Recombinant adenovirus with anti-tumor immune function as well as preparation method and application of recombinant adenovirus

A recombinant adenovirus, anti-tumor immunity technology, applied in anti-tumor drugs, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as drug resistance of monoclonal antibodies, and achieve the effect of immunotherapy

Pending Publication Date: 2022-04-19
ZHEJIANG MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the immune escape of tumor cells, there is a high probability of drug resistance in the treatment of monoclonal antibody drugs

Method used

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  • Recombinant adenovirus with anti-tumor immune function as well as preparation method and application of recombinant adenovirus
  • Recombinant adenovirus with anti-tumor immune function as well as preparation method and application of recombinant adenovirus
  • Recombinant adenovirus with anti-tumor immune function as well as preparation method and application of recombinant adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Screening of Polypeptide PP4

[0029] 1) Analyze the protein sequence of the avirulent mutant of diphtheria toxin, predict the CD4+ T cell epitope of CRM197 by NetMHCⅡpan4.0Server software, and screen out the peptides with medium and strong affinity, and obtain 9 peptides through preliminary screening, as shown in Table 1:

[0030] Table 1 Preliminary screening results of T cell active polypeptides of diphtheria toxin avirulent mutant protein

[0031]

[0032]

[0033] 2) Artificially synthesize the 9 polypeptides described in step 1), mix the obtained 9 polypeptides, emulsify them with an equal volume of Freund's complete adjuvant, and immunize 8 mice, kill the mice on the 10th day after immunization, and take the spleen to prepare a single nuclear lymphocytes.

[0034] 3) Take the mononuclear lymphocytes described in step 2), press 5×10 5 / well was added to a 96-well cell culture plate, and a blank control was set up at the same time. After culturin...

Embodiment 2

[0036] Example 2 Construction of recombinant adenovirus expressing CD137L-PP4 protein

[0037] 1) Artificially synthesized according to the nucleotide sequence encoding the PP4 polypeptide shown in SEQ ID NO.2, the obtained synthetic sequence was inserted into the pDC316 plasmid through the two restriction sites of MluI and HindIII, and the pDC316-CD137L-PP4 recombinant shuttle plasmid was obtained ( figure 2 ).

[0038] 2) Culture HEK293 cells with DEMM medium to grow to monolayer.

[0039] 3) The pDC316-CD137L-PP4 recombinant shuttle plasmid described in step 1) and the adenovirus backbone plasmid pBHGloxΔE1,3Cre were co-transfected with liposome Lipofectamine 2000 into the HEK293 cells described in step 2), and the cytopathic changes were observed under a microscope after transfection In other cases, when the cytopathic changes were completely shed, the primary virus culture medium was harvested.

[0040]4) The primary virus culture solution described in step 1) was coll...

Embodiment 3

[0041] Example 3 Preparation of recombinant adenovirus expressing CD137L-PP4 protein

[0042] 1) Culture HEK293 cells with DEMM medium to grow to monolayer.

[0043] 2) Infect the HEK293 cells described in step 1) with the recombinant adenovirus expressing CD137L-PP4 protein, and harvest the virus culture medium when the cytopathic changes are completely shed.

[0044] 3) Collect the supernatant of the virus culture solution described in step 2) after repeated freezing and thawing three times, and use 300kD membranous cells to carry out tangential ultrafiltration and concentrate 10 times to obtain a concentrated virus solution.

[0045] 4) Purify the concentrated virus solution described in step 3) first with SOURCE30Q anion exchange chromatography, and then with Superdex200pg molecular sieve chromatography to obtain purified virus solution.

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PUM

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Abstract

The invention provides a recombinant adenovirus with an anti-tumor immune function, a preparation method and application, an expression fusion protein CD137L-PP4 gene is inserted into an adenovirus vector to construct the recombinant adenovirus with the anti-tumor immune function, and the fusion protein CD137L-PP4 comprises a CD137L protein and PP4 polypeptide with a T cell activation function. The recombinant adenovirus can be expressed in vivo and can induce generation of specific anti-tumor cell immunity, so that immunotherapy of tumors is realized. Therefore, the compound can be used for preparing a medicine for inducing an organism to generate specific cellular immune response aiming at CD137L and / or inducing the organism to generate a medicine for inhibiting tumor cell proliferation.

Description

technical field [0001] The invention belongs to the field of medical biological products, and is particularly suitable for a preparation method and application of a recombinant adenovirus with anti-tumor immune function. Background technique [0002] CD137L (CD137 ligand), also known as 4-1BBL, belongs to the tumor necrosis factor superfamily member and is a type II transmembrane glycoprotein. 4-1BBL, which exists as a homotrimer and is characterized by an extended trefoil-like helical structure, exerts its biological function. As a high-affinity ligand of CD137, it is expressed on the surface of activated antigen-presenting cells (APC), including macrophages, B cells, dendritic cells, and a variety of tumor cells. As a pair of important co-stimulatory molecules, CD137L and CD137 regulate the immune response mediated by T cells by transmitting activation, proliferation or apoptosis signals between immune cells. [0003] Binding of CD137L to its receptor can initiate bidire...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N7/02C12N15/62C12N15/861A61K38/16A61K38/17A61P35/00
CPCC12N7/00C12N15/86C07K14/70575C07K14/33A61K38/16A61K38/1774A61P35/00C07K2319/55C12N2710/10021C12N2710/10043C12N2710/10051A61K2300/00Y02A50/30
Inventor 高孟吴洁朱赟沈钱通张柯欣安彤李思奇陈刚庄昉成陆绍红
Owner ZHEJIANG MEDICAL COLLEGE
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