Fusion protein of Her2 antibody and interleukin 2 and application thereof

A technology of interleukin and fusion protein, applied in the field of fusion protein of anti-Her2 antibody and interleukin 2, which can solve the problems of HF clinical application limitation, low biological activity and low expression level, and achieve biological activity and yield Improved effect

Inactive Publication Date: 2011-09-28
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, HF has the following defects: 1. Low biological activity; 2. Low expression level, which cannot be used for large-scale production
These defects have greatly restricted the clinical application of HF. It is urgent to carry out molecular modification of HF to overcome the bottleneck of large-scale production, so that HF ​​can be applied to the clinical treatment of Her2-positive breast cancer.

Method used

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  • Fusion protein of Her2 antibody and interleukin 2 and application thereof
  • Fusion protein of Her2 antibody and interleukin 2 and application thereof
  • Fusion protein of Her2 antibody and interleukin 2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Construction of Anti-Her2 Antibody-Interleukin 2 Fusion Protein Gene Expression Vector

[0033]Using the fusion protein gene vector pCID / HF containing anti-ErbB-2 scFv, Fc segment of human IgG1, and IL-2 as a template (the nucleotide sequence of the fusion protein in the vector is shown in SEQ ID No.5 in the sequence listing), Use primers P1 (its nucleotide sequence as shown in SEQ ID No.6 in the sequence listing) and P2 (its nucleotide sequence as shown in SEQ ID No.7 in the sequence listing) to PCR amplify the single Cloning of antibody heavy chain signal peptide gene sequence, ErbB2 antibody heavy chain variable region gene, Linker and light chain variable region gene and human antibody Fc gene fragment, the conditions are: denaturation at 95°C for 2 minutes; then denaturation at 94°C for 1 minute, annealing at 58°C 1min, 72°C extension for 2min, 30 cycles; finally 72°C extension for 10min. Insert the expression vector pCID with NheI and XhoI sites to obta...

Embodiment 2

[0046] The screening of embodiment 2 engineering cell lines and the expression of HFI

[0047] CHO / dhfr- cells were cultured in IMDM medium containing 10% FCS, 0.1mol / L hypoxanthine, and 0.016mol / L thymidine. According to the instructions of Lipofectamine reagent, the recombinant vector PCI / HFI was transfected into CHO / dhfr- cells. After the transfected cells were cultured for 48 hours, the transient expression of the fusion protein was detected by the sandwich ELISA method, and the method was the same as before. The transfected cells were cloned and cultured by limiting dilution method, and the culture medium was containing 10% dialyzed FCS, 10 -6 MTX in DMEM medium. During the screening process, at different time points, the antibody expression level of each cell clone was detected by sandwich ELISA method. The specific process is as follows: Coat a 96-well plate with goat anti-human IgG (2 μg / ml), overnight at 4°C. After blocking with 2% bovine serum albumin for 1 h, th...

Embodiment 3

[0051] Example 3 HFI and HF antigen binding activity and biological activity detection experiment

[0052] Collect 1×10 6 SKBR3 cells were washed with PBS buffer, added purified HFI and HF, incubated for 30 minutes with ice bath shaking, washed 3 times with PBS, added FITC-labeled goat anti-human IgG, incubated for 30 minutes under ice bath shaking, washed with PBS Three times, analyzed on the machine (Becton Dickinson Company), and the cells not incubated with the culture supernatant of transfected cells were used as negative control.

[0053] The cultured CTLL-2 cells were washed 3 times with 1640 medium, after counting, 3 × 10 4 Inoculate each well in a 96-well plate, add purified HFI and HF at the same time, and use the doubly diluted IL-2 standard as a positive control. After culturing for 18 hours, 10 μl of MTT (5 mg / ml) was added, and the culture was continued for 4 hours. The cells were lysed with 10% SDS-0.01mol / L HCl, and the A490 value was measured.

[0054] The...

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Abstract

The invention discloses a fusion protein of Her2 antibody and interleukin 2 and application thereof, and belongs to the technical fields of medical science and oncology phymatology. The fusion protein comprises CD16 monoclonal antibody heavy chain signal peptide, ErbB2 antibody heavy chain variable region, a Linker 1, a light chain variable region, a human antibody Fc segment, a Linker 2 and IL-2 mature peptide, and has the amino acid sequence shown as SEQ ID No.1. The fusion protein can inhibit Herceptin resistant breast cancer cell proliferation, and can kill Her2 high-expression breast cancer cells. The invention lays a foundation for clinical application of the fusion protein of the Her2 antibody and the interleukin 2 in the anti-tumor aspect.

Description

technical field [0001] The invention belongs to the technical field of medical oncology, and in particular relates to a fusion protein of anti-Her2 antibody and interleukin 2 and its application. Background technique [0002] The natural effector functions of antibodies are weak, and the presence of high concentrations of circulating IgG can interfere with the binding of antibodies to FcRs on the surface of effector cells. Therefore, enhancing the effector function of antibodies has always been an important direction in the field of antibody research. Since the 1980s, a series of research work on antibodies carrying effector molecules has been carried out at home and abroad, in an attempt to enhance the effector function of antibodies. In the late 1980s, the role of immunomodulatory factors in enhancing cellular immune responses and anti-idiotypic immune responses in tumor immunotherapy received great attention. Interleukin 2 (IL-2) is an important mediator molecule in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21C12N15/11A61K39/395A61P35/00A61K38/20
Inventor 施明郭宁
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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