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Heterogenetic antigen-Fc fusion protein capable of inducing antitumor immunity of organism and application thereof

A technology of fusion protein and anti-tumor activity, applied in the field of heterologous antigen-Fc fusion protein

Inactive Publication Date: 2010-02-03
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

Disis et al. confirmed that when immunizing rats with full-length rat neu protein, an immune response against rat neu protein could not be induced, but immunizing rats with Her2 intracellular segment protein, which is highly homologous to neu protein, could lead to Rats have strong cellular and humoral immune responses against Her2 and neu (Disis ML, Shiota FM, Cheever MA. Human HER-2 / neu protein immunity circumvents tolerance to rat neu: a vaccine strategy for self tumor antigens[J] . Immunology, 1998, 93(2): 192-199.)

Method used

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  • Heterogenetic antigen-Fc fusion protein capable of inducing antitumor immunity of organism and application thereof
  • Heterogenetic antigen-Fc fusion protein capable of inducing antitumor immunity of organism and application thereof
  • Heterogenetic antigen-Fc fusion protein capable of inducing antitumor immunity of organism and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. The immunomodulatory effect of fusion protein neu-Fc combined with GM-CSF and BCG

[0039] 1. Construction, expression and purification of neu-Fc

[0040] The extracellular domain of rat neu includes four domains, L1, S1, L2 and S2. The primers 5'-tgccatggtccgagtgtgctatggtctg-3' and 5'-agtttagcggccgcgtgggtgcagttgatggg-3' were designed according to the L2 and S2 domains of the extracellular domain of rat neu, and the coding sequences of the L2 and S2 domains of the extracellular domain of rat neu were amplified by PCR ( The upstream primer is located in the L2 domain of rat neu extracellular domain, and the downstream primer is located in the S2 domain of rat neu extracellular domain).

[0041] The total RNA of isolated peripheral blood mononuclear cells of healthy people was extracted, and the cDNA of human IgG1Fc segment was amplified by RT-PCR method. The cDNA of human IgG1 Fc segment was digested with NotI and XhoI and inserted into the vector pCI-neo (purchase...

Embodiment 2

[0058] Example 2. Immunomodulatory effect of heterologous protein neu-Fc combined with IFN-γ and BCG

[0059] 1. The combined effect of neu-Fc, IFN-γ and BCG on the secretion of cytokines

[0060] Adjust the density of PBMC to 2×10 with phenol red-free RPMI-1640 medium containing 20% ​​serum 6 / ml, added to 96-well cell culture plate, 100μl per well. In the experimental group, 100μl of MCF-7 cell culture supernatant was added. After 48-72 hours of culture, the culture solution was aspirated and divided into the following treatments: 1. Supplement RPMI-1640100μl; 2. Add neu-Fc containing a final concentration of 10nM 100μl of phenol red-free RPMI-1640 medium; 3. Add 100μl of phenol red-free RPMI-1640 medium with a final concentration of 40U / ml IFN-γ; 4. Add a final concentration of 40U / mlIFN-γ and 10nM neu -Fc-free phenol red RPMI-1640 medium 100μl; 5. Add 100μl of phenol red-free RPMI-1640 medium containing a final concentration of 300μg / ml BCG; 6. Add a final concentration of 300...

Embodiment 3

[0085] Example 3. Animal experiment

[0086] 1. Construction of EMT6 / Her2 cells

[0087] The mouse breast cancer cells EMT6 (purchased from ADCC, USA) derived from Balb / C mice were transfected with pcDNA3 / Her2 plasmid, and EMT6 cells expressing Her2 were selected under the pressure of 800μg / ml G418. After two limiting dilutions and pressure After screening, a clone of EMT6 cells stably expressing Her2 was obtained and named EMT6 / Her2. At the same time, EMT6 cells not transfected with pcDNA3 / Her2 plasmid were used as control. The anti-Her2 antibody (Ab20) was purchased from Neomarkers and FACS was used to detect the expression of Her2 in EMT6 / Her2 cells. The FACS test results are as follows Picture 8 Shown. among them, Picture 8 A is the FACS test result of EMT6 cells in the control group, Picture 8 B is the FACS test result of EMT6 / Her2 cells. The results showed that nearly 100% of EMT6 / Her2 cells express Her2 molecules.

[0088] 2. Animal experiment

[0089] 1) Determination o...

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Abstract

The invention discloses a fusion protein with antitumor activity which is protein of 1) or 2) as follows: 1) protein containing the amino acid sequence shown in sequence 2 of the sequence table; 2) protein which is related to antitumor and is derivative of 1) by replacing and / or losing and / or adding one or several amino acid residue in amino acid residue sequence shown in sequence 2 of the sequence table. The vaccine which adopts the fusion protein as active ingredient and GM-CSF and BCG or IFN-gamma and BCG as adjuvant has obvious inhibition for the growth of tumor; in tumor HE staining section, that a great number of inflammatory cells in tumor tissue and around the envelope are infiltrated can be observed so as to prove that effective antitumor immunologic reaction can be induced by combining the adjuvant and the fusion protein.

Description

Technical field [0001] The invention relates to a heterogeneous antigen-Fc fusion protein which can induce the body's anti-tumor immunity and its application. Background technique [0002] Breast cancer is one of the most common malignant tumors in women, accounting for 18% of female malignant tumors. Approximately 1.2 million women suffer from breast cancer each year and 500,000 die from breast cancer worldwide. In Western Europe, North America and other developed countries, the incidence of breast cancer is the first among female malignant tumors. Asia is a low-incidence area of ​​breast cancer, but in recent years, the incidence of breast cancer in my country has been increasing year by year, increasing from 17 per 100,000 in 1999 to 52 per 100,000 in 2003. three times. Surgery, chemotherapy and radiotherapy are conventional treatments for breast cancer. With the continuous advancement of the medical level, the treatment technology of primary tumors, especially the surgical t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A61K39/39A61K39/00A61P35/00
Inventor 郭宁解志刚施明
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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