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A kind of nano antibody against ca125 carbohydrate antigen and its application

A nanobody, carbohydrate antigen technology, applied in the direction of anti-animal/human immunoglobulins, antibodies, applications, etc., can solve the problems of poor tumor penetration and low targeting effect

Active Publication Date: 2019-10-25
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

[0008] The purpose of the present invention is to provide an anti-CA125 nanobody that can give full play to the superior performance of the nanobody, not only has excellent specific antigen binding ability, but also overcomes the inherent defects of traditional antibodies such as poor permeability to solid tumors and low targeting effect. , and further provide its application in the preparation of tumors, especially ovarian cancer therapeutic drugs and diagnostic preparations

Method used

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  • A kind of nano antibody against ca125 carbohydrate antigen and its application
  • A kind of nano antibody against ca125 carbohydrate antigen and its application
  • A kind of nano antibody against ca125 carbohydrate antigen and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Preparation of anti-CA125 nanobody

[0040] 1.1 Preparation of CA125 antigen

[0041] 1.1.1 Purification of CA125 in the ascites of patients with ovarian cancer by saturated ammonium sulfate precipitation: slowly add 228 g of ammonium sulfate to 2 L of ascites on a magnetic stirrer while stirring in an ice bath, so that the final concentration of ammonium sulfate is 20%, and stand at 4°C Overnight, centrifuge at 10,000 rpm for 10 minutes, and save the supernatant. Slowly add 524 g of ammonium sulfate to the supernatant while stirring in an ice bath on a magnetic stirrer to make the final concentration of ammonium sulfate 60%, leave it at 4° C. overnight, centrifuge at 10,000 rpm for 10 minutes, and retain the precipitate. Pipette and mix the precipitate with about 400mL PBS solution, centrifuge at 10000rpm for 10 minutes, and save the supernatant. Concentrate the supernatant to 200mL with a 100kD supershrink tube at 4000rpm, and divide into 50mL / tube.

[0...

Embodiment 2

[0077] Example 2. Affinity Activity Determination of Anti-CA125 Nanobody and Antigen

[0078] 2.1 Chip antigen coupling: Prepare the antigen with different pH sodium acetate buffer (pH 5.5, pH 5.0, pH 4.5, pH 4.0) to make 20μg / mL working solution, and prepare 50mM NaOH regeneration solution at the same time, use Biacore T100 The template method in the instrument of the protein interaction analysis system is used to analyze the electrostatic binding between the antigens at different pH conditions and the surface of the chip (GE Company), and the most neutral one is selected based on the signal increase of 5 times RL. The pH system and adjust the antigen concentration as the coupling conditions as needed. 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 amount. The coupling process takes approxi...

Embodiment 3

[0084] Example 3. Determination of ADCC activity induced by anti-CA125 Nanobody

[0085] Utilizing primers, using VHH-pMES4 as a template, PCR amplifies the nanobody gene. The primer sequences are as follows:

[0086] F: CCGAAATTCGAGTCTGGAGGAGG

[0087] R: GGAAGATCTCTGGGTCCCCTGGCCC

[0088] The PCR product and the fusion expression vector pFUSE-hIgG1-Fc were respectively digested with EcoRI and Bgl II restriction endonucleases (NEB) (for the schematic diagram of the vector, see Figure 10 , the vector carries the coding gene of the antibody constant region, and the amino acid sequence expressed by the coding gene is shown in SEQ ID NO.5). T4 ligase (NEB) was used to connect the double-enzyme-digested vector to the nanobody gene overnight, and after transforming into DH5α competent, the plasmid was extracted using an endotoxin-free large-scale extraction kit (Tiangen). Human 293 cells were transfected, and the anti-CA125 nanobody fused with the constant region sequence was ...

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Abstract

The invention discloses a nanobody against CA125 carbohydrate antigen. The nanobody has three unique complementarity determining regions CDR1, CDR2, and CDR3. application in reagents. The anti-CA125 nanobody provided by the invention has highly specific recognition and binding ability to CA125, has significant ADCC effect on ovarian cancer cells, and can realize precise imaging of tumors in mice.

Description

technical field [0001] The invention discloses an antibody, more specifically, the invention discloses a nanobody. Background technique [0002] The CA125 ovarian cancer-associated antigen was discovered in 1981 and is a related antigen of ovarian epithelial carcinoid. It is secreted by epithelial cells in the embryonic stage, and it is not secreted or rarely secreted under normal circumstances. However, when malignant lesions occur in the ovary, even if there is no clinical manifestation or pathologically difficult to identify, the CA125 value will increase, so it is a better ovarian Cancer diagnosis and screening indicators, and is closely related to the metastasis and prognosis of ovarian cancer. Initially, Bast et al. used the ovarian cell line OVCA433 immunogen to mediate the response of the murine monoclonal antibody OC125, and then recognized and confirmed its existence. The CA125 antigen is a glycoprotein with a molecular mass of 200ku, which has both membrane-boun...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/30C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00G01N33/574C12R1/19
CPCG01N33/57449C07K16/3069A61K2039/505C07K2317/732C07K2317/569C07K2317/565A61K51/1072A61P35/00C07K2317/33
Inventor 宋海鹏于建立刘原源
Owner 深圳市国创纳米抗体技术有限公司
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