar peptide CBLN1.

[0053] 5) This invention provides the possibility of using CBLN1 as an effective target to improve the response rate of immunotherapy for prostate cancer and to inhibit tumor metastasis. Compared with the murine CBLN1 antibody, the humanized CBLN1 antibody ZH003 of this invention has a more significant function of slowing down the progression of prostate cancer and can significantly improve the response rate of prostate cancer to immunotherapy αPD1 antibody, thereby improving the treatment effect of immunosuppressive prostate cancer patients.

[0054] 6) The humanized antibody ZH003 of the neurocerebellar peptide CBLN1 of the present invention can inhibit the metastasis of prostate cancer, showing a new use in the treatment of tumor metastasis. Attached Figure Description

[0055] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0056] Figure 1 is a protein electrophoresis diagram of the CBLN1 monoclonal antibody ZH-DT7792 prepared in Example 1; where Lane M1 is SDS-PAGE Marker, Lane 1 is BSA, and Lanes 2 and 3 are the reducing and non-reducing ZH-DT7792 antibodies, respectively.

[0057] Figure 2 shows the protein electrophoresis diagram of the CBLN1 monoclonal antibody ZH-DT7792 recognizing CBLN1; the left band represents control cells, and the right band represents CBLN1 overexpressing cells;

[0058] Figure 3 shows the functional experiment of the CBLN1 monoclonal antibody ZH-DT7792 blocking the action of CBLN1; group 1 is the control solvent group, group 2 is the CBLN1 protein group, and group 3 is the co-treatment group of CBLN1 protein and ZH-DT7792 antibody.

[0059] Figure 4 shows a flow cytometry analysis of neutrophils induced by the 4T1 breast cancer cell line to express the CBLN1 receptor NRXN1; the left figure is the control group, and the right figure is the 4T1 conditioned medium treatment group.

[0060] Figure 5 shows the in vivo experiment of CBLN1 monoclonal antibody ZH-DT7792 in the treatment of breast cancer; the left figure is a tumor morphology diagram, and the right figure is a tumor weight statistics diagram.

[0061] Figure 6 shows the in vivo experiment of CBLN1 monoclonal antibody ZH-DT7792 regulating the immune microenvironment of prostate cancer; where a is the flow cytometry analysis of immunosuppressive tumor-associated neutrophils (TAN), b is the flow cytometry analysis statistics of TAN, c is the flow cytometry analysis of cytotoxic CD8 T cells, and d is the flow cytometry analysis statistics of CD8 T cells.

[0062] Figure 7 shows the in vivo experiments of CBLN1 monoclonal antibody ZH-DT7792 in the treatment of castration-resistant prostate cancer and androgen receptor-dependent prostate cancer; in a, the left image is a statistical chart of tumor weight in castration-resistant prostate cancer, and the right image is a tumor morphology chart; in b, the left image is a statistical chart of tumor weight in androgen receptor-dependent prostate cancer, and the right image is a tumor morphology chart.

[0063] Figure 8 shows the electrophoresis results of the quality detection of the humanized CBLN1 antibody ZH003 in Example 7;

[0064] Figure 9 shows the results of the specific recognition of CBLN1 protein by humanized antibody ZH003 and initial mouse antibody in Example 8;

[0065] Figure 10 shows the synergistic effect of the humanized CBLN1 antibody ZH003 and the mouse antibody with the immunotherapy PD1 antibody in Example 9; the left figure is a tumor morphology diagram and the right figure is a tumor weight statistics diagram.

[0066] Figure 11 shows the mouse experimental results of the inhibition of tumor metastasis by the humanized CBLN1 antibody ZH003 and the mouse antibody in Example 9. Detailed Implementation

[0067] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0068] Example 1: Construction of monoclonal antibodies

[0069] Step 1: Expression and purification of CBLN1 immunogen. Due to the conservation of CBLN1 protein across different species, the full-length sequence of Homo Sapiens CBLN1 protein (amino acid sequence shown in SEQ ID NO. 8) was used as the immunogen expression template for successful immunization in mice. An original signal peptide (amino acid sequence shown in SEQ ID NO. 9) was added to the N-terminus to facilitate expression in the HEK293 / CHO in vitro culture system. A tag protein His (amino acid sequence shown in SEQ ID NO. 10) was added to the C-terminus to obtain recombinant CBLN1 protein (amino acid sequence shown in SEQ ID NO. 11) for subsequent expression and purification. Codon optimization (the optimized gene sequence is shown in SEQ ID NO. 12) ensured stable and high-yield expression and purification of the CBLN1 immunogen.

[0070] Step Two: Using the SingleB monoclonal antibody rapid development technology, the process involved animal immunization, monoclonal antibody preparation, and final high-throughput expression and purification. Mice were immunized using the CBLN1 protein expressed in Step One as an immunogen. Successful immunization was confirmed through antigen quality control, immunogen emulsification, animal (mouse) immunization, and ELISA titer detection. Serum from successfully immunized mice was collected for B cell enrichment and screening. Positive supernatant strains were identified and then subjected to high-throughput BGE expression and positive clone screening. Using prostate cancer cell lines as a validation system, positive antibody strains that specifically recognize CBLN1 and functionally block CBLN1 were selected. The gene sequence of the positive monoclonal antibody was sequenced, and after high-throughput expression and purification, the CBLN1 monoclonal antibody ZH-DT7792 was obtained. The quality of the obtained CBLN1 monoclonal antibody ZH-DT7792 was assessed using Western blotting, and the protein electrophoresis results are shown in Figure 1.

[0071] The CBLN1 monoclonal antibody ZH-DT7792 contains a light chain variable region and a heavy chain variable region. The light chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose respective amino acid sequences are shown below:

[0072] CDR1: QSLVHSNGNTY(SEQ ID NO.1);

[0073] CDR2: KVS;

[0074] CDR3: SQNTHVPWT (SEQ ID NO. 2).

[0075] The amino acid sequence of the light chain variable region (SEQ ID NO.6) is as follows:

[0076] The heavy chain variable region includes three complementarity-determining regions, CDR1, CDR2, and CDR3, whose amino acid sequences are shown below:

[0077] CDR1:GFNIKDYF(SEQ ID NO.3);

[0078] CDR2: IDPANGNT (SEQ ID NO.4);

[0079] CDR3: NAFHYGHYGRGYAVDY (SEQ ID NO. 5).

[0080] The amino acid sequence of the heavy chain variable region (SEQ ID NO.7) is as follows:

[0081] Example 2: Identification of CBLN1 monoclonal antibody ZH-DT7792

[0082] 2.1 Western blotting to identify CBLN1 protein recognized by ZH-DT7792

[0083] 1 ml of protein electrophoresis loading buffer was used to dilute 1.5 μg of CBLN1 protein and 293T cells overexpressing CBLN1. Western blotting analysis was performed using ZH-DT7792 purified antibody as the primary antibody, as shown in Figure 2. The left band represents the control 293T cells, and the right band represents the CBLN1 overexpressing 293T cells. The results show that ZH-DT7792 can specifically recognize CBLN1 protein.

[0084] 2.2 Fluo-4 calcium ion labeling assay to identify the functional blocking ability of ZH-DT7792

[0085] LNCaP prostate tumor cells in good condition were selected for passage, and after cell counting, cells were passaged at a rate of 5 × 10⁻⁶ cells / year. 5 Cells were seeded into 6-well plates at a density of cells per well. Each well was prepared according to the experimental group settings, with culture medium added as follows: Vehicle (1 μg / ml BSA), 1 μg / ml CBLN1 protein, and 1 μg / ml CBLN1 protein + 1 μg / ml ZH-DT7792 antibody. Cells were collected 10 minutes after drug stimulation. Intracellular calcium ion flux was labeled using the Fluo-4 calcium ion fluorescent probe according to the manufacturer's instructions. Flow cytometry was used for detection and statistical analysis. As shown in Figure 3, group 1 was the control solvent group, group 2 was the CBLN1 protein group, and group 3 was the group co-treated with CBLN1 protein and ZH-DT7792 antibody. The intracellular calcium ion labeling intensity was significantly reduced in group 3, indicating that ZH-DT7792 can antagonize the calcium ion influx induced by CBLN1 protein in prostate tumor cells.

[0086] Example 3: Breast cancer cell line 4T1 induces bone marrow-derived neutrophils to express CBLN1 receptor NRXN1.

[0087] Bone marrow cells from six-week-old C57 mice were cultured in vitro and then conditioned in 4T1 breast cancer cell line medium. Flow cytometry was used to detect the expression level of CBLN1 receptor NRXN1 on bone marrow-derived neutrophils. As shown in Figure 4, the left panel represents the control group, and the right panel represents the 4T1 conditioned medium treatment group. The results show that, compared to the control group (DMEM), the 4T1 conditioned medium induced bone marrow-derived neutrophils to express more CBLN1 receptor NRXN1.

[0088] Example 4: Application of CBLN1 monoclonal antibody ZH-DT7792 in the preparation of drugs for treating breast cancer

[0089] 4T1 breast cancer cell line was orthotopically transplanted into the mammary glands of BALB / c mice. Three days after surgery, the mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of the drug administration were 10 mg / kg, twice a week, for 3 weeks, during which the health status of the mice was regularly observed. Four weeks after inoculation, the mice were euthanized, and tumor tissue was collected. The therapeutic effect on breast cancer was evaluated by observing the tumor size. As shown in Figure 5, the left figure is a tumor morphology diagram, and the right figure is a tumor weight statistical diagram. It can be seen that the tumor weight of breast cancer in the IM3D5 treatment group was significantly reduced compared with the control group, indicating that ZH-DT7792 can slow down the progression of breast cancer.

[0090] Example 5: The therapeutic use of CBLN1 monoclonal antibody ZH-DT7792 in alleviating immunosuppression in the prostate cancer microenvironment.

[0091] Prostate cancer organoids from mice (PB-cre; Ptenf / f; Mychi) were orthotopically transplanted into the prostates of C57 / B6J mice. Three days after surgery, mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of the antibody treatment were 10 mg / kg, twice a week, for three weeks, during which the health status of the mice was regularly observed. Four weeks after inoculation, the mice were euthanized, and tumor tissue was collected. After digestion into single cells, tumor-associated neutrophils were labeled with CD45+CD11b+Ly6G+ (a classic surface marker combination for neutrophils), and cytotoxic CD8 T cells were labeled with CD45+CD3+CD8+ (a classic surface marker combination for cytotoxic CD8 T cells). The immunosuppressive nature of the tumor microenvironment was assessed by flow cytometry. As shown in Figure 6, a is the flow cytometry analysis plot of immunosuppressive tumor-associated neutrophils (TAN), b is the flow cytometry analysis statistical plot of TAN, c is the flow cytometry analysis plot of cytotoxic CD8 T cells, and d is the flow cytometry analysis statistical plot of CD8 T cells. It is evident that the proportion of immunosuppressive tumor-associated neutrophils decreased and the proportion of cytotoxic CD8 T cells increased in the IM3D5 treatment group, indicating that ZH-DT7792 can alleviate the immunosuppressive effect of the prostate cancer tumor microenvironment.

[0092] Example 6: Therapeutic use of CBLN1 monoclonal antibody ZH-DT7792 in castration-resistant prostate cancer and androgen receptor-dependent prostate cancer.

[0093] Clinically, androgen deprivation therapy and / or second-generation androgen inhibitors (ARSIs) are currently used to treat prostate cancer. 80% of prostate cancer patients (i.e., localized prostate cancer) respond well to this treatment regimen. However, castration-resistant prostate cancer patients do not benefit from conventional prostate cancer treatment regimens, suggesting that castration-resistant prostate cancer (CRPC) has a different pathogenesis and clinical treatment regimen than localized prostate cancer.

[0094] Organoids from castration-resistant prostate cancer CRPCs (Probasin-cre;Trp53f / f;Ptenf / f) mice were orthotopically transplanted into the prostates of C57 / B6J mice. Three days after surgery, mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of the antibody were 10 mg / kg, twice a week, for three weeks, during which the health status of the mice was regularly observed. Four weeks after inoculation, the mice were euthanized, and tumor tissue was collected. The therapeutic effect on castration-resistant prostate cancer was evaluated by observing tumor size. As shown in Figure 7a, the prostate cancer tumor weight in the IM3D5 treatment group was significantly reduced compared to the control group, indicating that ZH-DT7792 can slow the progression of castration-resistant prostate cancer.

[0095] Furthermore, some subtypes of castration-resistant prostate cancer exhibit androgen receptor signaling pathway reactivation during recurrence, defined as androgen receptor-dependent CRPC. Antibody treatment was evaluated by inoculating castrated mouse models with androgen receptor-dependent CRPC organoids of PB-cre;Ptenf / f;Mychi, following the same procedures as above. Tumor size was observed to assess the therapeutic effect on androgen receptor-dependent CRPC. As shown in Figure 7b, the prostate cancer tumor weight in the IM3D5 treatment group was significantly reduced compared to the control group, indicating that ZH-DT7792 can slow the progression of androgen receptor-dependent CRPC.

[0096] Example 7: Preparation of CBLN1 humanized antibody ZH003

[0097] 1. Initial murine antibody

[0098] Using the CBLN1 monoclonal antibody ZH-DT7792 as the initial murine antibody, the amino acid sequence of its heavy chain variable region is shown in SEQ ID NO.7:

[0099] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.6:

[0100] The heavy and light chain sequences were input into AlphaFold2 to construct the antibody structure in multimer mode. The antibody model was then input into GROMACS for energy minimization, and a new antibody structure was output. The amino acid sequences in the antibody structure model were renumbered using the Kabat nomenclature scheme.

[0101] 2. Select human antibody Germline gene

[0102] The variable region sequences of the heavy and light chains from mouse sources were distinguished as FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 according to the Kabat nomenclature scheme.

[0103] Antibody Germline templates were obtained using the intact variable region, FR1+FR2+FR3+FR4, and CDR1+CDR2+CDR3, respectively. Three sets of antibody Germline templates were obtained for each of the heavy chain and light chain variable regions.

[0104] Mouse antibody heavy chain FR1+FR2+FR3+FR4 sequence (SEQ ID NO.13):

[0105] Mouse antibody heavy chain CDR1+CDR2+CDR3 sequence (SEQ ID NO.14):

[0106] Mouse antibody light chain FR1+FR2+FR3+FR4 sequence (SEQ ID NO.15):

[0107] Mouse antibody light chain CDR1+CDR2+CDR3 sequence (SEQ ID NO.16):

[0108] 3. CDR porting

[0109] Under the same Kabat nomenclature scheme, the CDR of the murine antibody was transplanted to the CDR position of the corresponding human template Germline to construct a basic humanization framework.

[0110] 4. Generate a reverse mutation sequence

[0111] Based on the key amino acid positions reported in the paper Foote J, Winter G. Antibody framework residues affecting the conformation of the hypervariable loops. J Mol Biol. 1992 Mar 20; 224(2):487-99. doi:10.1016 / 0022-2836(92)91010-m.PMID:1560463, some mouse-derived amino acids were selectively retained to maintain the stability and binding activity of the CDR conformation, thus forming an optimized humanized framework.

[0112] 5. Energy calculation and sequence selection

[0113] All heavy and light chain combinations generated from the above two steps were systematically evaluated and compared with the initial murine CBLN1 monoclonal antibody ZH-DT7792 structure. First, the energy change was assessed based on each differential amino acid. Amino acids meeting the energy change requirements were selected and their combinations optimized, ultimately choosing the sequence corresponding to the most stable structure. A balance was struck between energy and humanization level to finally select candidate sequences.

[0114] The final selected humanized CBLN1 monoclonal antibody ZH003 sequence is shown below:

[0115] (1) Heavy chain

[0116] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.17:

[0117] The three complementary determinant regions are as follows:

[0118] CDR1: GFNIKDYF(SEQ ID NO.3),

[0119] CDR2: IDPANGNT(SEQ ID NO.4),

[0120] CDR3:NAFHYGHYGRGYAVDY(SEQ ID NO.5);

[0121] The four skeletal regions are as follows:

[0122] FR1: QVQLVQSGAEVKKPGASVKVSCKAS(SEQ ID NO.18),

[0123] FR2: IHWVRQAPGQGLEWIGW (SEQ ID NO.19),

[0124] FR3:KYDPKFQGRATITADTSASTAYMELSSLRSEDTAVYYC(SEQ ID NO.20),

[0125] FR4: WGQGTLVTVSS(SEQ ID NO.21);

[0126] The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.22:

[0127] The heavy chain constant region is the heavy chain constant region of human IgG1 antibody, and its amino acid sequence is shown in SEQ ID NO.23:

[0128] The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.24:

[0129] (2) Light chain

[0130] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.25:

[0131] The three complementary determinant regions are as follows:

[0132] CDR1: QSLVHSNGNTY(SEQ ID NO.1),

[0133] CDR2: KVS

[0134] CDR3: SQNTHVPWT(SEQ ID NO.2);

[0135] The four skeletal regions are as follows:

[0136] FR1: DVVMTQSPLSLPVTLGQQASISCRSS (SEQ ID NO.26),

[0137] FR2: LHWFQQRPGQSPRLLIY(SEQ ID NO.27),

[0138] FR3: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC (SEQ ID NO. 28),

[0139] FR4: FGQGTKVEIK(SEQ ID NO.29);

[0140] The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.30:

[0141] The constant region of the light chain is the constant region of the human κ-type light chain, and its amino acid sequence is shown in SEQ ID NO.31:

[0142] The nucleotide sequence of the light chain constant region is shown in SEQ ID NO.32:

[0143] 6. Express and purify antibodies

[0144] (1) Construction of humanized antibody expression vector:

[0145] The light and heavy chain amino acid sequences of the humanized antibody were optimized using the MaxCodon™ Optimization Program (V13) codon optimization software. The antibody light and heavy chain genes were synthesized as a whole gene and inserted into the expression vector pcDNA3.4 by double enzyme digestion (enzyme sites: EcoRI / BamHI). The successful construction of the expression vector was confirmed by enzyme digestion and sequencing. The vector was then transformed into the JM108 cloning strain, and plasmids were extracted.

[0146] (2) Transformation and culture of expression vectors:

[0147] Heavy and light chain expression plasmids containing the humanized antibody gene were added to prepared JM108 competent cells. After heat shock transformation, the transformed competent cells were added to LB liquid medium and incubated at 37°C with shaking for approximately 30 min. The cultured competent cells were then removed, and a portion of the suspension was spread onto LB agar plates containing ampicillin, and incubated overnight at 37°C. Single clones were picked from fresh culture plates and cultured in LB liquid medium at 37°C and 200 rpm for 8 h. The cultured cells were then inoculated at a 1 / 500 ratio into LB liquid medium and incubated overnight at 37°C. The cultured bacterial suspension was collected, centrifuged, and the supernatant was discarded for plasmid extraction to obtain the target plasmid.

[0148] (3) Antibody purification and identification

[0149] Transfected 293 cells in suspension culture. Cell seeding density was 2.0 × 10⁻⁶. 6 Cells / mL were cultured in suspension in an incubator. On the day of transfection, cells grew to 3.0-5.0 × 10⁶ cells / mL. 6 The cells / mL were diluted with fresh 293 cell culture medium to a density of 3.0 × 10⁻⁶ cells / mL. 6 cells / mL.

[0150] Perform transfection. Add DNA (light chain to heavy chain plasmid ratio of 1:1) and polyethyleneimine transfection reagent, mix well, and incubate at 37°C. Add the DNA-transfection reagent mixture to the cells to be transfected, and incubate at 37°C with 110 rpm and 8% CO2.

[0151] 24 hours after transfection, 5% feed was added for continued culture. Approximately 4-6 days post-transfection, the cell culture was harvested, centrifuged, and the supernatant or cells were collected. The supernatant was filtered through a 0.22 μm filter, dialyzed to buffer at 4°C, and then purified using a Protein A column. Samples with relatively high purity were collected and dialyzed into PBS buffer solution containing 5% Glycerol at 10 mM and pH 7.4. The samples were then aliquoted and frozen at -80°C.

[0152] The quality of the purified antibody was detected by SDS-PAGE gel electrophoresis, and the results are shown in Figure 8. In Figure 8, Lane M is the SDS-PAGE marker, Lane 5 is BSA, and Lanes 6 and 7 are the reduced and non-reduced humanized ZH003 antibodies, respectively. The results indicate that the ZH003 antibody has high purity.

[0153] Example 8: Identification of CBLN1 humanized antibody ZH003

[0154] Recombinant human CBLN1 protein: Using the full-length sequence of human CBLN1 protein as the target sequence, a signal peptide and a C-His tag were added. HEK293 / CHO was used as the expression host cell. Through codon optimization and gene synthesis, the recombinant CBLN1 protein was expressed and purified. Its amino acid sequence is shown in SEQ ID NO.11.

[0155] Dilute the above-mentioned human CBLN1 recombinant protein to 2 μg / ml with coating buffer (carbonate buffer, pH 9.6) and coat overnight. Discard the coating solution, wash the plate three times with PBST, 200 μL each time, and blot dry. Add 200 μL of 5% skim milk powder to each well as blocking solution and incubate at 37°C for 1 h.

[0156] Add 100 μL of sample to each well (ZH003 purified antibody and initial mouse antibody were diluted according to a concentration gradient; initial mouse antibody ZH-DT7792 was used as a positive control, and PBS was used as a negative control), with two replicates, and incubate at 37°C for 1 h. After washing the plate three times, add 100 μL of mAb-D-HRP working solution (1:2000, pre-diluted with blocking buffer) to each well and incubate at 37°C for 1 h. After washing the plate five times, add 100 μL of TMB substrate solution to each well and react at room temperature in the dark for 7-10 min. Add 50 μL of 0.64 M H2SO4 stop solution to each well and immediately read the absorbance at 450 nm.

[0157] The results, as shown in Figure 9, indicate that ZH003 has a high affinity for the CBLN1 protein.

[0158] Example 9: Therapeutic applications of ZH003 antibody in slowing prostate cancer metastasis and improving immunotherapy response rate.

[0159] 1. Human prostate cancer cells (PC3) were subcutaneously inoculated into humanized mice (a highly immunodeficient mouse model of functional human hematopoietic-immune system reconstructed in vivo, which can simulate human immune response and disease mechanisms at the in vivo level). When the subcutaneous tumor was visibly palpable (approximately 10 days after inoculation), the mice were randomly divided into a control IgG group, an αPD1 immunotherapy group, a mouse antibody ZH-DT7792 + αPD1 treatment group, and a ZH003 antibody + PD1 antibody treatment group. The dosage and frequency of administration were 10 mg / kg, twice a week, for 3 weeks, during which the health status of the mice was observed regularly. Four weeks after inoculation, the mice were euthanized, and tumor tissue was collected for evaluation by observing tumor size and mass.

[0160] The results are shown in Figure 10, where the left figure shows the tumor morphology and the right figure shows the tumor weight statistics. Figure 10 indicates that in a humanized mouse tumor-bearing model, the humanized antibody ZH003 exhibited a more significant function in slowing the progression of immunosuppressive prostate cancer than the mouse antibody, and could significantly improve the response rate of immunosuppressive prostate cancer to the immunotherapy αPD1 antibody. Clinically, prostate cancer patients respond poorly to immunotherapy αPD1, often showing almost no response. However, the humanized CBLN1 antibody ZH003 of this invention can synergistically interact with PD1, improving the treatment effect for immunosuppressive prostate cancer patients.

[0161] 2. Human prostate cancer cells (PC3-luciferase) were injected cardiacally into humanized mice. One week after inoculation, the mice were randomly divided into a control IgG group, a mouse antibody ZH-DT7792 treatment group, and a ZH003 antibody treatment group. The dosage and frequency of administration were 10 mg / kg, twice a week, for 3 weeks, during which the health status of the mice was observed regularly. Three weeks after inoculation, in vivo imaging was performed on the mice, and the treatment effect was evaluated by the intensity of the imaging signal. The results are shown in Figure 11, indicating that ZH003 can slow down and inhibit the metastasis of prostate cancer in a humanized mouse metastasis model.

[0162] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A CBLN1 monoclonal antibody, characterized in that, Includes variable regions of heavy chains and variable regions of light chains. The three complementary determinants of the variable region of the light chain are: CDR1: QSLVHSNGNTY CDR2: KVS CDR3: SQNTHVPWT; The three complementary determinants of the heavy chain variable region are: CDR1: GFNIKDYF CDR2: IDPANGNT CDR3: NAFHYGHYGRGYAVDY.

2. The CBLN1 monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the CBLN1 monoclonal antibody is shown in SEQ ID NO. 6; And / or, the amino acid sequence of the heavy chain variable region of the CBLN1 monoclonal antibody is shown in SEQ ID NO.

7.

3. The use of the CBLN1 monoclonal antibody as described in claim 1 or 2 in the preparation of a drug for treating breast cancer.

4. The use of the CBLN1 monoclonal antibody as described in claim 1 or 2 in the preparation of a drug for treating prostate cancer.

5. The application according to claim 4, characterized in that, The prostate cancer mentioned includes castration-resistant prostate cancer.

6. The application according to claim 4, characterized in that, The prostate cancer mentioned includes androgen receptor-dependent prostate cancer.

7. A humanized CBLN1 antibody, characterized in that, Including heavy chains and light chains; The heavy chain includes a heavy chain variable region, the amino acid sequence of which is shown in SEQ ID NO.17; The light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO.

25.

8. The CBLN1 humanized antibody according to claim 7, characterized in that, The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.

22.

9. The CBLN1 humanized antibody according to claim 7, characterized in that, The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.

30.

10. The CBLN1 humanized antibody according to claim 7, characterized in that, The heavy chain also includes a heavy chain constant region, the amino acid sequence of which is shown in SEQ ID NO.

23.

11. The CBLN1 humanized antibody according to claim 10, characterized in that, The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO.

24.

12. The CBLN1 humanized antibody according to claim 7, characterized in that, The light chain also includes a light chain constant region, the amino acid sequence of which is shown in SEQ ID NO.

31.

13. The CBLN1 humanized antibody according to claim 12, characterized in that, The nucleotide sequence of the constant region of the light chain is shown in SEQ ID NO.

32.

14. Use of a humanized CBLN1 antibody as described in any one of claims 7-14 in the preparation of a medicament for inhibiting prostate cancer metastasis.

15. An antibody composition comprising the humanized CBLN1 antibody as described in any one of claims 7-14, and a PD1 antibody.

16. Use of the antibody composition of claim 15 in the preparation of a medicament for treating immunosuppressive prostate cancer.

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