Anti-cancer surface antigen of p185 mono-antigen mosaic antibody and its preparation

A chimeric antibody and surface antigen technology, which is applied in the technical field to achieve the effect of saving time and cost and reducing workload

Inactive Publication Date: 2006-01-25
HEFEI HANKEMAB BIOTECH CO LTD
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AI Technical Summary

Problems solved by technology

[0013] Chinese "Journal of Immunology" (2000.16: 539-543) proposed a method for preparing anti-p185 monoclonal antibody hybridoma cell lines after immunization with surface epitope embedding method (SEM). Tumor cells with high expression of p185 have good membrane localization, and show a characteristic inhibitory effect on the growth of p185 high expression cells, but there is no research on the humanization of monoclonal antibodies obtained by surface epitope embedding method report

Method used

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  • Anti-cancer surface antigen of p185 mono-antigen mosaic antibody and its preparation
  • Anti-cancer surface antigen of p185 mono-antigen mosaic antibody and its preparation
  • Anti-cancer surface antigen of p185 mono-antigen mosaic antibody and its preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、A21

[0066] Example 1. Construction of A21 single chain antibody (A21ScFv)

[0067] The construction process of single chain antibody is as follows figure 1 Shown: Total RNA is extracted from hybridoma cells; reverse transcription (RT) and polymerase chain amplification reaction (PCR) are combined to obtain antibody light chain and heavy chain variable regions (VL and VH), respectively The gene sequence of the cloned gene is determined; based on the sequence determination, the primers used to construct the single-chain antibody are synthesized to add restriction endonuclease sites at both ends of the amplified fragment; the fragment overlap extension PCR is The SOE-PCR method connects the heavy and light chain variable regions through a linker peptide linker, and recombines to form a single-chain antibody fragment in the form of VL-linker-VH; then, the fragment is double-enzyme digested and recovered and the same is double-enzyme digested The treated vector (phagemid) named pCANTAB 5E...

Embodiment 2

[0177] Example 2. Construction and expression of scFv-Fc chimeric antibody molecule:

[0178] The construction of the anti-tumor surface antigen p185 monoclonal antibody chimeric antibody scFv-Fc in this example is to combine the single chain antibody scFv that has been experimentally proven to specifically bind to the p185 antigen and the constant region Fc of the human antibody IgG1 (hu IgG1). Gene operation is connected to form a recombinant single-chain antibody fusion gene, the chimeric antibody gene scFv-Fc. The gene is first cloned into the transient expression vector pSecTag and transfected into the transient expression cell COS7, and the transiently expressed chimeric antibody is measured. After confirming that it has the specific binding activity with p185 antigen, the fusion gene A21scFv-hu IgG1Fc or scFv-Fc gene is cloned into pEE14 vector plasmid DNA containing glutamine synthetase GS gene, and then transfected into mammalian cells CHO, by continuously increasing the ...

Embodiment 3

[0263] Example 3. Activity identification of scFv-Fc chimeric antibody molecule

[0264] Experimental steps:

[0265] 3.1 ELISA test for the antigen binding activity of chimeric antibody:

[0266] This experiment is basically the same as the ELISA experiment procedure of single-chain antibody in 1.5 of Example 1, except that the secondary antibody is changed to goat-anti-human HRP secondary antibody.

[0267] 3.2 ELISA analysis results of the antigen binding characteristics of chimeric antibodies:

[0268] Figure 6 The histograms of the ELISA analysis results of the antigen binding activity of the chimeric antibody are given. In the figure a and b: the supernatant of COS-7 transfected with pSectag recombinant vector containing A21ScFv-Fc (the coating antigen of a is T6-17 cell lysate , The coating antigen of b is NIH / 3T3 cell lysate); c and d: the supernatant of COS-7 transfected with pSectag vector alone (that is, without A21 ScFv-Fc) (the coating antigen of c is the same as th...

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Abstract

This invention relates to a mono-chain antibody-humanscFv-Fc embedded antibody and its preparation, specialized by hybrid tumor cell strain prepared by surface embedded immune method to transfect mammary animal cell CHO with GS low expression in form of mono-chain embedded antibody gene with pEE14 as recombination antibody carrier to screen CHO cell strains of recombination antibody. The embedded antibody molecule consists of two similar recombined mono-chains, of which each has an antigen combining region connected with heavy and light chains to form antibody to identify antigen. It is smaller a third than a complete antibody due to constant regions CHI structure zones of light and heavy chains removed, so that it is easier to penetrate into tumor and is not easy to be degraded like protein peptide too soon.

Description

Technical field: [0001] The present invention belongs to the technical field of anti-tumor surface antigen p185 single-chain antibody-human antibody constant region (scFv-Fc) chimeric antibody, and particularly relates to the cloning of monoclonal antibody genes, the recombination of antibody variable region genes and the combination with human antibody constant region genes Linking and constructing engineered antibodies, and transforming the mammalian-Chinese hamster ovary cell line CHO with the eukaryotic expression vector containing the engineered antibodies, so as to screen out a method for preparing chimeric antibody cell lines. Background technique: [0002] According to the medical branch of the American journal Nature (Nat Med, 2003, 9:129-134), monoclonal antibody technology has been successfully used to construct hybridomas against a specific antigen, and is used in the diagnosis and diagnosis of many diseases. Played a huge role in treatment. However, since all monoclo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K19/00C12P21/00
Inventor 刘兢程联胜
Owner HEFEI HANKEMAB BIOTECH CO LTD
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