阅读说明：本技术 一种抗ca125糖类抗原的高亲和力纳米抗体及其应用 (High-affinity nano antibody for anti-CA 125 carbohydrate antigen and application thereof ) 是由 宋海鹏 于建立 刘原源 于 2018-01-20 设计创作，主要内容包括：本发明公开了一种抗CA125糖类抗原的高亲和力纳米抗体,所述纳米抗体具有独特的3个互补决定区CDR1、CDR2、CDR3,本发明还公开了所述纳米抗体在制备肿瘤治疗药物和肿瘤诊断试剂中的应用。本发明提供的抗CA125纳米抗体对CA125具有高度特异的识别和结合能力,并且对卵巢癌细胞具有显著的ADCC效应,而且可以在小鼠体内实现肿瘤的精确成像。(The invention discloses a high-affinity nano antibody for resisting CA125 carbohydrate antigen, which has unique 3 complementary determining regions CDR1, CDR2 and CDR3, and also discloses application of the nano antibody in preparing tumor treatment medicines and tumor diagnosis reagents. The anti-CA 125 nano antibody provided by the invention has high specific recognition and binding capacity to CA125, has obvious ADCC effect on ovarian cancer cells, and can realize accurate imaging of tumors in mice.)

1. A high-affinity nanobody against CA125 carbohydrate antigen, characterized in that the variable region of said nanobody has 3 complementarity determining regions CDR1, CDR2 and CDR3, wherein the CDR1 sequence consists of the amino acid sequence described in SEQ ID No.1, the CDR2 sequence consists of the amino acid sequence described in SEQ ID No.2 and the CDR3 sequence consists of the amino acid sequence described in SEQ ID No. 3.

2. The nanobody of claim 1, wherein the variable region sequence of the nanobody consists of the amino acid sequence set forth in SEQ ID No. 4.

3. An antibody comprising the variable region of the nanobody of claim 2, wherein the antibody further comprises a constant region consisting of the amino acid sequence of SEQ ID No. 5.

4. A polynucleotide encoding the antibody of claim 3, the coding sequence being represented by SEQ ID No. 6.

5. An expression vector comprising the polynucleotide of claim 4.

6. The vector of claim 5, wherein said vector is pMES 4.

7. A host cell comprising the expression vector of claim 6, said cell being E.coli BL21(DE 3).

8. Use of the antibody of claim 3 for the preparation of a medicament for the treatment of tumors.

9. The use of claim 8, wherein the tumor is an ovarian tumor.

10. Use of the nanobody of claim 1 or 2 in the preparation of a tumor diagnostic reagent.

Technical Field

The invention discloses an antibody, and more particularly discloses a nano antibody.

Background

CA125 ovarian cancer associated antigen was found in 1981 to be an antigen associated with epithelial carcinoid of the ovary. The ovarian cancer cell is secreted by epithelial cells in an embryonic stage, and is not secreted or rarely secreted under normal conditions, but when the ovary has malignant lesion, even if the ovarian cancer cell does not show clinically or is difficult to identify pathologically, the CA125 value is increased, so that the ovarian cancer cell is a better ovarian cancer diagnosis and screening index and has close relation with the metastasis and prognosis of the ovarian cancer. The murine monoclonal antibody OC125 response was initially mediated by Bast et al via the ovarian cell line OVCA433 immunogen, and its presence was subsequently recognized and confirmed. The CA125 antigen is glycoprotein with the molecular mass of 200ku, has two attitudes of membrane-bound type and free type, and is one of the most comprehensively researched ovarian cancer serum markers so far. The serum CA125 concentration of 90% of the advanced ovarian cancer patients is increased in different degrees.

Although the ovarian cancer patients are first selected by surgery or chemotherapy, the prognosis is still not very ideal. Tumor recurrence, especially intra-abdominal recurrence, and chemotherapy resistance are often important factors affecting prognosis. At present, new therapeutic regimens for ovarian cancer, particularly for biological therapy, are rapidly evolving and have been applied in part in ex vivo and in vivo studies and clinical trials. Biological treatment for ovarian cancer includes various forms of cytokines, monoclonal antibodies, transgenic therapy, and the like. Among the approaches, ovarian cancer associated antigen CA125 monoclonal antibody therapy is of interest. The current technology for treating ovarian cancer by using the CA125 monoclonal antibody comprises two types of antigen-antibody compound mediated immune response of a human body and targeting induction of radioactive elements and antitumor drugs. The mediated immunity of the antigen-antibody complex depends on the stimulation of endogenous anti-tumor reaction of an organism, so as to achieve the purposes of removing focus and self-repairing regulation. The most representative antibodies used in this technology are anti-CA 125/anti-T cell surface molecule bispecific antibody (BsAb) and murine monoclonal antibody B43.13. The anti-CA 125/anti-T cell surface molecule bispecific antibody is combined with the CA125 antigen on the surface of the ovarian cancer cell, and then the anti-T cell surface molecule epitope is used for recognizing and inducing T cells, so that the cytotoxic effect of the T cells aiming at tumor tissues is generated, and the focus is killed. In addition, the therapy can stimulate the body to produce and maintain active immune status for a long time. Although the anti-CA 125/anti-T cell surface molecule BsAb shows excellent treatment effect, more problems still need to be solved, for example, the detailed in vivo pharmacokinetic characteristics are not clear; it is difficult to predict the in vivo application dose and the extent of cytotoxic effects after activating T-lymphocyte reaction; the preparation process has lower humanization level and obvious toxic and side effects; BsAb, tumor and T lymphocyte are required to be further improved in the aspects of specificity and affinity; the function of the Fc fragment of the antibody still needs to be improved; the permeability of antibody tumor tissue is not high, thereby bringing about the technical problems of weak tumor localization and high unintended clearance rate in vivo. The murine monoclonal antibody B43.13 initiates a classical idiotypic immune response by binding to the CA125 antigen to form an immune complex. The action mechanism is that the humanized anti-mouse antibody activates anti-idiotype chain reaction, thereby causing the humoral immune response of the polyclonal antibody aiming at the CA 125. The immune response therapy has good tolerance, no adverse reaction or non-compliance and other drug suspension situations, but cannot always keep the sensitive response state of the human body to the tumor cells and also cannot overcome the tumor immune escape mechanism. A targeting induction technology of radioactive elements and antitumor drugs is another CA125 monoclonal antibody treatment technology. After the ovarian cancer tumor cell debulking operation, residual cancer cells are still planted in the abdominal cavity. Generally, such seeded cells or cell clusters are not easily clinically detectable and have high chemotherapy resistance, but are rather vulnerable to radio-immune destruction. Based on the characteristics, the radiation immunotherapy for ovarian cancer develops rapidly. The existing radioimmunotherapy mainly selects a mouse monoclonal antibody containing anti-CA 125 epitope, and after isotope labeling, targeting positioning and radiotherapy are carried out. Several experiments demonstrated that the affinity of the CA125 monoclonal antibody to its antigen did not decrease after radioactive element labeling. However, the conventional direct radiolabelling of monoclonal antibodies has many drawbacks, especially the accumulation of radiotoxicity in the reticuloendothelial system and poor therapeutic effect on solid tumors. The reticuloendothelial system accumulates, and the target localization rate of the isotope labeled antibody is greatly weakened. For solid tumor, poor radio-immune efficacy is generally attributed to tumor tissue uptake concentration lower than the optimal radiotherapy concentration, low plasma clearance, bone marrow toxicity, and the like.

Based on the outstanding characteristics of CA125 in clinical diagnosis and the great application prospect in the field of tumor therapy, the development of specific binding antibodies against CA125 and the improvement of clinical diagnosis and therapeutic efficiency become urgent needs in the prior art. However, the traditional antibodies have some disadvantages, such as low affinity, low immune recognition efficiency, and difficulty in achieving ideal binding and neutralization effects for some antigens with high hiding degree.

In 1993, Hamers-Casterman et al found that a class of heavy chain-only dimers (H) was found in camelids (camels, dromedary and llamas) in vivo₂) The antibody of (1), which is mainly IgG₂And IgG₃In this type, such antibodies are also referred to as single domain antibodies or single domain antibodies (sdabs) because they lack a light chain and are thus referred to as Heavy chain-only antibodies (HCAbs), whose antigen-binding site consists of one domain, referred to as a VHH region. Since this type of antibody is a variable region sequence after removal of a constant region, the molecular weight is only 15kD, and the diameter is about 10 nm, and thus it is also called nanobody (Nbs). In addition, such single domain antibodies, called VNARs, are also observed in sharks. This heavy chain-only antibody was originally recognized only as a pathological form of a human B-cell proliferative disease (heavy chain disease). This type of antibody may be due to genomic level mutations and deletions that result in the inability of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the ability to bind to the light chain, thus forming a heavy chain dimer.

Nanobodies are comparable in affinity to their corresponding scFv, but surpass scfvs in solubility, stability, resistance to aggregation, refolding, expression yield, and ease of DNA manipulation, library construction, and 3-D structure determination, relative to scfvs of conventional four-chain antibodies.

Nanobodies have minimal functional antigen-binding fragments derived from HCabs in adult camelids, have high stability and high avidity for antigen binding, and can interact with protein clefts and enzymatic active sites, making their action similar to inhibitors. Therefore, the nano-antibody can provide a new idea for designing small molecule enzyme inhibitors from peptide-mimetic drugs. Due to the heavy chain only, nanobodies are easier to manufacture than monoclonal antibodies. The unique properties of nanobodies, such as stability in extreme temperature and pH environments, allow for large yields to be produced at low cost. Therefore, the nano antibody has great value in the treatment and diagnosis of diseases and has great development prospect in the antibody target diagnosis and treatment of tumors.

The invention aims to provide the anti-CA 125 nano antibody which can fully exert the excellent performance of the nano antibody, has excellent specific antigen binding capacity, overcomes the inherent defects of poor permeability, low targeting effect and the like of the traditional antibody solid tumor, and further provides the application of the anti-CA 125 nano antibody in the preparation of tumor, particularly ovarian cancer treatment drugs and diagnosis preparations.

Disclosure of Invention

Based on the above objects, the present invention provides a high affinity nanobody against CA125, the variable region of which has 3 complementarity determining regions CDR1, CDR2, and CDR3, wherein the CDR1 sequence consists of the amino acid sequence shown in SEQ ID No.1, the CDR2 sequence consists of the amino acid sequence shown in SEQ ID No.2, and the CDR3 sequence consists of the amino acid sequence shown in SEQ ID No. 3.

In a preferred technical scheme, the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID NO. 4.

Secondly, the invention also provides an antibody containing the variable region of the nano antibody, wherein the antibody also has a constant region, and the sequence of the constant region of the antibody consists of the amino acid sequence shown in SEQ ID NO. 5.

Thirdly, the invention also provides a polynucleotide for coding the antibody sequence, and the sequence of the polynucleotide is shown by SEQ ID NO. 6.

Fourth, the present invention provides an expression vector comprising the above polynucleotide.

In a preferred embodiment, the vector is pMES 4.

Fifth, the present invention provides a host cell comprising the above expression vector, said cell being E.coli BL21(DE 3).

Sixth, the invention also provides the application of the antibody in preparing tumor treatment medicines.

In a preferred embodiment, the tumor is an ovarian tumor.

Finally, the invention provides the application of the nano antibody in preparing a tumor diagnosis reagent.

The anti-CA 125 nanobody provided by the invention has specific recognition and binding capacity to CA125 antigen due to the unique CDR1, 2 and 3 region sequences. But not with other non-specific cross-reactive proteins. The nano antibody provided by the invention has a remarkable ADCC effect, can induce the cracking of OVCAR-3 expressing CA125 tumor cells, does not have the effect on A431 cells not expressing CA125, and shows an application prospect in the preparation of tumor treatment medicines. Moreover, the anti-CA 125 nano antibody provided by the invention can realize accurate imaging of tumors in mice, and shows application prospects in diagnostic reagent preparation and in-vivo imaging technologies.

Drawings

FIG. 1 shows the identification pattern of CA125 through SDS-PAGE and Western blot detection and affinity purification;

FIG. 2 shows molecular sieve purified CA125 identification patterns detected by SDS-PAGE and Western blot;

FIG. 3 is a schematic diagram of the structure of the pMES4 expression vector;

FIG. 4 shows the total RNA electrophoresis identification map;

FIG. 5 is the first round of PCR amplification antibody variable region gene electrophoresis identification map;

FIG. 6 is a second round of PCR amplification antibody variable region gene electrophoresis identification map;

FIG. 7 shows the electrophoretic identification map of the product of the pMES4 vector double enzyme digestion reaction;

FIG. 8 shows an electrophoretic identification map of colony PCR for identifying transformants;

FIG. 9 is a nano antibody expression SDS-PAGE identification map;

FIG. 10 is a schematic diagram of the construction of the fusion expression vector;

FIG. 11 is a SDS-PAGE profile of nanobody purification;

figure 12 graph of the ADCC effect of nanobodies.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of the present invention.