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Anti-cancer surface antigen of p185 monoantigen embedded antibody and its preparation

A technology of chimeric antibodies and surface antigens, applied in chemical instruments and methods, hybrid peptides, anti-animal/human immunoglobulins, etc.

Inactive Publication Date: 2005-05-11
HEFEI HANKEMAB BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 monoantigen embedded antibody and its preparation
  • Anti-cancer surface antigen of p185 monoantigen embedded antibody and its preparation
  • Anti-cancer surface antigen of p185 monoantigen embedded antibody and its preparation

Examples

Experimental program
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Effect test

Embodiment 1、A21

[0066] Embodiment 1, Construction of A21 single-chain antibody (A21ScFv)

[0067] The single-chain antibody construction process is as follows: figure 1 Shown: total RNA was extracted from hybridoma cells; antibody light and heavy chain variable regions (VL and VH) were obtained by combining reverse transcription (RT) and polymerase chain amplification reaction (PCR) The gene of the clone is determined, and the sequence of the cloned gene is determined; on the basis of 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 variable regions of the heavy chain and the light chain through a connecting peptide linker, and recombines to form a single-chain antibody fragment in the form of VL-linker-VH; then, the fragment is recovered by double enzyme digestion and the same with double enzyme...

Embodiment 2

[0180] Example 2, construction and expression of scFv-Fc chimeric antibody molecules:

[0181] The construction of the anti-tumor surface antigen p185 monoclonal antibody chimeric antibody scFv-Fc described in this example is to combine the single-chain antibody scFv that can specifically bind to the p185 antigen and the constant region Fc of the human antibody IgG1 (hu IgG1) through the The gene operation is connected to form a recombinant single-chain antibody fusion gene, that is, 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 determined After confirming that it has the activity of specifically binding to the p185 antigen, the fusion gene A21scFv-hu IgG1Fc, that is, the scFv-Fc gene, is cloned into the pEE14 vector plasmid DNA containing the glutamine synthetase GS gene, and then transfected into mammalian cell...

Embodiment 3

[0269] Example 3, Activity Identification of scFv-Fc Chimeric Antibody Molecules

[0270] Experimental steps:

[0271] 3.1 ELISA identification experiment of chimeric antibody antigen binding activity:

[0272] This experiment is basically the same as the ELISA experiment procedure of the single-chain antibody in 1.5 of Example 1, except that the secondary antibody is changed to goat-anti-human HRP secondary antibody.

[0273] 3.2 Antigen-binding properties of chimeric antibodies ELISA analysis results:

[0274] Image 6 The histogram of the ELISA analysis results of the antigen-binding activity of the chimeric antibody is given. In the figure a and b: the supernatant of COS-7 transfected with the 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 t...

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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 invention belongs to the technical field of anti-tumor surface antigen p185 single-chain antibody-human antibody constant region (scFv-Fc) chimeric antibody technology, and in particular relates to the cloning of monoclonal antibody genes, gene recombination of antibody variable regions and integration with human antibody constant region genes A method of linking and constructing an engineered antibody, and transforming the eukaryotic expression vector containing the engineered antibody into the mammalian-Chinese hamster ovary cell line CHO, so as to screen the cell line that can be used to prepare the chimeric antibody. Background technique: [0002] According to the medical branch of the American "Nature" magazine (Nat Med, 2003, 9: 129-134), monoclonal antibody technology has been successfully used to construct hybridomas targeting a specific antigen, and has been used in the diagnosis and treatment of many diseases. Played a huge role in treat...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K19/00C12P21/00
Inventor 刘兢程联胜
Owner HEFEI HANKEMAB BIOTECH CO LTD
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