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Method for detecting anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate neutralizing antibody in human serum

A technology of epidermal growth factor and monoclonal antibody, which is applied in the field of antibody-drug conjugate neutralizing antibody detection, can solve the problems of sensitivity discount and low sensitivity, and achieve the effects of eliminating interference, improving sensitivity, and improving neutralizing activity

Inactive Publication Date: 2019-01-29
上海有临医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, if a neutralizing antibody detection method is developed based on this cell, considering its relatively low sensitivity to ADC drugs, EC should be selected. 50 When the concentration is used as the fixed concentration of the drug, the sensitivity of the corresponding method will be greatly reduced
At the same time, the presence of serum and drugs in the sample will also bring challenges to the reliability of the test results

Method used

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  • Method for detecting anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate neutralizing antibody in human serum
  • Method for detecting anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate neutralizing antibody in human serum
  • Method for detecting anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate neutralizing antibody in human serum

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Experimental program
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Effect test

Embodiment 1

[0010] SK-BR-3 cell culture and proliferation inhibition experiment

[0011] Use culture flasks or culture dishes of appropriate specifications to culture SK-BR-3 cells (ATCC, HTB-30) TM ). at 75cm 2 Take the culture flask (Corning, 430641) as an example. The cell subculture process is as follows: Discard the medium, and wash the flask wall (including the cell layer) once with sterile phosphate buffered saline (hereinafter referred to as PBS). Add 1.5–2 mL of 0.25% Trypsin-EDTA (Gibco, 25200056) solution to the bottle and place at 37 °C, 5% CO 2 Digest for about 3 minutes in the incubator. Take out the culture bottle and observe the state of the cells under an inverted microscope. When the edge of the cells becomes oval and starts to fall off, pat the bottle wall to help the cells fall off, and then add 6-8 mL of complete medium to stop the digestion. Complete medium was prepared by mixing RPMI-1640 (Gibco, 31800022) medium with 10% FBS (Gibco, 10099141). Discard the exce...

Embodiment 2

[0015] Preparation of anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate positive control antibody

[0016] Using cynomolgus monkeys, the anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate was used as the immunogen, referring to the classic preparation procedure of polyclonal antibody, and the positive control antibody was prepared by immunization method.

[0017] TiterMax® Gold Adjuvant (Sigma, T2684) was used as an immune adjuvant, mixed with the antigen to prepare an emulsion preparation, and multi-point subcutaneous injection was used to immunize cynomolgus monkeys (about 4-6kg), and the immune dose was 400μg. The anti-drug antibody titer in the serum was monitored 7 days after the initial immunization, and the F(ab)'2 fragment of the drug molecule was used as an antigen to boost the immunization 14 days later. Two weeks after the second immunization, venous blood was collected to evaluate the antibody titer. If t...

Embodiment 3

[0028] Extraction of ADA in Human Serum by Solid Phase Extraction

[0029] The composition of human serum samples is very complex, and drugs and other endogenous components may interfere with sample detection. For biological sample analysis methods, diluent dilution is usually used to reduce interference, and the minimum dilution factor (Minimum Required Dilution, MRD) that can eliminate the matrix effect is explored, but for cell culture-based neutralizing antibody methods, there is often little effect. Krista M. McCutcheon et al. adopted Protein A / G column chromatography to purify immunoglobulins in serum, so as to eliminate the influence of non-specific interfering substances in patient serum on the cell-based neutralizing antibody method (Journal of Immunological Methods, 358 , 2010), however, this method cannot eliminate blood drug interference. Solid phase extraction (SPE) is used to extract the ADA components in a directional manner, which can eliminate matrix interfer...

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Abstract

A method for detecting an anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugate neutralizing antibody in human serum includes steps of: 1) with SK-BR-3 cell line being a target cell, researching bioactivity of a medicine; 2) immunizing a machin with the medicine as an antigen and enhancing the immunization with a medicine F(ab)'2 fragment to prepare a positive polyclonalantibody; 3) pretreating a serum sample by using SPE extraction to eliminate serum interference, thus increasing stability of the method and drug tolerance capability; 4) developing a neutralizing antibody detection method on the basis of the medicine activity method, and applying the detection method to detection on the neutralizing antibody in the pretreated serum sample. The method is sourcedfrom cell level-based bioactivity method and can directly reflect the molecular mechanism of action (MoA) of the medicine. By using the SK-BR-3 cell that is more sensitive to medicine action, the method is improved in sensitivity. The positive control antibody is prepared by immunizing a non-human primate animal, so that comparability of the method with an actual sample is better. The serum sampleis detected after the pretreatment, so that the detection results are more stable and reliable.

Description

technical field [0001] The invention relates to a method for detecting neutralizing antibodies of antibody-drug conjugates, in particular to a method for detecting neutralizing antibodies of anti-human epidermal growth factor receptor 2 monoclonal antibody-MMAE conjugates in human serum based on a cell-level analysis method . Background technique [0002] Antibody-drug conjugate (ADC, Antibody-drug conjugate) is a new type of therapeutic protein drug, which consists of three parts, an antibody targeting a specific target, a chemically modified linker (linker) and a cytotoxic Small molecule drugs are formed by covalent binding. The ADC binds to the overexpressed antigen target on the surface of tumor cells through its antibody molecular structure region, and reaches a specific site, and the specially designed linker ensures its stability before reaching the specific site, thereby specifically releasing small molecules poison. After binding to the antigen, the ADC stimula...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N33/53
CPCG01N33/5011G01N33/53
Inventor 陈秋宇谭青乔
Owner 上海有临医药科技有限公司
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