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Soluble NK-CAR fusion protein as well as preparation method and application of soluble NK-CAR fusion protein in drug for mediating immune cells to kill tumor cells

A technology of NK-CAR and fusion protein, which is applied in the direction of anti-tumor drugs, DNA/RNA fragments, drug combinations, etc., can solve the problems of enhanced cytotoxic activity, achieve the effects of prolonging metabolic half-life, facilitating protein purification, and saving production time

Active Publication Date: 2022-04-08
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, most CAR-NK cell studies have used CAR structures designed for CAR-T cells. Although these CARs originally designed for T cells also exerted anti-tumor activity after being applied to NK cells, there are reports that contain NK cells with 2B4 (NK-specific co-stimulatory domain) have significantly enhanced cytotoxic activity against tumor cells. Compared with NK cells carrying conventional 4-1BB, the cells proliferate faster and can produce more cytokines, while the cells Less apoptosis, suggesting that NK cell-specific activation signals affect NK-CAR performance

Method used

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  • Soluble NK-CAR fusion protein as well as preparation method and application of soluble NK-CAR fusion protein in drug for mediating immune cells to kill tumor cells
  • Soluble NK-CAR fusion protein as well as preparation method and application of soluble NK-CAR fusion protein in drug for mediating immune cells to kill tumor cells
  • Soluble NK-CAR fusion protein as well as preparation method and application of soluble NK-CAR fusion protein in drug for mediating immune cells to kill tumor cells

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment one: the construction of pCMV5.1-MS-Ig recombinant plasmid

[0049] The nucleotide sequence of the extracellular region of MICA was queried from IPD-IMGT / HLA Database, the anti-CD20 single-chain antibody sequence was preserved in our laboratory, and the IgG Fc nucleotide sequence was queried from PubMed. Such as figure 1 As shown, the above sequences were spliced ​​in sequence, and the recombinant sequences were synthesized by BGI. The synthesized sequence and the pCMV5.1 plasmid were subjected to double enzyme digestion at the same time, and the digested product was run on agarose gel, and the gel was cut to recover the digested recombinant sequence and the corresponding band of the empty plasmid vector, and then ligated with T4 ligase. The product was transformed into DH5α Escherichia coli competent cells, and the positive monoclonals were screened by smearing resistance plates, the recombinant plasmids were verified by enzyme digestion, and sent to the c...

Embodiment 2

[0052] Example 2: Expression of soluble NK-CAR MS-Ig

[0053] Recover and culture HEK293 suspension cells in large quantities, and extract a large amount of pCMV5.1-MS-Ig recombinant plasmid at the same time, mix PEI and plasmid at a volume ratio of 3:1, incubate at 25°C for 15 minutes, and transfect at a final concentration of 1 μg / ml plasmid , 7 days after transfection, the cells were centrifuged, and the supernatant was taken to verify the expression of recombinant protein by western blotting. The result is as figure 2 As shown, a disulfide bond is formed between the Fc ends of two monomers of the fusion protein, so that the protein is in a dimer state under natural conditions. After the protein is denatured, the disulfide bond is destroyed and the protein returns to a monomer. "Denatured PAGE" corresponds to the immunoblotting results of proteins denatured by heating and running 10% reducing gel, and "Natural PAGE" corresponds to the immunoblotting results of proteins no...

Embodiment 3

[0054] Example 3: Purification of soluble NK-CAR MS-Ig

[0055] Draw an appropriate amount of SPA microsphere packing into the protein purification column, first balance the column with the company's matching balance solution at a flow rate of 30ml / min, dilute the cell culture supernatant collected after the above expression with the balance solution at a ratio of 1:4, and place it on ice and pass the supernatant through the column at a flow rate of 2ml / min. After passing through the column, wash the column with a balance solution at a flow rate of 30ml / min. The flow rate of min was eluted, and the collection tube was used for collection. The collection tube was added with an appropriate amount of neutralizing solution in advance to restore the pH value of the collected protein eluate to 7.4. Concentrate the eluted protein solution with a protein ultrafiltration tube, replace the protein solvent with PBS, detect the concentration of the concentrated protein, and store it at -8...

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Abstract

The invention discloses a soluble NK-CAR fusion protein, a preparation method and an application of the soluble NK-CAR fusion protein in a drug for mediating immune cells to target tumor cells. The fusion protein is composed of an MICA extracellular region, a single-chain antibody and a human IgG Fc terminal from an amino terminal to a carboxyl terminal. A nucleotide sequence for coding the protein is inserted into an eukaryotic expression vector and transfected to an HEK293 suspension cell for protein expression, then purification is carried out through an SPA column, the protein is in a dimer state in an aqueous solution, a CD20 single-chain antibody of the protein can be combined with a CD20 antigen on a target cell, an MICA terminal is combined with an NKG2D receptor on an NK cell to activate the NK cell to play a role in specifically killing the target cell, and the target cell is killed. The invention lays a foundation for development and application of biological medicines for specifically killing tumor target cells by mediating NK cells and CD8 + T cells.

Description

technical field [0001] The invention belongs to the field of tumor immunotherapy, and in particular relates to a soluble NK-CAR fusion protein, a preparation method and an application in mediating immune cells to target and kill tumor cells. Background technique [0002] In recent years, breakthroughs have been made in immunotherapy represented by chimeric antigen receptor (CAR) T cells technology and PD-1 / PD-L1 antibody, opening up a new path for tumor treatment. However, accumulating evidence suggests that some cancers have developed multiple strategies to evade CD8 + T cells, such tumors can be preferentially attacked by natural killer cells-NK cells. NK cells are derived from multipotent hematopoietic progenitor cells in the bone marrow, and are an important innate lymphocyte. Studies have shown that the occurrence of malignant tumors is closely related to primary NK cell immunodeficiency is associated with a higher risk of cancer in individuals with lower NK cell toxic...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K38/17A61K39/395A61P35/00
CPCY02A50/30
Inventor 邹义洲刘荣娇罗奇志罗伟光万玲
Owner CENT SOUTH UNIV
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