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4D5-targeting mouse-derived monoclonal antibody as well as preparation method and application thereof

A monoclonal antibody, mouse-derived technology, applied in the field of immunodetection, can solve the problems of indistinguishable, low sensitivity, poor specificity, etc.

Active Publication Date: 2020-09-01
BEIJING DCTY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these existing detection reagents still have some shortcomings, and the detection effect is unsatisfactory
Among them, anti-Protein L has poor specificity in detecting HER2CAR, and cannot distinguish different target CARs (such as CD19CAR, CD22CAR, etc.) constructed from different murine scFvs.
The antigen protein detection method relies on the binding of HER2 protein to the 4D5ScFv part of the CAR. Since the affinity of the scFv to HER2 is significantly lower than that of the intact antibody Herceptin to HER2, the sensitivity is also low

Method used

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  • 4D5-targeting mouse-derived monoclonal antibody as well as preparation method and application thereof
  • 4D5-targeting mouse-derived monoclonal antibody as well as preparation method and application thereof
  • 4D5-targeting mouse-derived monoclonal antibody as well as preparation method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Establishment of idiotype antibody hybridoma cell line targeting 4D5 antibody

[0047] 1. Test materials

[0048] 1.1 Animals and Cells

[0049] SPF grade BALB / C mice (female 6-8 weeks old)

[0050] SP2 / 0 cells

[0051] 1.2 Main reagents

[0052]

[0053]

[0054] 1.3 Main instruments and consumables

[0055] I×70 inverted microscope Labofuge stratos centrifuge

[0056] Type 381 CO 2 Incubator Ultra-clean bench

[0057] Pure water equipment Cell culture plate (96 and 24 well plate)

[0058] 1.4 Reagent preparation

[0059] 7.5% NaHCO 3 Solution: weigh analytically pure NaHCO 3 Dissolve 7.5g in 100mL triple-distilled water, filter and sterilize with a 0.22μm microporous membrane, dispense into vials, and freeze at -20°C for later use.

[0060] Cell culture medium: DMEM culture stock solution, the specific preparation method is operated according to the instructions, sterilized by filtration with a 0.22 μm microporous membrane, and stored at 4°C for later...

Embodiment 2

[0132] Preparation and sequence determination of anti-4D5 single chain antibody 5D1

[0133] The anti-4D5 single-chain antibody 5D1 is composed of the heavy chain variable region sequence of the anti-4D5 monoclonal antibody 5D1, the light chain variable region sequence and the linking sequence connecting the heavy chain variable region sequence and the light chain variable region sequence .

[0134] The preparation method of the anti-4D5 single-chain antibody 5D1 is as follows:

[0135] By TRIZOL reagent (Thermo Fisher), according to the supplier's instructions, extract the total RNA of 5D1 hybridoma cells, as follows: 10 6 After adding TRIZOL reagent to homogenize each cell, add chloroform and centrifuge at 12,000×g centrifugal force for 10min at 2-8°C, take the supernatant and add 0.5mL isopropanol; refrigerate and centrifuge at 12,000×g high-speed centrifugal force for 10min. Wash the RNA pellet once with 75% ethanol, dry the RNA pellet, and dissolve the RNA in RNase-free...

Embodiment 3

[0141] Preparation of recombinant vector

[0142] Arrange the expression sequence in the form of IL-2 signal peptide-5D1 single-chain antibody region, and clone it into the pFUSE vector. Specifically, the sequences of each fragment are as follows:

[0143] Nucleotide sequence of IL-2 signal peptide: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACGAATTCG (SEQ ID No. 9).

[0144] The amino acid sequence of the heavy chain variable region (the bold sequence is the CDR region):

[0145] QVQLEQSGAEFVRPGALVRLSCKASGFNIKDYYIHWVKQRPEQGLEWIGWIYPENDDTIYDPKFQGKASLTADTSSNTAYLQLSLTSEDSAVYFCVRSGSDSSFDYWGQGTTLTVSS (SEQ ID No. 1)

[0146] The nucleotide sequence of the heavy chain variable region (the bold sequence is the nucleotide sequence of the CDR region):

[0147] CAAGTCCAACTTGAACAATCAGGAGCAGAATTTGTGAGACCTGGAGCACTTGTGAGACTTTCATGCAAAGCATCAGGATTTAACATCAAAGATTACTACATCCACTGGGTGAAACAAAGACCTGAACAAGGACTTGAATGGATCGGATGGATCTACCCTGAGAATGACGATACAATCTACGATCCTAAATTTCAAGGAAAGGCTTCGCTTACAGCAGAT...

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Abstract

The invention provides a 4D5-targeting mouse-derived monoclonal antibody and application thereof, and belongs to the technical field of immuno-detection. The 4D5-targeting mouse-derived monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1; and the amino acid sequence of the light chain variable region is shown as SEQ ID No.2. Western blot experimental results show that an obvious band is formed by the HRP-labeled mouse-derived monoclonal antibody subjected toin-vitro recombinant expression and 4D5, and indicate that the mouse-derived monoclonal antibody has the characteristic of targeting 4D5. The invention also discloses an application of the 4D5-targeting mouse-derived monoclonal antibody or recombinant vector in the preparation of a kit for detecting a 4D5 antibody or 4D5 chimeric antigen receptor positive cells. Compared with other detection methods, the mouse-derived monoclonal antibody 5D1 has the strongest 4D5-CAR structure binding capacity, and the detection sensitivity is greatly improved.

Description

technical field [0001] The invention belongs to the technical field of immune detection, and in particular relates to a mouse monoclonal antibody targeting 4D5 and its application. Background technique [0002] With the development of tumor immunotherapy, chimeric antigen receptor (chimeric antigen receptor, CAR)-T cell immunotherapy, which combines the advantages of antibodies and immune cells, has received great attention. CAR is mainly composed of an antigen-binding region outside the cell membrane and an intracellular signal transduction region through a hinge region and a transmembrane region. The extramembrane antigen-binding region has the function of specifically recognizing and binding target cell surface antigens, which constitutes a single-chain variable region domain (SingleChain Variable Fragment, scFv) derived from a monoclonal antibody, composed of a heavy chain variable region (VH) and light chain variable region (VL). The intracellular signal transduction ...

Claims

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Application Information

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IPC IPC(8): C07K16/32C12N15/13C12N15/85C12N15/62G01N33/68G01N33/577G01N33/569
CPCC07K14/55C07K16/32C07K2317/56C07K2319/02C12N15/85C12N2800/107G01N33/56966G01N33/577G01N33/6854G01N2333/82G01N2469/10
Inventor 焦顺昌陈静远袁翰李斯慧冯向辉
Owner BEIJING DCTY BIOTECH CO LTD
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