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Anti-HER2 dual-targeting antibodies, and preparation method and application thereof

A dual-targeted antibody, antibody technology, applied in the biological field, can solve the problem of difficult to kill tumors and other problems

Inactive Publication Date: 2015-05-13
张帆
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

More importantly, due to the complexity of tumorigenesis, it is difficult to kill tumors more effectively and overcome tumor recurrence by relying only on monoclonal antibodies targeting a single site

Method used

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  • Anti-HER2 dual-targeting antibodies, and preparation method and application thereof
  • Anti-HER2 dual-targeting antibodies, and preparation method and application thereof
  • Anti-HER2 dual-targeting antibodies, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1. Trastuzumab and Pertuzumab Antibody heavy chain variable region, Pertuzumab Cloning of light chain variable region gene

[0062] Use Trastuzumab or Pertuzumab antibody heavy chain as a template to clone Trastuzumab and Pertuzumab heavy chain variable region genes and Pertuzumab light chain variable region gene by PCR respectively. The reaction conditions are: 95°C for 15 minutes; 94°C for 50 seconds, 58°C for 50 seconds , 72°C for 50 seconds, 30 cycles; 72°C for 10 minutes to obtain the PCR products Trastuzumab HV, Pertuzumab HV and Pertuzumab LV. Antibody signal peptide amino acid sequence MGWSCIILFLVATATGVHS. The correct cloned fragments were obtained by sequencing. SEQ ID NO: 2 shows the amino acid sequence of Trastuzumab heavy chain variable region, its nucleotide sequence is SEQ ID NO: 1; SEQ ID NO: 3 is the nucleotide sequence of Pertuzumab heavy chain variable region, its amino acid sequence is SEQ ID NO: 4; SEQ ID NO: 6 shows the amino acid sequ...

Embodiment 2

[0063] Example 2. Antibody light chain constant region, heavy chain constant region 1, Fc area cloning

[0064] Lymphocyte separation medium (product of Dingguo Biotechnology Development Co., Ltd.) was used to separate healthy human lymphocytes, and total RNA was extracted with Trizol reagent (product of Invitrogen Company). According to the literature (Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov;22(1 Pt 1):197-207.) and literature (The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10;10(13):4071-9.) RT-PCR was used to amplify the antibody light chain constant region, heavy chain constant region CH1 and Fc region genes. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, ...

Embodiment 3

[0065] Example 3. Antibody Fc Area knob Construction of mutants

[0066] The Fc region of the antibody obtained in Example 2 was introduced into the mutation point by overlapping PCR method: T366W, S354C (Schaefer WW, Regula JTJ, Böhner MM, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci U S A. 2011;108(27):11187–11192.). The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed that the correct clone was obtained. SEQ ID NO:14 shows the amino acid sequence of Fc-knob, and its nucleotide sequence is SEQ ID NO:13.

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Abstract

The invention discloses anti-HER2 dual-targeting antibodies, a preparation method thereof and application of the anti-HER2 dual-targeting antibodies in the preparation of antibody tumor drugs. More specifically, the invention discloses the dual-targeting antibodies Tras-Per CrossMab and Tras-Permut CrossMab with structures and functions similar to the structures and functions of complete antibodies, and amino acid sequences and the preparation method thereof, and application of the dual-targeting antibodies in the preparation of antitumor drugs. According to the invention, the functions similar to the functions of the complete antibodies are retained; HER2 target protein is dually targeted; and antitumor activity of the anti-HER2 dual-targeting antibodies is better than the antitumor activity of single-targeting anti-HER2 antibodies-Trastuzumab and Pertuzumab.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention discloses a class of anti-HER2 dual-targeting antibody, its preparation method and its application in the preparation of antibody tumor drugs. The anti-HER2 dual-targeting antibody disclosed in the present invention is a HER2 dual-targeting antibody that has a structure and function similar to that of a full antibody. Antibody Trastuzumab, Pertuzumab better anti-tumor activity. Background technique [0002] Tumor, especially malignant tumor, is a disease that seriously endangers human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend. The curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor. Although conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently us...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K16/30C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
Inventor 张帆赵磊杨俊兰
Owner 张帆
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