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Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application

A polypeptide complex and composition technology, which is applied in the direction of drug combination, medical preparations containing active ingredients, carrier-bound antigen/hapten components, etc., to achieve the effect of convenient source of antigen, flexible design, and clear antigen

Active Publication Date: 2010-07-21
江苏得康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0035] 6. Insufficiency of the current DC vaccine
The above-mentioned simple antigen polypeptide-loaded DC vaccines, or simple HSP70 antigen-polypeptide complex vaccines all have deficiencies

Method used

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  • Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application
  • Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application
  • Dendritic cell vaccine carrying recombinant human HSP70 polypeptide complexes, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Example 1: Dendritic cell vaccine loaded with four HSP70-melanoma antigen polypeptide complexes

[0089] 1. Preparation of melanoma antigen polypeptide and rhHSP70 protein

[0090] The known HLA-A201-restricted melanoma antigen polypeptide fragments were artificially synthesized, and the names and sequences were GP100: IMDQVPFSV; MART1: AAGIGILTV; MAGE3: FLWGPRALV and Try: YMDGTMSQV. Fully dissolved in DMSO, the peptide concentration was 5 mg / mL; rhHSP70 was dissolved in PBS, the concentration was 0.1 μg / mL.

[0091] 2. Preparation of rhHSP70-polypeptide complex

[0092] Take 5μg (5μl) polypeptide and 10μg (100μl) rhHSP70 polypeptide respectively in 1.5mL EP tube, add 400μl containing 10nM ATP, 1mM KCl, 2mM MgCl 2 Mix well and incubate at 20°C for 40 minutes, add 100nM ADP and continue to incubate at 20°C for 40 minutes. Transfer the solution to a 0.5mL microdialysis tube (containing a 14kD molecular weight ultrafiltration membrane) overnight, remove unbound polypept...

Embodiment 2

[0106] Example 2: Liver cancer dendritic cell vaccine loaded with two kinds of HSP70-liver cancer antigen polypeptide complexes

[0107] 1. Antigen polypeptide synthesis: Most patients with primary liver cancer highly express alpha-fetoprotein (AFP) and are combined with chronic persistent HBV infection. For this type of liver cancer patients, AFP and HBV can be used as target antigens, such as AFP: GVALQTMKQ; HBsAg: FLLTRILTI; Polypeptide dissolution and concentration are the same as in Example 1;

[0108] 2. Preparation of rhHSP70-polypeptide complex: the operation steps are the same as in Implementation 1, and two complexes of HSP70-AFP and HSP70-HBsAg are prepared;

[0109] 3. Induction of dendritic cells in vitro

[0110] Collect fresh heparin anticoagulated primary liver cancer patients (HLA-A201 + ) peripheral blood or HLA-A201 + Peripheral mononuclear cells (PBMCs) were obtained from the peripheral blood of normal healthy persons by Ficoll density gradient centrifug...

Embodiment 3

[0115] Example 3: Dendritic cell vaccine loaded with two kinds of HSP70-breast cancer antigen polypeptide complex

[0116] 1. Antigen peptide synthesis: Most breast cancer patients highly express MUC1 and Her2-neu related antigens; for this type of breast cancer patients, this can be used as a target antigen to synthesize peptides, such as MUC1: SLADPAHGV: Her2-neu: KIFGSLAFL; peptide dissolution and Concentration is with embodiment 1;

[0117] 2. Preparation of rhHSP70-polypeptide complex: the operation steps are the same as in Implementation 1, and two complexes of HSP70-MUC1 and HSP70-Her2-neu are prepared;

[0118] 3. Induction of CD34-DCs in vitro

[0119] HLA-201+ breast cancer patients undergo hematopoietic mobilization with GM-CSF, collect mononuclear cells (PBMCs) with a cell harvester, add CD34 monoclonal antibody magnetic beads to separate and purify CD34+ hematopoietic precursor cells (purity>90%), and suspend them In X-VIVO medium containing 5% (v / v) mixed human...

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Abstract

The invention discloses a dendrite-shaped cell vaccine for loading a recombination human heat shock protein 70 polypeptide composite, a method for preparing the same and application thereof. The preparation method comprises: preparing a dendrite-shaped cell by designing and synthesizing an antigen polypeptide, forming the composition by the antigen polypeptide and the recombination human heat shock protein 70(rhHSP70), and loading the composite on the dendritic cell (DC) to obtain the vaccine. The vaccine has the advantages of multi-target points, double adjuvants and high efficacy, and can be used for resisting tumors and treating infectious diseases. The preparation process is simple and fast, and low in cost.

Description

Technical field: [0001] The present invention belongs to the field of biopharmaceuticals, and more specifically relates to forming a complex between an antigen polypeptide and recombinant human heat shock protein 70 (rhHSP70), and then loading the complex on dendritic cells (DC) to prepare a dendritic cell polypeptide vaccine. For the treatment of tumors and infectious diseases. Background technique: [0002] 1. Current status of the incidence and treatment of malignant tumors and infectious diseases [0003] (1) Tumor incidence and treatment progress [0004] Tumors and infectious diseases are globally recognized major diseases that seriously threaten human life and health. At the 18th International Anti-Cancer Union Conference, a research report published by the World Health Organization showed that the global cancer situation will become increasingly serious, and the number of new patients will increase from the current 10 million to 15 million per year in the next 20 y...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61P35/00A61K39/385A61K39/29A61K39/04A61K39/21A61K39/00A61P31/00A61K39/39
Inventor 时宏珍
Owner 江苏得康生物科技有限公司