阅读说明：本技术 一种抗癌胚抗原的高亲和力纳米抗体及其应用 (High-affinity nano antibody for resisting carcinoembryonic antigen and application thereof ) 是由 宋海鹏 于建立 古一 李飞 王欢 刘原源 周宇航 黄琪 李婧婵 于 2016-12-04 设计创作，主要内容包括：本发明公开了一种抗癌胚抗原CEA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中的应用。本发明提供的抗CEA的纳米抗体对CEA抗原具有特异的识别和结合能力,并且对于肿瘤细胞MKN-45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 carcinoembryonic antigen CEA, which has unique 3 complementarity determining regions CDR1, CDR2 and CDR3, and also discloses application of the nano antibody in preparing a tumor treatment drug. The anti-CEA nanobody provided by the invention has specific recognition and binding capacity to CEA antigen, has obvious ADCC effect on tumor cells MKN-45, and can realize accurate imaging of tumor in a mouse body.)

1. A nanobody against carcinoembryonic antigen, the variable region of which has 3 complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR1 sequence consists of the amino acid sequence set forth in SEQ ID No.13, and the CDR2 sequence consists of the amino acid sequence set forth in SEQ ID No.14, and the CDR3 sequence consists of the amino acid sequence set forth in SEQ ID No. 15.

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. 16.

3. An antibody comprising the variable region of the nanobody of claim 2, wherein the antibody further comprises a constant region, and wherein the sequence of the constant region of the antibody consists of the amino acid sequence set forth in SEQ ID No. 17.

4. A polynucleotide molecule encoding the sequence of the antibody of claim 3, said polynucleotide molecule having the sequence shown in SEQ ID No. 18.

5. An expression vector comprising the polynucleotide molecule 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.

8. The host cell of claim 7, wherein the cell is E.coli BL21(DE 3).

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

Technical Field

The invention discloses a nano antibody, belonging to the field of immunology.

Background

Carcinoembryonic antigen (CEA, also known as CEACAM-5 or CD66e) is a glycoprotein with a molecular weight of about 180 kDa. CEA is a member of the immunoglobulin superfamily and contains 7 domains linked to the cell membrane via a Glycosylphosphatidylinositol (GPI) anchor. The 7 domains include a single N-terminal Ig variable domain and 6 domains homologous to Ig constant domains (a1-B1-a 2-B2-A3-B3). CEA was originally classified as a protein expressed only in fetal tissues and has now been identified in several normal adult tissues. Overexpression of CEA is observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Thus, CEA has been identified as a tumor associated antigen. CEA is readily cleaved from the cell surface and shed from the tumor into the bloodstream, either directly or via the lymphatic system. Because of this property, serum CEA levels have been used as clinical markers to diagnose and screen for cancer. Furthermore, CEA has also been used as a tumor marker, and immunological assays to measure elevated CEA in the blood of cancer patients have been used clinically for the prognosis and control of cancer.

More importantly, CEA has become a potentially useful tumor-associated antigen for targeted therapy. There have been reported 2 major approaches to cancer treatment using CEA-targeted immunotherapy. One method uses an anti-CEA antibody to elicit the lytic activity of immune cells, particularly by antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), to eliminate CEA-expressing tumor cells. Another approach is to specifically target CEA-expressing tumor cells by conjugating the anti-CEA antibody or antibody fragment to an effector molecule such as a drug, toxin, radionucleotide, immunomodulator or cytokine, to exert the therapeutic effect of the effector molecule.

Various monoclonal antibodies have been generated against CEA. Chester et al have isolated single chain anti-CEA antibodies from phage display libraries for use in radioimmunoassay and radioimmunotherapy (U.S. Pat. No.5,876,691), followed by humanization of the antibodies (U.S. Pat. No.7,232,888). Radiolabeled anti-CEA antibodies have been used in clinical trials in patients with colorectal cancer.

In camelids (camels, dromedary and llamas) there is a class of heavy chain-only dimeric antibodies H₂It is mainly of the IgG2 and IgG3 type. Such antibodies are also referred to as single domain antibodies or single domain antibodies (sdabs) because they lack a light chain, and thus are referred to as heavy chain-only antibodies (HCAbs), while their 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, originally recognized only as a pathologic form of human B-cell proliferative disease (heavy chain disease), may be due to mutations and deletions at the genomic level resulting in the failure of the heavy chain CH1 domain to be expressed, such that the expressed heavy chain lacks CH1 and thus lacks the light chainThereby 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.

The nanobody has the smallest functional antigen-binding fragment derived from HCabs in adult camelids, has high stability and high affinity for antigen binding, and can interact with protein cleft and active sites of enzyme, making its action similar to that of 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 mabs. 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.

In view of the fact that CEA is more over-expressed in some solid tumors such as colorectal cancer, pancreatic cancer, lung cancer, gastric cancer, hepatoma, breast cancer and thyroid cancer, the development of anti-CEA nanobodies is a new need in the field of antibody technology to fully utilize the super-strong antigen recognition capability of nanobodies, and particularly to recognize some epitopes hidden in crevices or cavities. However, the existence of some structural defects such as low affinity, easy aggregation, short serum half-life and the like due to the low molecular weight of the nanobody prevents the further application of the nanobody. The invention aims to provide an anti-CEA nano antibody which can fully exert the excellent performance of the nano antibody and overcome the inherent defects of the nano antibody, and further exert the application of the nano antibody in CEA detection of human samples and the pharmaceutical field.

Disclosure of Invention

Based on the above object, the present invention provides a nanobody against carcinoembryonic antigen, the variable region of which has 3 complementarity determining regions CDR1, CDR2, CDR3, wherein the CDR1 sequence consists of the amino acid sequence set forth in SEQ ID No.13, the CDR2 sequence consists of the amino acid sequence set forth in SEQ ID No.14, and the CDR3 sequence consists of the amino acid sequence set forth in SEQ ID No. 15.

In a preferred technical scheme, the variable region sequence of the nanobody consists of the amino acid sequence shown in SEQ ID NO. 16.

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. 17.

Thirdly, the invention also provides a polynucleotide molecule for coding the sequence of the antibody, and the sequence of the polynucleotide molecule is shown by SEQ ID NO. 18.

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

In a preferred embodiment, the vector is pMES 4.

Fifth, the present invention provides a host cell containing the above expression vector.

In a preferred embodiment, the cell is E.coli BL21(DE 3).

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

The nanometer anti-CEA antibody provided by the invention has unique CDR1, 2 and 3 region sequences, so that the antibody has specific recognition and binding capacity to CEA antigen and does not react with other non-specific cross-reactive proteins. The nano antibody provided by the invention has a remarkable ADCC effect, can induce the cracking of tumor cells MKN-45 expressing CEA, does not have the effect on the MKN-74 cells not expressing CEA, and shows an application prospect in the preparation of tumor treatment drugs. Moreover, the anti-CEA nano antibody provided by the invention can realize accurate imaging of tumors in a mouse body, and shows application prospects in diagnostic reagent preparation and in-vivo imaging technologies.

Drawings

FIG. 1 is a schematic diagram of the structure of the pMES4 expression vector;

FIG. 2 shows the electrophoretic identification of total RNA extracted;

FIG. 3 shows the first round of PCR amplification of antibody variable region gene electrophoresis identification map;

FIG. 4 shows the second round of PCR amplification of antibody variable region gene electrophoresis identification map;

FIG. 5 shows the electrophoretic identification chart of the product of the double digestion reaction with pMES4 vector;

FIG. 6 shows the electrophoretic identification chart of the transformant identified by colony PCR;

FIG. 7 is an SDS-PAGE identification chart of the nanobody;

FIG. 8 is a schematic diagram of the construction of the fusion expression vector;

FIG. 9 shows a double restriction enzyme electrophoresis identification chart of the nanobody and the fusion expression vector;

FIG. 10 is a SDS-PAGE pattern of nanobody purification;

FIG. 11. Nanobody ADCC effect curves;

FIG. 12 is a diagram showing the imaging localization result of the labeled nanobody on the mouse transplanted tumor

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.