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A kind of anti-carcinoembryonic antigen nanobody and its application

A nanobody and antigen technology, applied in the field of immunology, can solve the problems of low affinity, short serum half-life, hindering the application of nanobodies, etc.

Active Publication Date: 2020-07-14
深圳市国创纳米抗体技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low molecular weight of Nanobodies, some structural defects such as low affinity, easy aggregation, and short serum half-life hinder the further application of Nanobodies.

Method used

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  • A kind of anti-carcinoembryonic antigen nanobody and its application
  • A kind of anti-carcinoembryonic antigen nanobody and its application
  • A kind of anti-carcinoembryonic antigen nanobody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Construction and Screening of Anti-CEA Nanobody Phage Display Library

[0043] 1.1 Immunization of alpaca: Select a healthy adult alpaca, mix the recombinant protein CEA and Freund's adjuvant at a ratio of 1:1, and immunize the alpaca by subcutaneous multi-point injection on the back at 6-7μg / Kg , a total of four immunizations, immunization interval of 2 weeks. Afterwards, the peripheral blood of the alpaca was collected for the construction of a phage display library.

[0044] 1.2 Separation of camel-derived lymphocytes: analyze lymphocytes from the collected camel-derived anticoagulated whole blood according to routine procedures in this technical field, every 2.5×10 7 Add 1mL RNA isolation reagent to each living cell, take 1mL for RNA extraction, and store the rest at -80°C.

[0045] 1.3 Extraction of total RNA: extract total RNA according to routine procedures in this technical field, and adjust the concentration to 1 μg / μL with RNase-free water (for the...

Embodiment 2

[0077] Example 2. Preparation of anti-CEA nanobody

[0078] 2.1 Amplification of original nanobody strain TG1 and transformation of nanobody recombinant plasmid into Escherichia coli BL21(DE3)

[0079] Inoculate the original strain TG1 glycerolbacterium containing Nanobody nucleic acid in 5 mL of fresh LB-A medium at a ratio of 1:1000, and culture overnight at 37°C and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the instructions. After verification, transform 1 ul of the above plasmid into 100 ul of competent cells, mix gently, place on ice for 30 min, heat shock in a water bath at 42°C for 90 s, and cool in an ice bath for 3 min. Add 600ul LB medium to the centrifuge tube, shake and culture at 37°C for 60min. Take 100ul of the supernatant, spread it on the LB-A plate with a triangle spreader, and culture it upside down at 37°C overnight.

[0080] 2.2 Induced expression and extraction of nanobodies

[0081]The above monocl...

Embodiment 3

[0084] Example 3 Affinity activity of anti-CEA nanobody and CEA antigen

[0085] 3.1 Chip antigen coupling

[0086] The antigen was prepared into a 20ug / mL working solution with different pH sodium acetate buffers (pH 5.5, pH 5.0, pH 4.5, pH 4.0), and a 50mM NaOH regeneration solution was prepared at the same time, and used in the Biacore T100 protein interaction analysis system instrument The template method is used to analyze the electrostatic binding between the antigen and the surface of the chip (GE company) under different pH conditions, and the signal increase amount reaches 5 times RL as the standard, select the most suitable neutral pH system and adjust it as needed The antigen concentration was used as the condition during coupling. The chip was coupled according to the template method that comes with the instrument: select the blank coupling mode for channel 1, select the Target coupling mode for channel 2, and set the target to the designed theoretical coupling am...

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Abstract

The invention discloses a nanobody against carcinoembryonic antigen CEA. The nanobody has three unique complementarity determining regions CDR1, CDR2, and CDR3. The invention also discloses the application of the nanobody in the preparation of tumor therapeutic drugs . The anti-CEA nanobody provided by the present invention has specific recognition and binding ability to the CEA antigen, and has a significant ADCC effect on the tumor cell MKN-45, and can realize precise imaging of tumors in mice.

Description

technical field [0001] The invention discloses a nanobody and belongs to the field of immunology. Background technique [0002] 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 seven domains linked to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. The seven domains include a single N-terminal Ig variable domain and six domains homologous to Ig constant domains (A1-B1-A2-B2-A3-B3). CEA, originally classified as a protein expressed only in fetal tissues, has now been identified in several normal adult tissues. Overexpression of CEA has been observed in many types of cancer, including colorectal, pancreatic, lung, gastric, hepatoma, breast and thyroid cancers. Therefore, 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00
CPCC07K16/3007C07K2317/732C07K2317/569C07K2317/565C12N15/70A61P35/00C07K2317/22A61K51/1048C07K2317/33A61K2039/505
Inventor 宋海鹏于建立古一李飞王欢刘原源周宇航黄琪李婧婵
Owner 深圳市国创纳米抗体技术有限公司
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