Anti-CD44v6 and anti-CD3 bispecific antibody, minicircle DNA containing anti-CD44v6 and anti-CD3 bispecific antibody expression cassette, and applications of anti-CD44v6 and anti-CD3 bispecific antibody and minicircle DNA

A dual-targeting antibody and specific technology, applied in the direction of antibodies, medical preparations with non-active ingredients, anti-tumor drugs, etc., can solve the problems of inability to mediate T cell killing, inability to bind T cells, etc.

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

The antibody can only bind to CD44v6 antigen and CD44v6-positive cells, it cannot bind to T cells, and cannot mediate T cell killing; it can only be targeted and killed by ADCC (antibody-dependent cell-mediated cytotoxicity) or C...

Method used

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  • Anti-CD44v6 and anti-CD3 bispecific antibody, minicircle DNA containing anti-CD44v6 and anti-CD3 bispecific antibody expression cassette, and applications of anti-CD44v6 and anti-CD3 bispecific antibody and minicircle DNA
  • Anti-CD44v6 and anti-CD3 bispecific antibody, minicircle DNA containing anti-CD44v6 and anti-CD3 bispecific antibody expression cassette, and applications of anti-CD44v6 and anti-CD3 bispecific antibody and minicircle DNA
  • Anti-CD44v6 and anti-CD3 bispecific antibody, minicircle DNA containing anti-CD44v6 and anti-CD3 bispecific antibody expression cassette, and applications of anti-CD44v6 and anti-CD3 bispecific antibody and minicircle DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0149] The construction of the minicircle parent plasmid with the nucleotide sequence encoding the CD44v6×CD3BsAb antibody comprises the following steps:

[0150] 1) Design BsAb antibody gene expression cassette

[0151] 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.

[0152] In a specific embodiment, the BsAb antibody gene expression cassette of Example 1 of the present invention includes sequentially connected:

[0153] Nucleotide sequence (Murine Ig kapa-chain signal peptide, SEQ ID NO: 17)-BsAb antibody gene sequence (V L CD44v6-linker1-V H CD44v6-linker2-V H CD3-linker3-V L CD3; the specific nucleotide sequence corresponds to CD44v6LCVR-linker1-CD44v6HCVR-linker2-CD3HCVR-linker3-CD3LCVR in Table 2)-the nucleotide sequence (SEQ ID NO:18)-encoding histid...

Embodiment 6

[0190] Steps of Example 6 "A Method for Constructing a Microcircular DNA Recombination Mother Plasmid pMC.Bab Containing Genetic Engineering Antibody Gene Expression Cassette"; to obtain the pMC with the nucleotide sequence encoding CD44v6×CD3BsAb antibody in Example 2 of the present invention .BESXP minicircle parent plasmid.

[0191] 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 CD44v6×CD3BsAb" of 2.

[0192] B)

[0193] 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 and...

Embodiment 2

[0196] Construction of microcircle DNA with nucleotide sequence encoding CD44v6×CD3BsAb antibody

[0197] 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 CD44v6 antigen, can specifically recognize and bind to CD44v6 antigen and CD44v6 overexpression tumor cells including liver cancer and other CD44v6-positive tumor diseases, can further bind to T cell receptors, and can mediate T cells to provide killing effects, such that CD44v6 infections can be cleared, and great application valuescan be provided in diagnosis, treatment, prevention and detection of CD44v6-related diseases. The present invention further provides minicircle DNA having a nucleotide sequence encoding the bispecificantibody. According to the present invention, the bispecific antibody and the minicircle DNA can be used for preparing antibody pharmaceutical compositions or CD44v6-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-CD44v6 and CD3 specific dual-targeting antibody, a microcircle DNA containing the expression cassette of the dual-targeting antibody, and applications. Background technique [0002] CD44 molecule is an integral membrane protein on the cell surface and belongs to the adhesion molecule family. Its normal function is to act as a receptor to recognize hyaluronic acid (HA) and collagen I and IV, and is mainly involved in immune recognition, lymphocyte aggregation and cell-cell, cell - Specific adhesion process between substrates. It can be divided into standard type (CD44s) and splice variant type (CD44v). Currently, it is believed that CD44 splicing variant 6 (CD44v6) is closely related to the degree of tumor progression, invasion, and metastasis. It is mainly involved in the heterogeneous adhesion of tumor cells to host cells and host matrix, enablin...

Claims

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Application Information

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IPC IPC(8): C07K16/46A61K39/395A61K47/68A61P35/00
Inventor 刘运洪陈志英何成宜钟育健张婧
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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