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An anti-erbb2 antibody and its application in the treatment of breast cancer

An antibody and antigen technology, applied in the field of biomedicine, can solve the problem that the biological activity of antibodies needs to be further improved.

Active Publication Date: 2021-01-12
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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  • An anti-erbb2 antibody and its application in the treatment of breast cancer
  • An anti-erbb2 antibody and its application in the treatment of breast cancer
  • An anti-erbb2 antibody and its application in the treatment of breast cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Preparation and purification of anti-ErbB2 antibody

[0029] Recombinant human ErbB2 extracellular domain (ErbB2-ECD) protein was used as immunogen, and Balb / c healthy female mice homologous to SP2 / 0 were selected as immunized animals. The immune antigen was dissolved in PBS, and mixed evenly with an equal volume of Freund's adjuvant to form a water-in-oil emulsion. Subcutaneous multi-point immunization was used for the first immunization, and the immunization dose was 100 μg / bird. 7-10 days after the initial immunization, use incomplete Freund's adjuvant to mix and emulsify with the same volume of immune antigen, and immunize mice with the same route and dosage. Afterwards, the immunization was boosted several times every 7-10 days, so that the antibody titer of the mouse serum reached more than 10,000. One week after the last booster immunization, blood was collected from eyeballs, and serum titers were determined by ELISA. If the mouse serum titer does ...

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 ELISA (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, lambda light chain, and the anti-NSE purified in Example 2 Antibody samples E1-7E2 and E2-4F6 were added to the sample wells respectively, 50 μl per well, without incubation. Add 1X goat anti-mouse IgA+IgM+IgG-HRP into the sample wells, 50 μl per well, mix gently, and incubate for 1 h. Remove the liquid in the well and add 1XPBST to wash the well 3 times, and absorb excess water with absorbent paper. Add chromogenic solution, 100 μl per well, and develop color for 15 minutes at room temperature in the dark. Add 100 μl stop solution to stop the color reaction. Detect the OD value at 450nm by microplate reader, the...

Embodiment 3

[0032] Example 3. Antibody humanization

[0033] The positive hybridoma cells obtained in Example 1 were expanded and cultured, and an appropriate amount of cells were used for TRNzol-A + After lysis, each 1ml of TRNzol-A + Add 200 μl of chloroform, vortex for 15 seconds, and let stand for 3 minutes. Centrifuge at 13,000rpm at 4°C for 10 minutes. The cell solution is divided into three layers: the upper colorless aqueous phase, the middle layer and the lower yellow organic phase. Transfer the aqueous phase containing RNA to a centrifuge tube, and add an equal volume of isopropyl to the aqueous phase. alcohol, mix well, and let stand at room temperature for 25 minutes. Centrifuge at 13,000 rpm at 4°C for 10 minutes, discard the waste liquid to obtain the RNA pellet that sinks to the bottom. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved with DEPC water. Subsequently, the first strand of cDNA was synthesized using the TransScript One-Step gDNA Rem...

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Abstract

The present invention relates to an antibody that specifically binds to ErbB2. The antibody can bind to ErbB2 protein with higher affinity, and has the ability to inhibit the proliferation of SK-BR3 breast cancer cells with high ErbB2 expression and at the same time inhibit the low expression of ErbB2 induced by Heregulin β MCF‑7 breast cancer cell proliferation and other biological activities.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-ErbB2 antibody and its application in treating various hematological malignancies and solid tumors. Background technique [0002] In recent years, the incidence of clinical cancer in the world, breast cancer ranks first in the incidence of female malignant tumors, and it is second only to lung cancer in the incidence of cancer in the world. Obviously, breast cancer is the largest malignant tumor that endangers women's health, and surgery is the key to treatment. The main means of breast cancer, chemotherapy and radiotherapy are recognized as adjuvant therapy. However, in recent years, with the deepening of research on targeted therapy and anti-tumor methods, as well as the development of molecular pathology and genotyping, monoclonal antibody drugs against breast cancer have been rapidly developed. Wherein trastuzumab (trade name Herceptin, Herceptin) is the first antibody dru...

Claims

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Application Information

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Patent Type & Authority Patents(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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