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HBV specific double-targeted antibody as well as preparation method and application thereof, minicircle DNA containing double-targeted antibody expression box and application of minicircle DNA

A dual-targeted antibody, specific technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as BsAb infectious diseases that have not yet been seen, and achieve the effect of eliminating chronic HBV infection

Inactive Publication Date: 2017-01-25
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although BsAb has begun to be used for tumor treatment, the applicant has not yet seen the report of BsAb being used for the treatment of infectious diseases including HBV

Method used

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  • HBV specific double-targeted antibody as well as preparation method and application thereof, minicircle DNA containing double-targeted antibody expression box and application of minicircle DNA
  • HBV specific double-targeted antibody as well as preparation method and application thereof, minicircle DNA containing double-targeted antibody expression box and application of minicircle DNA
  • HBV specific double-targeted antibody as well as preparation method and application thereof, minicircle DNA containing double-targeted antibody expression box and application of minicircle DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] The design of the specific dual targeting antibody (BsAb) provided by the invention comprises the following steps:

[0174] a) first search for HBV T cell antigen (HBsAg and HBcAg) epitopes in the immune epitope database (Immune Epitope Database; IEDB);

[0175] b) Select several dominant antigenic epitopes (Dominant T cell epitope) and synthesize HBV antigenic peptides:

[0176] There are 21 HBcAg T cell epitopes in IEDB (some epitopes such as Figure 6 ); select HLA-A*02:01 (this HLA type is the most widely distributed in the population, especially Chinese, more than 60%; it makes BsAb applicable to a wider range) restricted antigenic epitopes, and the top ranked Antigen epitope (references>10; assays>10).

[0177] Finally, one HBcAg epitope was selected, the sequence is: FLPSDEFPSV

[0178] A total of 37 HBsAg T cell epitopes (some epitopes such as Figure 7 ), according to the same principle, two HBsAg epitopes were selected, and the sequences were: WLSLLVPFV; F...

Embodiment 2

[0202] The construction of the minicircle parent plasmid with the nucleotide sequence encoding the BsAb antibody comprises the following steps:

[0203] 1) Design BsAb antibody gene expression cassette

[0204] In addition to the nucleotide sequence encoding the BsAb of Example 1, the BsAb antibody gene expression cassette of Example 2 of the present invention also includes a coding sequence for a promoter, a secretion signal peptide, a protein purification tag, and a polyA tailing signal.

[0205] In a specific embodiment, the BsAb antibody gene expression cassette of Example 2 of the present invention includes sequentially connected:

[0206] Nucleotide sequence (SEQ ID NO: 21) encoding the signal peptide of the immunoglobulin κ chain - the nucleotide sequence (SEQ ID NO: 22) encoding the Flag tag - the gene sequence of the BsAb antibody (antiHBV-V L —antiHBV-V H —antiCD3-V H —antiCD3-V L ; The specific nucleotide sequence corresponds to SEQ ID NO: 17 in Table 2? -linke...

Embodiment 6

[0219] Steps of Example 6 "A Method for Constructing a Minicircular DNA Recombination Mother Plasmid pMC.Bab Containing a Genetic Engineering Antibody Gene Expression Cassette"; to obtain the pMC.BESXP having the nucleotide sequence encoding the BsAb antibody in Example 2 of the present invention Minicircle parent plasmid.

[0220] The only difference between step 1-2) of the present invention and the above PCT application is that step 1-2) of embodiment 2 of the present invention replaces the "gene sequence of BiTE" in embodiment 1 or 6 of the above PCT application with the embodiment of the present invention 2 "BsAb Antibody Gene".

[0221] B)

[0222] Directly adopt the method of seamless cloning, design about 15 bp homologous sequences at both ends of the BsAb antibody gene expression cassette and both ends of the pMC.BESXP empty vector or p2ФC31 empty vector, and insert the synthesized BsAb antibody gene expression cassette directly into p2ФC31 Between the attB and attP...

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Abstract

The invention provides a specific double-targeted antibody as well as a method and application thereof. The specific double-targeted antibody has a first antigen combination structural domain with the specificity combined with human CD3 and a second antigen combination structural domain with the specificity combined with an HBV (Hepatitis B Virus), can specifically identify and be combined with HBV antigen positive cells, can be also combined with TCR (T Cell Receptor), mediates T cells to develop the killing effect so as to clear away chronic HBV infection, and has extremely large application value in diagnosis, treatment, prevention and detection of HBV related diseases. The invention further provides a minicircle DNA with a nucleotide sequence for coding the double-targeted specific antibody. The specific double-targeted antibody and the minicircle DNA which are provided by the invention can be used for preparing an antibody medicine composition or an HBV diagnosis reagent.

Description

technical field [0001] The invention relates to the fields of biomedicine and diagnosis and treatment, in particular to a hepatitis B virus-specific dual-targeting antibody, a preparation method and application thereof, and a microcircle DNA containing the expression cassette of the dual-targeting antibody and its application. Background technique [0002] Hepatitis B virus (HBV), a member of the hepadnaviridae family, causes acute and chronic hepatitis. Approximately 350 million people in the world's population, especially 5-8% of the population in Korea and China are chronic HBV patients, and HBV is the main cause of liver disease and liver cancer. Although the development of anti-HBV vaccines has made it possible to prevent hepatitis B, many people still suffer from chronic hepatitis due to HBV infection. HBV infection can cause hepatitis, cirrhosis and liver cancer, and the incidence of liver cancer in patients with chronic hepatitis is 300 times higher than that of nor...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/42A61K39/395A61P31/20C12N15/13
Inventor 陈志英谌平何成宜徐文娟
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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