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Anti-CEACAM5 nanometer antibody

A nanobody and expression carrier technology, which is applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of low CEA production and lack of specificity in diagnosis, etc. Achieve low immunogenicity, high stability, and high throughput

Active Publication Date: 2020-12-04
THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Carcinoembryonic antigen (CEA) is a protein found in colon cancer and fetal intestinal tissue. It was used as a marker for tumor detection earlier in clinical practice. The production of CEA in normal human somatic cells is small, while in malignant It is expressed in various organs of tumor patients and can be used for the diagnosis and identification of tumors, but the diagnosis of this tumor standard lacks specificity

Method used

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  • Anti-CEACAM5 nanometer antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Construction of a nanobody library against CEACAM5 protein.

[0032] The recombinant CEACAM5 protein was mixed with Freund's adjuvant to immunize alpacas for 4 times. After the 4 times of immunization, 50 mL alpaca peripheral blood lymphocytes were extracted and total RNA was extracted; then the VHH region of the antibody was cloned by Nest-PCR technology, and the cloned product was inserted into phage by homologous recombination to obtain a phage display library.

Embodiment 2

[0033] Example 2: Affinity panning and library amplification after panning.

[0034] (1) Affinity panning

[0035] 1) Dilute the recombinant CEACAM5 protein with carbonate buffer solution with a pH value of 9.6 to a final concentration of 5 μg / mL, add 100 μL / well into the wells of the enzyme label, coat 8 wells per target molecule, and coat overnight at 4°C ;

[0036] 2) Discard the coating solution, wash with PBS 3 times, add 300 µL of 3% BSA-PBS blocking solution to each well, and block for 1 hour at 37°C;

[0037] 3) Wash 3 times with PBS, add 100 µL phage library, and incubate at 37°C for 1 hour;

[0038] 5) Aspirate unbound phage, wash 6 times with PBST and 2 times with PBS;

[0039] 6) Add 100 µL Gly-HCl eluate, incubate at 37°C for 8 min, and elute the specifically bound phage; transfer the eluate to a 1.5 mL sterile centrifuge tube, and quickly neutralize the buffer with 10 µL Tris-HCl and;

[0040] 7) Take 10 µL for gradient dilution, measure the titer, and calcu...

Embodiment 3

[0049] Example 3: Identification and analysis of specific phage clones.

[0050] (1) Phagemid rescue

[0051] 1) From the plate with the eluate titer in the first round of panning, randomly pick 96 single clones with a sterilized toothpick and inoculate them in 1mL 2×YT-A, culture at 37°C and 220 r / min for 8 h with shaking;

[0052] 2) Take 200 µL of the above culture, add M13K07 phage at the ratio of cell:phage = 1:20, let stand at 37°C for 15 minutes, and shake at 220 r / min for 45 minutes;

[0053] 3) Add 800 µL of 2×YT-AK, culture at 30°C overnight with shaking;

[0054] 4) The next day, centrifuge at 12,000 rpm for 2 minutes, and take the supernatant for monoclonal ELISA identification.

[0055] (2) Identification of positive phage clones

[0056] 1) Dilute the recombinant CEACAM5 protein with carbonate buffer solution with a pH value of 9.6 to a final concentration of 2 μg / mL, add 100 μL / well to the enzyme-labeled well, and coat overnight at 4°C;

[0057] 2) Discard t...

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Abstract

The invention provides a nanometer antibody. The antibody comprises: (1) an amino acid sequence shown as SEQ ID NO: 1 or 2 or 3 or 4 or 5 or 6; and (2) the sequences have high sequence homology, suchas more than 80% of sequence homology. According to the embodiment of the invention, nucleic acid specifically encodes the nanometer antibody, and the nanometer antibody can specifically target CEACAM5 and has high water solubility, high tolerance, high stability, high antigen binding property, low immunogenicity and relatively strong tissue penetrating power.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a nanobody and its preparation method and application, and the nanobody can be applied to the research and development of CEACAM5 protein detection reagents and therapeutic antibodies. Background technique [0002] Carcinoembryonic antigen (CEA) is a protein found in colon cancer and fetal intestinal tissue. It is used as a marker for tumor detection earlier in clinical practice. The production of CEA in normal human somatic cells is small, while in malignant It is expressed in various organs of tumor patients and can be used for the diagnosis and differentiation of tumors, but the diagnosis of this tumor standard lacks specificity. High levels of CEA mainly appear in various tumors such as pancreatic cancer, colorectal cancer, breast cancer, gastric cancer, and small cell lung cancer. The sensitivity of CEA in the serum of tumor patients is related to whether ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21G01N33/574A61K39/395A61P35/00C12R1/19
CPCC12N15/70C07K2317/569C07K2317/567C07K2317/565C07K2317/22C07K16/3007C07K2317/92C07K2317/94
Inventor 单鸿李丹梅超明杨帆
Owner THE FIFTH AFFILIATED HOSPITAL SUN YAT SEN UNIV
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