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Novel bifunctional antibody conjugate, preparation method and application thereof

A technology of bifunctional antibodies and conjugates, applied in the direction of antibodies, drug combinations, anti-tumor drugs, etc., can solve the problems of unsatisfactory chemical coupling efficiency of bispecific antibodies

Inactive Publication Date: 2015-06-17
SHANGHAI YUYAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, so far, the chemical conjugation efficiency of bispecific antibodies has not been satisfactory

Method used

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  • Novel bifunctional antibody conjugate, preparation method and application thereof
  • Novel bifunctional antibody conjugate, preparation method and application thereof
  • Novel bifunctional antibody conjugate, preparation method and application thereof

Examples

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preparation example Construction

[0102] A typical coupling method such as figure 2 shown. A preferred preparation method of anti-CD3×anti-HER2 bispecific antibody comprises the following steps:

[0103] (1) Antibody dissolution: Dissolve commercial anti-CD3 monoclonal antibody in reaction buffer A (150mM NaCl, 2mM EDTA˙Na 2 , 100mM phosphate buffer, pH8.0); dissolve the anti-medical HER2 monoclonal antibody in reaction buffer B (150mM NaCl, 2mM EDTA˙Na 2 , 100mM phosphate buffer, pH 7.2).

[0104] (2) Dissolution of coupling agent: Quickly weigh Traut’s reagent and Sulfo-SMCC and dissolve them in ultrapure water respectively to prepare a mother liquor.

[0105] (3) Coupling of anti-CD3 monoclonal antibody and Traut's reagent: Mix the prepared Traut's reagent mother solution and anti-CD3 monoclonal antibody at a molar ratio of 5:1 to 10:1; place the well-mixed mixture in On a constant temperature reaction shaker, keep the temperature at 15-28°C and react for 60 minutes.

[0106] (4) Coupling of anti-HER2...

Embodiment 1

[0127] Preparation of anti-CD3×anti-HER2 bispecific antibody

[0128] Experimental steps:

[0129] Take 132 μg of mouse anti-human CD3 monoclonal antibody and dissolve it in reaction buffer A (150 mM NaCl, 2 mM EDTA·Na 2 , 100mM phosphate buffer, pH8.0), weighed 1.2mg of Traut's reagent and dissolved it in 120μL of reaction buffer A, and prepared it as a 10mg / mL stock solution. Anti-CD3 monoclonal antibody was mixed at a molar ratio of 10:1 and incubated at room temperature for 1 hour;

[0130] Take 30 μg of mouse anti-HER2 monoclonal antibody and dissolve it in reaction buffer B (150 mM NaCl, 2 mM EDTA·Na 2 , 100mM phosphate buffer, pH7.2), weighed 1.5mg of Sulfo-SMCC and dissolved it in 300μL of reaction buffer B to prepare a mother solution, diluted 10 times to 0.5mg / mL when used, and mixed Sulfo-SMCC with anti-HER2 Monoclonal antibodies were mixed at a molar ratio of 20:1, 40:1, and 60:1 and incubated at room temperature for 1 hour;

[0131] The Zeba desalting column was...

Embodiment 2

[0137] Preparation of anti-CD3×anti-HER2 bispecific antibody

[0138] Different from Example 1, the specific steps are as follows:

[0139] Take mouse anti-human CD3 monoclonal antibody OKT3 antibody 133μg dissolved in reaction buffer A (150mM NaCl, 2mM EDTA·Na 2 , 100mM phosphate buffer, pH 8.0), weighed 1.3mg of Traut's reagent and dissolved it in 130μL of reaction buffer A to make 10mg / mL stock solution, diluted 50 times to 0.2mg / mL when used, and mixed Traut's reagent with anti CD3 monoclonal antibody was mixed at a molar ratio of 5:1 or 10:1 and incubated at room temperature for 1 hour;

[0140] Take 31 μg of mouse anti-HER2 monoclonal antibody and dissolve in reaction buffer B (150mM NaCl, 2mM EDTA·Na 2 , 100mM phosphate buffer, pH7.2), weighed 1.0mg of Sulfo-SMCC dissolved in 100μL of reaction buffer B, prepared as a mother solution, diluted 20 times to 0.5mg / mL when used, mixed Sulfo-SMCC with anti-HER2 Monoclonal antibodies were mixed at a molar ratio of 4:1 or 20:...

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Abstract

The invention provides a novel bifunctional antibody conjugate, a preparation method and application thereof. The bifunctional antibody conjugate provided by the invention includes the structure of: a first antibody specifically directed at immune cells and a second antibody specifically directed at tumor markers, with the two antibodies coupled together through a coupling arm. The bifunctional antibody conjugate can significantly improve the tumor-killing activity after combination with an immune cell (like CIK). The invention also provides application of the novel bifunctional antibody conjugate in treatment of tumors and other diseases. The invention also provides an anti-HER2 positive cancer bispecific antibody and a preparation process. According to the invention, the bispecific antibody with high coupling rate and high activity can be simply and quickly produced, and the CIK tumor-killing activity can be improved by 2-6 times.

Description

technical field [0001] The invention relates to the technical field of biomedicine and antibody chemical coupling. More specifically, the present invention relates to novel bifunctional antibody conjugates and their preparation methods and uses. Background technique [0002] According to the statistics of the World Health Organization, the number of people who die from malignant tumors in the world has exceeded 7 million every year, and it will gradually become the number one killer threatening human survival. [0003] Cellular immunotherapy is an emerging and brand-new anti-tumor treatment method with significant curative effect, and has been recognized as the most active and promising treatment method in the comprehensive treatment mode of tumors in the 21st century. [0004] As a popular technology in the field of cell therapy today, bispecific antibody therapy combines the advantages of antibody and cell therapy. It has been applied to cancer, autoimmune diseases and st...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P35/00
Inventor 葛微王有朝何庆仲丁航海
Owner SHANGHAI YUYAN BIOTECH CO LTD
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