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

A fusion protein, tumor technology, applied in anti-tumor drugs, medical preparations containing active ingredients, hybrid peptides, etc.

Inactive Publication Date: 2011-10-26
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. 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 region of rat neu includes four domains: L1, S1, L2 and S2. Primers 5'-tgccatggtccgagtgtgctatggtctg-3' and 5'-agtttagcggccgcgtgggtgcagttgatggg-3' were designed according to the L2 and S2 domains of the rat neu extracellular region, and the coding sequences of the L2 and S2 domains of the rat neu extracellular region were amplified by PCR ( The upstream primer is located in the L2 domain of the extracellular region of rat neu, and the downstream primer is located in the S2 domain of the extracellular region of rat neu).

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

Embodiment 2

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

[0059] 1. Effect of combined action of neu-Fc, IFN-γ and BCG on cytokine secretion

[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. The experimental group was added with 100 μl of MCF-7 cell culture supernatant, cultured for 48-72 hours, the culture medium was aspirated, and divided into the following treatments: 1. Supplement 100 μl of RPMI-1640; 2. Add neu-Fc with a final concentration of 10 nM 100 μl of phenol red-free RPMI-1640 medium; 3. Add 100 μl of phenol red-free RPMI-1640 medium containing a final concentration of 40 U / ml IFN-γ; 4. Add 100 μl of a final concentration of 40 U / ml IFN-γ and 10 nM neu -Fc 100 μl of phenol red-free RPMI-1640 medium; 5. Add 100 μl of phenol red-free RPMI-1640 medium containing BCG with a final concentration of 300 μg / ml; 6...

Embodiment 3

[0085] Embodiment 3, animal experiments

[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 the pcDNA3 / Her2 plasmid, and the EMT6 cells expressing Her2 were screened under the pressure of 800 μg / ml G418. After screening, EMT6 cell clone stably expressing Her2 was obtained, which was named EMT6 / Her2. Meanwhile, EMT6 cells not transfected with pcDNA3 / Her2 plasmid were used as control. Anti-Her2 antibody (Ab20) was purchased from Neomarkers Company and FACS was used to detect the expression of Her2 in EMT6 / Her2 cells, and the FACS detection results were as follows: Figure 8 shown. in, Figure 8 A is the FACS detection result of EMT6 cells in the control group, Figure 8 B is the FACS detection result of EMT6 / Her2 cells. The results showed that nearly 100% of EMT6 / Her2 cells expressed Her2 molecules.

[0088] 2. Animal experiments

[0089] 1) Determination of the ...

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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 capable of inducing anti-tumor immunity of a body 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. About 1.2 million women in the world suffer from breast cancer every year, and 500,000 people die from breast cancer. In developed countries such as Western Europe and North America, the incidence of breast cancer ranks 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, from 17 / 100,000 in 1999 to 52 / 100,000 in 2003, an increase three times. Surgery, chemotherapy and radiotherapy are the routine treatment methods for breast cancer. With the continuous improvement of medical level, the treatment technology of primary tumor, especially the effec...

Claims

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Application Information

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Patent Type & Authority Patents(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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