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Immune complex as well as preparation method and application thereof

A technology of immune complexes and hepatitis B, which is applied in the direction of non-active ingredients of polymer compounds, drug combinations, drug delivery, etc., can solve the problem of poor tumor inhibitory effect, achieve a wide range of safe doses, and reduce toxicity

Pending Publication Date: 2020-04-17
河南省生物工程技术研究中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, when OX40 is used alone to inhibit tumors, its inhibitory effect on tumors is also poor.

Method used

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  • Immune complex as well as preparation method and application thereof
  • Immune complex as well as preparation method and application thereof
  • Immune complex as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Expression and purification of hepatitis B core virus-like particles

[0024] 1.1 Download the hepatitis B virus antigen (HBc) sequence from the NCBI database (NCBI accession number is ABC97614.1), obtain the first 144 amino acid HBc-144 sequence of HBc, and design the nucleotide encoding HBc-144 by combining the E. coli codon preference Sequence; insert the gene sequence encoding the RGD sequence between the gene sequence encoding the amino acids at positions 78-79 of the main immune region of HBc-144, and insert the gene sequence encoding the RGD sequence into the gene sequence encoding HBc-144 at both ends of the gene sequence The gene sequences are respectively connected to the gene sequence encoding the connecting peptide (GGGGSGGGGSGGGGSGGGGS); the gene sequence of 6 histidines is inserted at the end of the gene sequence encoding the HBc-144 amino acid (C-terminal end). The above-mentioned nucleotide sequence encoding the hepatitis B core virus-like particl...

Embodiment 2

[0057] Example 2 Preparation of the immune complex of the present invention

[0058] 2.1 Loading of oligodeoxynucleotides containing unmethylated CPG dinucleotides

[0059] (1) Dissolve 10 mg of CPG powder in 10 ml of deionized water to prepare a CPG stock solution with a concentration of 1 mg / ml, and store it at -20°C for use.

[0060] (2) Take 5 mg of hepatitis B core virus-like particles and add them to 3M urea for 2 hours at room temperature for depolymerization. Then add the above CPG stock solution to make the mass of CPG and hepatitis B core virus-like particles 1:1-1:10. After addition, the polymer was dialyzed and polymerized in 20 mm phosphate buffer (pH 7.4) containing 150 mm of sodium chloride in an ice bath for 2 hours.

[0061] (3) Centrifuge the dialysate at 10000 rpm for 10 min. The supernatant is further used DEAE stream Flow chromatography column to remove free CPG. The samples collected through the column at 280nm absorbance were detected by an ultraviolet detect...

Embodiment 3

[0063] Example 3 Preparation of the immune complex of the present invention

[0064] 3.1 Loading of oligodeoxynucleotides containing unmethylated CPG dinucleotides

[0065] (1) Dissolve 10 mg of CPG powder in 10 ml of deionized water to prepare a CPG stock solution with a concentration of 1 mg / ml, and store it at -20°C for use.

[0066] (2) Take 5 mg of hepatitis B core virus-like particles and add them to 3M urea for 2 hours at room temperature for depolymerization. Then add the above CPG stock solution to make the mass of CPG and hepatitis B core virus-like particles 1:10. After addition, the polymer was dialyzed and polymerized in 20 mm phosphate buffer (pH 7.4) containing 150 mm of sodium chloride in an ice bath for 2 hours.

[0067] (3) Centrifuge the dialysate at 10000 rpm for 10 min. The supernatant is further used DEAE stream Flow chromatography column to remove free CPG. The samples collected through the column at 280nm absorbance were detected by an ultraviolet detector t...

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Abstract

The invention relates to the technical field of tumor immunotherapy, in particular to an immune complex as well as a preparation method and application thereof. The immune complex comprises an OX40 antigen or an OX40 antibody, a hepatitis B core virus-like particle and CPG; and the CPG is encapsulated in the hepatitis B core virus-like particle. The immune complex can be used for inhibiting tumors, the safety is high, and the use amounts of the CPG and the OX40 can be significantly reduced.

Description

Technical field [0001] The invention relates to the technical field of tumor immunotherapy, in particular to an immune complex and its preparation method and application. Background technique [0002] Containing unmethylated CPG dinucleotide oligodeoxynucleotides can directly activate B cells, dendritic cells, macrophages, antigen presenting cells, etc.; indirectly activate T cells and NK cells; induce Th1 type as The main immune system. As a highly effective and low-toxic immune adjuvant, it has strong potential value in the treatment of tumors, immunodeficiency diseases, infectious diseases, and allergic diseases. A large number of studies on CPG ODN stimulating the body's own anti-tumor immune response indicate that as an immune adjuvant, CPGODN can enhance the anti-tumor effect of certain monoclonal antibodies, tumor vaccines, chemotherapy drugs or radiotherapy. CPGODN alone has a significant inhibitory effect on lymphoma, malignant melanoma, glioma, cervical cancer, and re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395A61K31/7088A61K9/08A61K9/19A61K47/42A61P35/00
CPCA61K39/001117A61K39/39558A61K31/7088A61K9/08A61K9/0019A61K9/19A61K47/42A61P35/00A61K2300/00
Inventor 王云龙蒋丹丹李玉林王继创张怡青
Owner 河南省生物工程技术研究中心
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