A CBLN1 monoclonal antibody and its application

Abstract

The present invention discloses a CBLN1 monoclonal antibody and its application; the CBLN1 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises three complementary determining regions CDR1, CDR2, and CDR3, the amino acid sequence of CDR1 is as shown in SEQ ID NO.1, the amino acid sequence of CDR2 is KVS, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.2; the amino acid sequences of the three complementary determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are as shown in SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5, respectively. The present invention also discloses a method for preparing the monoclonal antibody and its application in preparing drugs for treating breast cancer and prostate cancer, especially castration-resistant prostate cancer and androgen receptor-dependent prostate cancer. The CBLN1-specific monoclonal antibody can functionally block CBLN1 and improve the inhibitory nature of the tumor immune microenvironment.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, in particular to a CBLN1 monoclonal antibody and application thereof. Background Art

[0002] For patients with early-stage prostate cancer, active monitoring, radical resection and adjuvant radiotherapy are usually used clinically. However, for mid-to-late and metastatic prostate cancer, due to its complex and diverse pathogenesis, there is still no optimal diagnosis and treatment plan. Androgen deprivation therapy targeting the AR signaling pathway is the first-line option for the clinical treatment of prostate cancer, but most patients will relapse and become castration-resistant prostate cancer CRPC after receiving this treatment. In addition, the immunosuppressive tumor microenvironment of prostate cancer makes it impossible for immune checkpoint therapy to achieve significant therapeutic effects in prostate cancer patients.

[0003] As for breast cancer, most clinical anticancer drugs are still targeted therapies against tumor cells themselves, but many patients still eventually relapse and develop metastatic breast cancer. Immunotherapy has entered clinical practice for breast cancer patients as early as 1998, and these drugs have improved the overall survival of patients with early or advanced breast cancer. However, after treatment, tumor tissues usually develop intrinsic or acquired drug resistance mechanisms, eventually leading to disease progression, and there is no effective subsequent treatment option. Therefore, it is urgent to develop new and effective targeted therapies for prostate cancer and breast cancer.

[0004] Studies have shown that the peripheral autonomic nervous system plays an important role in tissue growth, homeostasis maintenance, and organ function. Since tumor tissues also need key regulatory signals during organ development to maintain growth and survival, studies have shown that targeting the intratumoral nervous system can effectively control tumor progression. Both the breast and prostate are organs rich in nerve infiltration, 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 occurrence and development of prostate cancer. It is particularly important to note that the density of infiltrated nerves in tumor tissues is positively correlated with the malignancy of the disease and is related to the PSA expression level of tumor tissues, indicating the potential role of the nervous system in the progression of androgen receptor-dependent CRPC prostate cancer and castration-resistant prostate cancer CRPC. Neurochemical signaling is the main way in which the peripheral nervous system exerts its regulatory role. In addition to classical neurotransmitters, there are currently no studies exploring the possible role of neurosecretory proteins in prostate cancer and breast cancer and related targeted therapies.

[0005] CBLN1 is a highly conserved neurosecretory protein expressed and secreted by cerebellar granule cells. The lack of expression of this protein directly leads to defects in the construction of neural networks under physiological conditions. Most of the CBLN1 antibodies currently on the market are polyclonal recognition antibodies that can only recognize CBLN1 protein and do not have the effect of targeted blocking of CBLN1 protein function in vivo or in vitro. Summary of the invention

[0006] The purpose of the present invention is to provide a CBLN1 monoclonal antibody and its application in view of the above-mentioned deficiencies in the prior art, and to provide a use of the CBLN1 antibody in the preparation of a drug for treating breast cancer and prostate cancer.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] 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,

[0009] The three complementarity determining regions of the light chain variable region of ZH-DT7792 are:

[0010] CDR1: QSLVHS NGNTY (SEQ ID NO.1)

[0011] CDR2:KVS

[0012] CDR3: SQNTHVPWT (SEQ ID NO.2)

[0013] The three complementarity determining regions of the ZH-DT7792 heavy chain variable region are:

[0014] CDR1:GFNIKDYF (SEQ ID NO.3)

[0015] CDR2: IDPANGNT (SEQ ID NO.4)

[0016] CDR3: NAFHYGHYGRGYAVDY (SEQ ID NO. 5).

[0017] 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;

[0018] 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.

[0019] In a second aspect, 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 CBLN1 recombinant protein as an immunogen; the CBLN1 recombinant protein is obtained by using the full-length sequence of human CBLN1 protein as the target sequence, adding a signal peptide and a C-His tag, using HEK293 / CHO as the expression host cell, through codon optimization and gene synthesis, expression and purification.

[0020] As some specific embodiments of the present invention, the amino acid sequence of the human CBLN1 protein is shown as SEQ ID NO.8, the amino acid sequence of the signal peptide is shown as SEQ ID NO.9, and the amino acid sequence of the C-His tag is shown as SEQ ID NO.10.

[0021] As some specific embodiments of the present invention, the amino acid sequence of the CBLN1 recombinant protein is shown as SEQ ID NO.11; the gene sequence of the CBLN1 recombinant protein after codon optimization is shown as SEQ ID NO.12.

[0022] As some specific embodiments of the present invention, the preparation method of the CBLN1 monoclonal antibody ZH-DT7792 specifically comprises the following steps:

[0023] Step 1: Select Balb / c mice as immunization objects, use CBLN1 recombinant protein as immunogen, collect a small amount of blood from mice after multiple rounds of immunization, and determine the serum titer by indirect ELISA; when the serum titer reaches the B cell isolation standard, take a certain amount of immunogen to perform the final impact immunization on the mice;

[0024] Step 2: Collect peripheral blood of target mice to sort memory B cells, obtain antibody heavy and light chain gene fragments by single-cell gene amplification technology and construct expression plasmids, collect supernatant after transient expression, and detect positive cell supernatant by indirect ELISA;

[0025] Step 3: Sequence the antibody heavy and light chain plasmids corresponding to the positive cell supernatant, construct the expression plasmid and transiently express it, then collect and purify it to obtain the CBLN1 recombinant protein specific monoclonal antibody ZH-DT7792.

[0026] In a third aspect, the present invention provides a use of a neurocerebellar peptide CBLN1 monoclonal antibody ZH-DT7792 in the preparation of a drug for treating breast cancer.

[0027] The inventors found that the breast cancer cell line 4T1 can induce tumor-associated neutrophils to express the CBLN1 receptor NRXN1. By intraperitoneally injecting CBLN1 monoclonal antibodies into 4T1 breast cancer tumor-bearing mice, the tumor size and mass were analyzed to clarify the possible use of CBLN1 monoclonal antibodies in the treatment of breast cancer.

[0028] In a fourth aspect, the present invention provides a use of a neurocerebellar peptide CBLN1 specific monoclonal antibody ZH-DT7792 in the preparation of a drug for treating prostate cancer.

[0029] As some specific embodiments of the present invention, the prostate cancer includes castration-resistant prostate cancer. By intraperitoneally injecting CBLN1 monoclonal antibody into Probasin-cre;Trp53f / f;Ptenf / f castration-resistant prostate cancer tumor-bearing mice, analyzing the tumor size and mass, the possible use of CBLN1 monoclonal antibody in the treatment of castration-resistant prostate cancer is clarified.

[0030] As some specific embodiments of the present invention, the prostate cancer includes androgen receptor-dependent prostate cancer. By intraperitoneally injecting CBLN1 monoclonal antibody into Probasin-cre; Ptenf / f; Mychi androgen receptor-dependent prostate cancer-bearing mice, analyzing the tumor size and mass, the possible use of CBLN1 monoclonal antibody in the treatment of androgen receptor-dependent prostate cancer is clarified.

[0031] The present invention provides an application of CBLN1 monoclonal antibody ZH-DT7792 in alleviating the inhibitory effect of the immune microenvironment of prostate cancer. By intraperitoneally injecting CBLN1 monoclonal antibody into Probasin-cre;Ptenf / f;Mychi orthotopic prostate cancer-bearing mice, the main immune cell composition in the immune microenvironment is analyzed, and the possible application of CBLN1 monoclonal antibody in alleviating the inhibitory effect of the immune microenvironment of prostate cancer is clarified.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The CBLN1 recombinant 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;

[0034] 2) The CBLN1 monoclonal antibody ZH-DT7792 of the present invention can be used to prepare a drug for treating breast cancer;

[0035] 3) The CBLN1-specific monoclonal antibody ZH-DT7792 of the present invention has the effect of functionally blocking CBLN1 in prostate cancer and alleviating the immunosuppression of the tumor microenvironment of prostate cancer; and can be used to prepare drugs for treating castration-resistant prostate cancer and androgen receptor-dependent prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0037] Figure 1 The protein electrophoresis diagram of the CBLN1 monoclonal antibody ZH-DT7792 prepared in Example 1; wherein Lane M1 is SDS-PAGE Marker, Lane 1 is BSA, and Lanes 2 and 3 are respectively reduced and non-reduced ZH-DT7792 antibodies;

[0038] Figure 2 This is a protein electrophoresis diagram for detecting the recognition of CBLN1 by the CBLN1 monoclonal antibody ZH-DT7792; the left band is the control cell, and the right band is the CBLN1 overexpressing cell;

[0039] Figure 3 This is a functional experiment in which the CBLN1 monoclonal antibody ZH-DT7792 blocks the effect of CBLN1; Group 1 is the control solvent group, Group 2 is the CBLN1 protein group, and Group 3 is the CBLN1 protein and ZH-DT7792 antibody co-treatment group;

[0040] Figure 4 This is a cell flow cytometry analysis of breast cancer cell line 4T1 inducing neutrophils to express CBLN1 receptor NRXN1; the left picture is the control group, and the right picture is the 4T1 conditioned medium treatment group;

[0041] Figure 5 This is an in vivo experiment of using CBLN1 monoclonal antibody ZH-DT7792 to treat breast cancer. The left picture shows the tumor morphology, and the right picture shows the tumor weight statistics.

[0042] Figure 6 This is an in vivo experiment of CBLN1 monoclonal antibody ZH-DT7792 regulating the immune microenvironment of prostate cancer; wherein, a is a flow cytometry analysis of immunosuppressive tumor-associated neutrophils TAN, b is a flow cytometry statistical graph of TAN, c is a flow cytometry analysis of cell-killing CD8 T cells, and d is a flow cytometry statistical graph of CD8 T cells;

[0043] Figure 7This is an in vivo experiment of using CBLN1 monoclonal antibody ZH-DT7792 to treat castration-resistant prostate cancer and androgen receptor-dependent prostate cancer; the left picture of a is a statistical graph of tumor weight for castration-resistant prostate cancer, and the right picture of a is a tumor morphology graph; the left picture of b is a statistical graph of tumor weight for androgen receptor-dependent prostate cancer, and the right picture of b is a tumor morphology graph. DETAILED DESCRIPTION

[0044] The present invention is described in detail below in conjunction with specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, several variations and improvements may be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0045] Example 1: Construction of monoclonal antibodies

[0046] Step 1: Expression and purification of CBLN1 immunogen. Since CBLN1 protein is somewhat conserved among different species, in order to successfully immunize mice, Homo Sapiens The full-length sequence of CBLN1 protein (amino acid sequence as shown in SEQ ID NO.8) is used as an immunogen expression template. The original signal peptide (amino acid sequence as shown in SEQ ID NO.9) is added to the N-terminus for smooth expression in the HEK293 / CHO in vitro culture system, and the tag protein His (amino acid sequence as shown in SEQ ID NO.10) is added to the C-terminus to obtain the recombinant CBLN1 protein (amino acid sequence as shown in SEQ ID NO.11) for subsequent expression and purification. Stable and high-yield expression and purification of CBLN1 immunogens are achieved through codon optimization (the gene sequence after codon optimization is shown in SEQ ID NO.12).

[0047] Step 2: Apply SingleB monoclonal antibody rapid development technology, through animal immunization, monoclonal antibody preparation and final high-throughput expression and purification. Use the CBLN1 protein expressed in step 1 as the immunogen to immunize mice, and determine the successful immunization of mice through antigen quality control, immunogen emulsification, animal (mouse) immunization and ELISA titer detection. Collect the serum of successfully immunized mice for B cell enrichment screening, and after determining the positive supernatant strain, use BGE high-throughput expression and positive clone screening. Using the prostate cancer cell line as the verification system, select the positive antibody strain that can specifically recognize CBLN1 and has the effect of functionally blocking CBLN1, sequence the positive monoclonal antibody gene sequence, and obtain the CBLN1 monoclonal antibody ZH-DT7792 after high-throughput expression and purification. Western bloting was used to identify the quality of the obtained CBLN1 monoclonal antibody ZH-DT7792, and the protein electrophoresis results are as follows: Figure 1 shown.

[0048] The CBLN1 monoclonal antibody ZH-DT7792 comprises a light chain variable region and a heavy chain variable region. The light chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0049] CDR1: QSLVHS NGNTY (SEQ ID NO.1);

[0050] CDR2:KVS;

[0051] CDR3: SQNTHVPWT (SEQ ID NO. 2).

[0052] The amino acid sequence of the light chain variable region (SEQ ID NO.6) is:

[0053] DVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSSEGTDFTLKISRVEAEDLGVYFCSQNTHVPWTFGSGTKLEIK.

[0054] The heavy chain variable region includes three complementary determining regions CDR1, CDR2 and CDR3, and their respective amino acid sequences are shown below:

[0055] CDR1:GFNIKDYF (SEQ ID NO.3);

[0056] CDR2: IDPANGNT (SEQ ID NO.4);

[0057] CDR3: NAFHYGHYGRGYAVDY (SEQ ID NO. 5).

[0058] The amino acid sequence of the heavy chain variable region (SEQ ID NO.7) is:

[0059] EVQLQQSGAEVVRSGASVELSCTASGFNIKDYFMHWVKQRPEQGLEWIGWIDPANGNTKYAPKFQGKATMTADTSSNTAYLQLSSLTSEDTAVYYCNAFHYGHYGRGYAVDYWGQGISVTVSS.

[0060] Example 2 Identification of CBLN1 monoclonal antibody ZH-DT7792

[0061] 2.1 Western blotting to identify ZH-DT7792 to recognize CBLN1 protein

[0062] Take 1 ml of protein electrophoresis loading buffer to dilute 1.5 μg of CBLN1 protein and 293T cells overexpressing CBLN1, and perform Western blotting analysis using ZH-DT7792 purified antibody as the primary antibody. Figure 2 As shown, the left band is the control 293T cells, and the right band is the 293T cells overexpressing CBLN1. The results show that ZH-DT7792 can specifically recognize CBLN1 protein.

[0063] 2.2 Fluo-4 calcium ion labeling experiment to identify the functional blocking ability of ZH-DT7792

[0064] Prostate tumor cells LNCaP cells with better cell status were selected for subculture. After cell counting, the cells were counted and the number of cells was calculated as 5×10 5 Cells were seeded into 6-well plates at a cell density of 100 cells / well, and culture medium was added to each well according to the experimental group settings, namely Vehicle (1μg / ml BSA), 1μg / ml CBLN1 protein, and 1μg / ml CBLN1 protein + 1μg / ml ZH-DT7792 antibody. Cells were collected 10 minutes after drug stimulation, and intracellular calcium ion flow was labeled with Fluo-4 calcium ion fluorescent probe according to the instructions, and flow cytometry was performed for statistical analysis. Figure 3 As shown, Group 1 is the control solvent group, Group 2 is the CBLN1 protein group, and Group 3 is the CBLN1 protein and ZH-DT7792 antibody co-treatment group; the intracellular calcium flux labeling intensity in Group 3 was significantly reduced, and the results showed that ZH-DT7792 can antagonize the calcium flux influx of prostate tumor cells caused by CBLN1 protein.

[0065] Example 3: Breast cancer cell line 4T1 induces bone marrow-derived neutrophils to express the CBLN1 receptor NRXN1

[0066] Bone marrow cells from six-week-old C57 mice were cultured in vitro and then cultured with conditioned medium from the breast cancer cell line 4T1. Flow cytometry was used to detect the expression level of CBLN1 receptor NRXN1 on bone marrow-derived neutrophils. Figure 4 As shown, the left picture is the control group, and the right picture is the 4T1 conditioned medium treatment group. The results show that compared with the control group DMEM, the conditioned medium of the breast cancer cell line 4T1 can induce bone marrow-derived neutrophils to express more CBLN1 receptor NRXN1.

[0067] Example 4: Application of CBLN1 monoclonal antibody ZH-DT7792 in the preparation of drugs for treating breast cancer

[0068] The breast cancer cell line 4T1 was orthotopically transplanted into the mammary gland of BALB / c mice. Three days after the operation, the mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of administration were: 10 mg / kg, twice a week for 3 weeks. The health status of the mice was regularly observed during this period. The mice were euthanized 4 weeks after inoculation, and the tumor tissues were taken to evaluate the effect of breast cancer treatment by observing the tumor size. Figure 5 As shown, the left picture is the tumor morphology picture, and the right picture is the tumor weight statistics picture; it can be seen that the breast cancer tumor weight of the IM3D5-treated group was significantly reduced compared with the control group. The results showed that ZH-DT7792 can slow down the progression of breast cancer.

[0069] Example 5: Therapeutic use of CBLN1 monoclonal antibody ZH-DT7792 in alleviating immunosuppression in the prostate cancer microenvironment

[0070] Mouse PB-cre; Ptenf / f; Mychi prostate cancer organoids were taken and orthotopically transplanted into the prostate of C57 / B6J mice. Three days after the operation, the mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of administration were: 10 mg / kg, twice a week for 3 weeks. The health status of the mice was observed regularly during the period. Four weeks after inoculation, the mice were euthanized, and the tumor tissues were taken and digested into single cells. Tumor-associated neutrophils were labeled with CD45+CD11b+Ly6G+ (classic surface marker combination of neutrophils), and cytotoxic CD8 T cells were labeled with CD45+CD3+CD8+ (classic surface marker combination of cytotoxic CD8 T cells). The immunosuppressive nature of the tumor microenvironment was evaluated by flow cytometry. Figure 6 As shown, a is the flow cytometry analysis of immunosuppressive tumor-associated neutrophils TAN, b is the flow cytometry statistics of TAN, c is the flow cytometry analysis of cell-killing CD8 T cells, and d is the flow cytometry statistics of CD8 T cells. It can be seen that the proportion of immunosuppressive tumor-associated neutrophils in the IM3D5-treated group decreased, and the proportion of cell-killing CD8 T cells increased. The results show that ZH-DT7792 can alleviate the immunosuppression of the prostate cancer tumor microenvironment.

[0071] Example 6: Therapeutic use of CBLN1 monoclonal antibody ZH-DT7792 in castration-resistant prostate cancer and androgen receptor-dependent prostate cancer

[0072] Currently, androgen deprivation therapy and / or the second-generation androgen inhibitor ARSI are used to treat prostate cancer in clinical practice. 80% of prostate cancer (i.e., localized prostate cancer) patients have a good response to this treatment regimen, while patients with castration-resistant prostate cancer cannot benefit from conventional prostate cancer treatment regimens, suggesting that castration-resistant prostate cancer CRPC has a different pathogenesis and clinical treatment regimen from localized prostate cancer.

[0073] The castration-resistant prostate cancer CRPC organoids of Probasin-cre;Trp53f / f;Ptenf / f mice were orthotopically transplanted into the prostate of C57 / B6J mice. Three days after the operation, the mice were randomly divided into a control group and a ZH-DT7792 antibody treatment group. The dosage and frequency of administration were: 10 mg / kg, twice a week for 3 weeks. The health status of the mice was regularly observed during this period. Four weeks after the inoculation, the mice were euthanized, and the tumor tissues were obtained. The treatment effect of castration-resistant prostate cancer was evaluated by observing the tumor size. Figure 7 As shown in middle a, it can be seen that the prostate cancer tumor weight in the IM3D5-treated group was significantly reduced compared with the control group. The results showed that ZH-DT7792 can slow down the progression of castration-resistant prostate cancer.

[0074] In addition, some subtypes of castration-resistant prostate cancer experience reactivation of the androgen receptor signaling pathway during recurrence, which is defined as androgen receptor-dependent CRPC. Antibody therapy is evaluated by inoculating PB-cre; Ptenf / f; Mychi androgen receptor-dependent CRPC organoids in a castrated mouse model. The implementation steps are the same as above, and the tumor size is observed to evaluate the therapeutic effect of androgen receptor-dependent CRPC. Figure 7 As shown in middle b, the prostate cancer tumor weight in the IM3D5-treated group was significantly reduced compared with the control group. The results showed that ZH-DT7792 could slow down the progression of androgen receptor-dependent CRPC.

[0075] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may 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: including the heavy chain variable region and the light chain variable region, The three complementarity determining regions of the light chain variable region are: CDR1:QSLVHS NGNTY CDR2:KVS CDR3: SQNTHVPWT; The three complementarity determining regions of the heavy chain variable region are: CDR1:GFNIKDYF CDR2: IDPANGT 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. Use of the CBLN1 monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating breast cancer.

4. Use of the CBLN1 monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating castration-resistant prostate cancer.

5. Use of the CBLN1 monoclonal antibody according to claim 1 or 2 in the preparation of a drug for treating androgen receptor-dependent castration-resistant prostate cancer.

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