Anti-GPC3 and anti-CD3 bispecific antibody, minicircle DNA containing anti-GPC3 and anti-CD3 bispecific antibody expression cassette, and applications of anti-GPC3 and anti-CD3 bispecific antibody and minicircle DNA

A dual-targeting antibody and specific technology, applied in applications, antibodies, anti-tumor drugs, etc., can solve problems such as inability to bind T cells and inability to mediate T cell killing

Inactive Publication Date: 2018-01-09
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

However, Codrituzumab can only bind to GPC3 antigen and GPC3-positive cells, it cannot bind to T cells, and cannot mediate T cell killing; it can only target and kill GPC3 antigen-positive tumor cells through ADCC or CDC

Method used

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  • Anti-GPC3 and anti-CD3 bispecific antibody, minicircle DNA containing anti-GPC3 and anti-CD3 bispecific antibody expression cassette, and applications of anti-GPC3 and anti-CD3 bispecific antibody and minicircle DNA
  • Anti-GPC3 and anti-CD3 bispecific antibody, minicircle DNA containing anti-GPC3 and anti-CD3 bispecific antibody expression cassette, and applications of anti-GPC3 and anti-CD3 bispecific antibody and minicircle DNA
  • Anti-GPC3 and anti-CD3 bispecific antibody, minicircle DNA containing anti-GPC3 and anti-CD3 bispecific antibody expression cassette, and applications of anti-GPC3 and anti-CD3 bispecific antibody and minicircle DNA

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Experimental program
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Effect test

Embodiment 1

[0148] The construction of the minicircle parent plasmid with the nucleotide sequence encoding the GPC3×CD3BsAb antibody comprises the following steps:

[0149] 1) Design BsAb antibody gene expression cassette

[0150] In addition to the nucleotide sequence encoding BsAb, the BsAb antibody gene expression cassette of Example 1 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.

[0151] In a specific embodiment, the BsAb antibody gene expression cassette of Example 1 of the present invention includes sequentially connected:

[0152] Nucleotide sequence (Murine Ig kapa-chain signal peptide, SEQ ID NO: 17)-BsAb antibody gene sequence (V L GPC3-linker1-V H GPC3-linker2-V H CD3-linker3-V L CD3; specific nucleotide sequence corresponding to GPC3LCVR-linker1-GPC3HCVR-linker2-CD3HCVR-linker3-CD3LCVR-nucleotide sequence (SEQ ID NO: 18)-double stop codon in Table 2-encoding hist...

Embodiment 6

[0189]Steps of Example 6 "A method for constructing a microcircular DNA recombination mother plasmid pMC.Bab containing a genetically engineered antibody gene expression cassette"; to obtain the pMC with the nucleotide sequence encoding the GPC3×CD3BsAb antibody in Example 2 of the present invention .BESXP minicircle parent plasmid.

[0190] 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 "Gene sequence of GPC3×CD3BsAb" of 2.

[0191] B)

[0192] Directly adopt the method of enzyme digestion-ligation, design two restriction sites of SmaI-ApaI at both ends of the BsAb antibody gene expression cassette and pMC.BESXP empty vector or p2ФC31 empty vector, and synthesize the BsAb antibody gene expression cassette by enzyme digestion- Insert the attB...

Embodiment 2

[0195] Construction of Microcircle DNA with Nucleotide Sequence Encoding GPC3×CD3BsAb Antibody

[0196] Referring to the description of the PCT application (application number: PCT / CN2014 / 083741) submitted by the inventor in 2014:

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Abstract

The present invention provides a bispecific antibody, a method and applications thereof. The bispecific antibody has a first antigen binding domain capable of specifically binding to human CD3 and a second antigen binding domain capable of specifically binding to GPC3 antigen, can specifically recognize and bind to GPC3 antigen and GPC3 overexpression tumor cells including liver cancer and other GPC3-positive tumor diseases, can further bind to T cell receptors, and can mediate T cells to provide killing effects, such that GPC3 infections can be cleared, and great application values can be provided in diagnosis, treatment, prevention and detection of GPC3-related diseases. The present invention further provides minicircle DNA having a nucleotide sequence encoding the bispecific antibody. According to the present invention, the bispecific antibody and the minicircle DNA can be used for preparing antibody pharmaceutical compositions or GPC3-positive tumor cell diagnosis reagents.

Description

technical field [0001] The invention relates to the fields of biomedicine and diagnosis and treatment, in particular to an anti-GPC3 and CD3 specific dual-targeting antibody, a microcircle DNA containing the expression cassette of the dual-targeting antibody and applications thereof. Background technique [0002] The GPC3 (glypican 3) gene is located in the Xq26.1 region of the human chromosome, and the relative molecular mass of the encoded GPC3 protein is about 70Kda, consisting of 580 amino acids. The core protein of GPC3 consists of two parts, a 40kDa amino-terminal (N-) fragment of GPC3 and a 30kDa carboxy-terminal (C-) fragment of GPC3. In 1996, Pilia et al. first reported that patients with GPC3 gene mutation and loss of function developed overgrowth syndrome (SGBS), which is an X-linked abnormal disease, mainly manifested as prenatal and postpartum fetal overgrowth, accompanied by There are widespread visceral and skeletal abnormalities and an increased likelihood o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C12N15/13G01N33/574A61K39/395A61P35/00A61K35/17
Inventor 刘运洪陈志英何成宜钟育健
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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