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Anti-MMAE monoclonal antibody, coding sequence and application thereof

A monoclonal antibody and coding technology, applied in the field of biochemistry, can solve problems such as toxic and side effects, and achieve the effects of reducing toxic and side effects, good binding activity, and accelerating metabolism

Active Publication Date: 2021-03-09
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the clinical application of ADC, small molecular toxicants such as MMAE can be non-specifically shed from the ADC molecule, causing toxic side effects

Method used

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  • Anti-MMAE monoclonal antibody, coding sequence and application thereof
  • Anti-MMAE monoclonal antibody, coding sequence and application thereof
  • Anti-MMAE monoclonal antibody, coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1: Preparation of anti-MMAE monoclonal antibody cDNA

[0023] (1) MMAE small-molecule antigen (obtained from Nanjing Lianning Biology) was mixed with complete Freund's adjuvant in equal volumes, and 4 4-5 week-old BALB / C female mice (obtained from the Southern Model Animal Center) were immunized. For the first immunization, the volume of each injection is 0.5ml;

[0024] (2) From the third week, mix 1 mg MMAE small molecule antigen with incomplete Freund's adjuvant in equal volume, immunize once every 3 weeks, the last immunization is 3-5 days before fusion, and immunize 5 times in total to stimulate B cells express antigen-specific antibodies;

[0025] (3) Antibody titer detection. For each mouse that has completed four immunizations, blood must be collected from the orbit, and then titer detection is performed. The detection time is generally 7-10 days after the fourth immunization.

[0026] (4) Myeloma cell preparation. One week before fusion, cryopreser...

Embodiment 2

[0033] Example 2: Cloning of Monoclonal Antibody Genes

[0034] (1) Acquisition of light and heavy chain genes: use mouse-specific primers to amplify the cDNA prepared in Example 1, clone the amplified DNA sequences of VH and VL into pUC19 vectors, and then carry out Transformation plated, picked clones for sequencing analysis.

[0035] (2) Analysis of the light and heavy chain variable region genes of the antibody: analyze the VH and VL according to the sequencing results, and finally obtain two light chain sequences and one heavy chain sequence (SEQ ID NO:5), combined with relevant biological information The final light chain sequence (SEQ ID NO: 6) was determined by chemical analysis, the nucleotide sequence of the heavy chain variable region was SEQ ID NO: 3, and the nucleotide sequence of the light chain variable region was SEQ ID NO: 4.

[0036] (3) Clone the light and heavy chain genes into the expression vector: clone the VH and VL sequences obtained from the analysi...

Embodiment 3

[0037] Example 3: Expression and purification of anti-MMAE antibody in eukaryotic expression system

[0038] (1) Cloning the correctly sequenced light and heavy chain genes into eukaryotic expression vectors pCAT1.0 and pCAT2.0, respectively;

[0039] (2) The cloned vector is further sequenced and identified, and then the correct clone is extracted into a plasmid;

[0040] (3) Culture HEK293F suspension cells, culture the cells in Freestyle 293 medium, when the density reaches 1×10 6 / ml, cell transfection was performed when the survival rate was >90%;

[0041] (4) Transfection method: the mass ratio of PEI / plasmid is 3:1, transfect according to the ratio of 1 μg plasmid / 1ml HEK293F cells;

[0042] (5) 5-7 days after transfection, collect the cell supernatant, perform affinity purification with Protein G resin to obtain high-purity antibody protein, and replace the elution buffer with PBS with an ultrafiltration column. The amino acid sequence of the heavy chain variable re...

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PUM

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Abstract

The invention relates to an anti-MMAE monoclonal antibody, a coding sequence and application thereof. The heavy chain variable region of the anti-MMAE monoclonal antibody has an amino acid sequence defined by SEQ ID NO: 1, and the light chain variable region has an amino acid sequence defined by SEQ ID NO: 2. The monoclonal antibody provided by the invention shows good binding activity with TROP2-ADC, and can be used as a detection reagent for MMAE coupled antibody drugs or as a scavenging drug for free MMAE in plasma.

Description

technical field [0001] The present invention relates to the field of biochemistry, specifically, the present invention relates to an anti-MMAE monoclonal antibody, its coding sequence and application. Background technique [0002] In the treatment of tumors, the use of small molecular compounds with anticancer activity is limited due to their toxicity to normal tissues, and coupling them with monoclonal antibodies can well solve the toxicity of small molecular compounds to normal cells The problem. Therefore, antibody-compound drug conjugates (Antibody Drug Conjugate, ADC) came into being. [0003] In the antibody-compound drug conjugate (ADC), there have been reports of monomethyl auristatin E (MMAE) conjugated antibody drug in the prior art, which has great efficacy in the treatment of Hodgkin's lymphoma and systemic degenerative disease. Good results have been shown in cell lymphoma. Therefore, in the prior art, there is a need for related reagents for detecting conjug...

Claims

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Application Information

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IPC IPC(8): C07K16/44C12N15/13G01N33/68G01N33/577A61K38/07A61K47/68A61P35/00
CPCC07K16/44G01N33/94G01N33/68G01N33/577A61K38/07A61K47/6811A61P35/00C07K2317/56C07K2317/51C07K2317/515C07K2317/92
Inventor 王春河谭小蓉陈艺丽王桂凤王琪
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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