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Fusion protein and application thereof in preparation of tumor drugs

A fusion protein and amino acid technology, applied in the field of tumors, can solve the problems that hinder the clinical application of IL-2, short half-life, toxic and side effects, etc., and achieve the effect of having immune memory, weak toxic and side effects, and preventing recurrence and metastasis

Pending Publication Date: 2022-07-08
THE GBA NAT INST FOR NANOTECHNOLOGY INNOVATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its half-life in vivo is very short, only 5 to 7 minutes; and IL-2 in an effective therapeutic dose will have certain toxic and side effects on the body; it will also activate Treg cells in the body and lead to immunosuppression of the body
This series of problems seriously hinders the clinical application of IL-2 as an immunomodulator in tumor immunotherapy

Method used

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  • Fusion protein and application thereof in preparation of tumor drugs
  • Fusion protein and application thereof in preparation of tumor drugs
  • Fusion protein and application thereof in preparation of tumor drugs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 pHLIP-IL2 de novo synthesis (de novo synthesis) and purification

[0058] 1. Synthesis of pHLIP-IL2 de novo and construction of expression vector

[0059] The C-terminus of the IL-2 amino acid sequence (shown in SEQ ID NO: 1) was linked to the N-terminus of the pHLIP amino acid sequence (shown in SEQ ID NO: 2) by a 2 amino acid peptide (GS). The corresponding cDNA was synthesized according to the designed amino acid sequence (as shown in SEQ ID NO: 3), cloned into the pET30a plasmid between the EcoRI site and the XhoI site by PCR, and the expression vector was constructed and verified successfully to obtain the expression vector.

[0060] 2. Construction of recombinant strains

[0061] Transform the constructed expression vector into the expression strain. Take out the BL21 competent bacteria stored at -80°C and thaw slowly on ice, add the previously synthesized expression vector, and place on ice for 30 minutes. Subsequently, the competent cells were heat-...

Embodiment 2

[0080] Example 2 pHLIP-IL2 chemical coupling synthesis

[0081] 1. Experimental method

[0082] Sulfo-SMCC was used as the linker. Sulfo-SMCC has both a Sulfo-NHS ester group and a maleimide group as a cross-linking agent. Under the condition of pH 7~9, the NHS group can react with the primary amine group of the protein to form an amide bond. Under the condition of pH 6.5~7.5, the maleimide group can react with the sulfhydryl group of the protein to form a thioether bond.

[0083] In order to achieve the purpose of covalent coupling, the pHLIP polypeptide sequence was designed, and a cysteine ​​was added to the N-terminus of the pHLIP original sequence (as shown in SEQ ID NO: 2) for subsequent connection with the maleimide group. reaction. The redesigned synthetic peptide was synthesized by solid-phase peptide synthesis, and the purity was verified by HPLC and the molecular weight was verified by MS.

[0084] Dissolve 1 mg of IL-2 lyophilized powder (as shown in SEQ ID NO:...

Embodiment 3

[0087] Example 3 Detection of pHLIP-IL2 acidic pH response characteristics by flow cytometry

[0088] 1. Experimental method

[0089] 1. Fluorescently label the pHLIP-IL2 fusion protein.

[0090] Dip a 20-fold molar excess of fluorescent dye Cy5.5-NHS into 100 μl DMSO with a 1 ml pipette tip, dissolve it in 100 μl DMSO, add 900 μl PBS Buffer, dilute it by pipetting evenly, take 500 μl and add 1 ml of 0.5 mg / ml pHLIP- IL2 fusion protein solution, react at 4°C for 4 hours. Then, the excess Cy5.5-NHS in the protein was removed by ultrafiltration with a 3kD ultrafiltration tube at 12000rpm for 30 minutes, and the Cy5.5-labeled pHLIP-IL2 fusion protein was obtained.

[0091] 2. The pHLIP-IL2 fusion protein reacts with cells

[0092] One tube of B16 cells was recovered, cultured in 10% FBSDMEM medium, and plated in six-well plates one day in advance after passage and expansion, and 105 cells were plated in each well. After one day of culture, the original medium was removed, was...

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Abstract

The invention discloses a fusion protein and application thereof in preparation of tumor drugs, the fusion protein contains an amino acid sequence of a cytokine IL-2 and an amino acid sequence of an acid-responsive cell-penetrating peptide pHLIP, and the amino acid sequence of the cytokine IL-2 is at the N terminal of the amino acid sequence of the acid-responsive cell-penetrating peptide pHLIP. The fusion protein can be used as a tumor targeting immunomodulator and can be positioned to the surface of a tumor cell membrane through pHLIP, so that IL-2 is enriched in tumor tissue, CD8 + T cells and NK cells around a tumor are stimulated, proliferation of the cells is promoted, the anti-tumor activity of the cells is enhanced, the killing power of the cells to the tumor cells is enhanced, and immunotherapy is achieved. Compared with traditional treatment means such as surgical treatment, radiotherapy and chemotherapy, the immunotherapy has weak toxic and side effects, can be used for metastatic cancer treatment, has immune memory ability, and prevents tumor recurrence and metastasis.

Description

technical field [0001] The present invention relates to the field of tumor technology, in particular, to a fusion protein and its application in the preparation of tumor drugs. Background technique [0002] As an emerging tumor treatment method, tumor immunotherapy can better kill tumor tissue by regulating its own immune system. Compared with traditional treatment methods, immunotherapy has less toxic and side effects and can be used for the treatment of metastatic cancer. At the same time, it has the ability of immune memory to prevent tumor recurrence and metastasis. [0003] Interleukin 2 (IL-2), also known as T-Cell Growth Factors (TCGF), can promote the proliferation of effector T cells and NK cells and enhance the tumor cell killing ability of these cells. Cytokines can play a role in regulating the immune function of the body, and have the effects of inhibiting tumor growth and metastasis. However, its half-life in vivo is very short, only 5 to 7 minutes; and IL-2 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K38/20A61K47/64A61P35/00
CPCC07K14/55A61K38/2013A61K47/64A61P35/00C07K2319/10
Inventor 李素萍聂广军李博朝李梦瑶
Owner THE GBA NAT INST FOR NANOTECHNOLOGY INNOVATION
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