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Anti-ErbB2 antibody and application thereof in treatment of breast cancer

An antibody and amino acid technology, applied in the direction of antibodies, antineoplastic drugs, diseases, etc., can solve the problem that the biological activity of antibodies needs to be further improved

Active Publication Date: 2020-10-09
SHANGHAI RUOTUO BIOSCIENCES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although ErbB2 antibodies such as trastuzumab and pertuzumab have been used in the clinical treatment of breast cancer, the biological activity of these antibodies needs to be further improved

Method used

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  • Anti-ErbB2 antibody and application thereof in treatment of breast cancer
  • Anti-ErbB2 antibody and application thereof in treatment of breast cancer
  • Anti-ErbB2 antibody and application thereof in treatment of breast cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Anti-ErbB2 antibody preparation and purification

[0029] Recombinant human ErbB2 extracellular domain (ErbB2-ECD) protein was used as an immunogen, and Balb / c healthy female mice homologous to SP2 / 0 were selected as immunized animals. The immune antigen is dissolved in PBS and mixed with an equal volume of Freund's adjuvant to form a water-in-oil emulsion. The first immunization was subcutaneous multi-point immunization, and the immunization dose was 100 μg / mouse. 7-10 days after the initial immunization, use incomplete Freund's adjuvant and an equal volume of immune antigen to emulsify, and immunize the mice with the same route and dose. After that, boost the immunization several times every 7-10 days to make the antibody titer of the mouse serum reach more than 10,000. One week after the last booster immunization, blood was taken from the eyeball, and the serum titer was determined by ELISA. If the mouse serum titer does not reach 10,000, the booster immun...

Embodiment 2

[0030] Example 2. Identification of mouse anti-ErbB2 antibody subtypes

[0031] The subtype of the anti-ErbB2 monoclonal antibody obtained in Example 1 was identified by the ELISA method (the kit was purchased from Proteintech). The ELISA plate provided in the kit has been pre-coated with specific antibodies against mouse IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, kappa light chain, and lambda light chain. The anti-NSE purified in Example 2 Antibody samples E1-7E2 and E2-4F6 were added to the sample wells, 50μl per well, without incubation. Add 1X goat anti-mouse IgA+IgM+IgG-HRP to the sample wells, 50μl per well, mix gently, and incubate for 1h. Remove the liquid from the wells and add 1XPBST to wash the wells 3 times, absorb the excess water with absorbent paper. Add the color developing solution, 100μl per well, and avoid light at room temperature for 15min. Add 100μl stop solution to stop the color reaction. Detect the OD value at 450nm by a microplate reader, the results are as...

Embodiment 3

[0032] Example 3. Humanization of antibodies

[0033] The positive hybridoma cells obtained in Example 1 were expanded and cultured, and an appropriate amount of cells was used for TRNzol-A + After lysis, every 1ml TRNzol-A + Add 200 μl of chloroform, vortex for 15 seconds, and leave for 3 minutes. Centrifuge at 13000rpm and 4℃ for 10 minutes. The cell solution is divided into three layers: the upper colorless water phase, the middle layer and the lower yellow organic phase. The RNA-dissolved water phase is transferred to a centrifuge tube, and an equal volume of isopropyl is added to the water phase. Alcohol, mix well, and let stand at room temperature for 25 minutes. Centrifuge at 13000 rpm and 4°C for 10 minutes, discard the waste solution to obtain the RNA precipitate that sinks to the bottom. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in DEPC water. Subsequently, the first strand of cDNA was synthesized using TransScript One-Step gDNA Remova...

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Abstract

The invention relates to an antibody specifically binding to CCR4. The antibody can be combined with ErbB2 protein with higher affinity, and has biological activities of inhibiting proliferation of SK-BR3 breast cancer cells with high expression of ErbB2, inhibiting proliferation of MCF-7 breast cancer cells with low expression of ErbB2 induced by modulin beta and the like.

Description

Technical field [0001] The present invention relates to the field of biomedicine, in particular to anti-ErbB2 antibodies and their application in the treatment of various hematological malignancies and solid tumors. Background technique [0002] In the global incidence of clinical cancer in recent years, breast cancer ranks first in the incidence of female malignant tumors, and it is second only to lung cancer in the global cancer incidence. Obviously breast cancer is the number one malignant tumor that harms women’s health, and surgery is the treatment. The main methods of breast cancer, chemotherapy and radiotherapy are recognized adjuvant treatment methods. However, in recent years, with the continuous in-depth research on targeted therapy and anti-tumor methods, the development of molecular pathology and genotyping, and the rapid development of monoclonal antibody drugs for breast cancer. Among them, trastuzumab ((brand name Herceptin, Herceptin) is the first antibody drug u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/32A61K39/395A61P35/00A61P15/14
CPCA61P15/14A61P35/00C07K16/32C07K2317/24C07K2317/56C07K2317/565C07K2317/73C07K2317/92
Inventor 彭菲顾超
Owner SHANGHAI RUOTUO BIOSCIENCES CO LTD
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