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Anti-MMAE monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody and antibody technology, applied in the field of biomedicine, can solve the problems of difficult MMAE analysis and high requirements for personnel and instruments

Pending Publication Date: 2022-03-15
SUZHOU IBIO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At the same time, in the existing ligand conjugates, there are many reports of MMAE-related conjugated drugs, but the detection of the existing small molecule MMAE is mainly through the method of LC-MS, and the analysis of MMAE in tissue samples is difficult. Larger, and for the extraction and analysis of trace MMAE, the requirements for personnel and instruments are relatively high

Method used

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  • Anti-MMAE monoclonal antibody as well as preparation method and application thereof
  • Anti-MMAE monoclonal antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Antigen Preparation

[0027] The company synthesizes MMAE small molecules in-house, and labels a cysteine ​​(MMAE-CYS) to facilitate coupling with carrier proteins for immunization or detection. Carrier protein coupling, immune antigen preparation, because MMAE molecules are relatively small and highly toxic, it is not suitable for direct immunization, MMAE-CYS is coupled with carrier protein, choose Thermo Scientific maleamide-activated keyhole blue (KLH) For the protein kit, follow the procedure provided by the kit to perform coupling with the carrier protein.

Embodiment 2

[0028] Example 2: Animal immunization

[0029] The antigen for immunization (MMAE-CYS-KLH) prepared in Example 1 was emulsified with complete Freund's adjuvant, and immunized with 4-6 week-old female Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. ), subcutaneously injected into the abdomen, 6 points per mouse, and the dose was 100 μg / only. Immunization was boosted once every 14 days, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 50 µg per mouse. Seven days after the third booster immunization, the polyantibody titer against the immunogen in the mouse serum was detected by indirect ELISA (wavelength 450nm). The dose is 50µg / only.

Embodiment 3

[0030] Embodiment 3: hybridoma cell preparation

[0031] 1. Fusion: Blood was collected from the mouse that reached the immune standard in Example 2, and the spleen was taken to prepare a cell suspension, which was mixed with mouse myeloma cells F0 at a ratio of 10:1, and centrifuged at 1200 rpm for 5 minutes. After discarding the supernatant, put the centrifuge tube into a 37°C water bath, slowly add 1 mL of PEG1500 (Roche Company) within 1 minute, and mix the cells evenly. After standing in warm water for 1 minute, add 10 mL of serum-free IMDM (Hyclone Company), mix well, and centrifuge at 1000 rpm for 5 minutes. After the supernatant was discarded, 10 mL of IMDM medium containing 10% serum (Excell Company) was added, the cells were gently blown up, and the prepared HAT complete medium (Sigma Company) was added to mix well, and the 96-well plate was spread. Put the culture plate with cells in 37°C, 5% CO 2 cultured in an incubator.

[0032] 2. Screening and subcloning: EL...

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Abstract

The invention provides an anti-MMAE monoclonal antibody as well as a preparation method and application thereof. The antibody comprises antigen complementary determining regions aaCDR1, aaCDR2 and aaCDR3 of a heavy chain variable region, and antigen complementary determining regions aaCDR1, aaCDR2 and aaCDR3 of a light chain variable region. The anti-MMAE monoclonal antibody disclosed by the invention can be applied to research in related ligand coupling drugs, and MMAE at a nanogram level or lower concentration can be detected.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to an anti-MMAE monoclonal antibody and its preparation method and application. Background technique [0002] Monomethyl auristatin E (MMAE; SGD-1010), a synthetic derivative of dolastatin 10, is a potent mitotic inhibitor by inhibiting tubulin polymerization. MMAE is widely used as a cytotoxic component to make antibody drug conjugates (ADCs) for cancer research. In the treatment of tumors, the use of small molecular compounds with anticancer activity is limited due to their toxicity to normal tissues, and when combined with targeting ligands (monoclonal antibodies, polypeptides, fusion proteins, etc.), the Active small molecules brought to the target area can effectively control the toxicity of small molecule compounds to normal cells. Therefore, ligand-drug conjugates, such as ADC (Antibody Drug Conjugate, ADC), PDC (peptidedrug conjugate) and other XDCs, came i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N5/20C12P21/08A61K39/395A61K47/64A61K47/68A61P35/00G01N33/68G01N33/577C12R1/91
CPCC07K16/18A61K39/3955A61K47/64A61K47/68A61P35/00G01N33/68G01N33/577C07K2317/56C07K2317/565A61K2039/505
Inventor 黄保华王中波
Owner SUZHOU IBIO TECH CO LTD
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