Application of CIK (cytokine induced killer) cell loaded by anti-CD3/anti-CD133 bispecific antibody

A bispecific antibody and cell technology, applied in the direction of antibodies, medical preparations with non-active ingredients, anti-tumor drugs, etc., can solve the problems of limited therapeutic effect and lack of specific targeted killing of CIK cells.

Inactive Publication Date: 2014-05-14
黄 建华
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the adoptive immunotherapy of tumors, cytokine induced killer cells (cytokine induced killer, CIK), as a new generation of cells, have both the strong tumoricidal activity of T lymphocytes and the non-MHC-restricted tumor killing characteristics of NK cells, and have been clinically obtained. Great progress has been made, but the therapeutic effect is still limited because CIK cells lack the ability to specifically target and kill tumor stem cells

Method used

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  • Application of CIK (cytokine induced killer) cell loaded by anti-CD3/anti-CD133 bispecific antibody
  • Application of CIK (cytokine induced killer) cell loaded by anti-CD3/anti-CD133 bispecific antibody
  • Application of CIK (cytokine induced killer) cell loaded by anti-CD3/anti-CD133 bispecific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: Preparation of bispecific antibody

[0062] 1. Take the mouse anti-human CD3 monoclonal antibody—OKT3 antibody (1mg) and dissolve it in 500 μl of the cross-linking agent Traut's reagent (2-iminothiolane HCl; Pierce, Rockford, IL) at a 5-fold molar concentration, and act for 1 hour at room temperature; use PD -10 column (Pharmacia, Uppsala, Sweden) filtration, removes uncrosslinked monoclonal antibody;

[0063] 2. Take the mouse anti-human CD133 / 1 monoclonal antibody (1 mg) and dissolve it in 500 μl of the cross-linking agent Sulfo-SMCC (sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate) at a 4-fold molar concentration, and act for 1 hour at room temperature , filtered with a PD-10 column (Pharmacia, Uppsala, Sweden) to remove uncrosslinked monoclonal antibodies;

[0064] 3. After mixing the cross-linked OKT3 and the mouse anti-human CD133 / 1 monoclonal antibody overnight at 4°C, an anti-CD3 / anti-CD133 bispecific antibody was prepared.

[0065] ...

Embodiment 2

[0067] Example 2: Equipping CIK cells with anti-CD3 / anti-CD133 bispecific antibody

[0068] 1. Culture of CIK cells

[0069] Take 10ml of peripheral venous blood from healthy volunteers, anticoagulate with heparin, dilute with normal saline 1:1, add to the upper layer of lymphocyte separation medium, collect mononuclear cells (PBMC) by centrifugation, and use RPMI1640 culture medium containing 5% fetal bovine serum Adjust the cell density to 2 x 10 5 IFN-γ was added at a final concentration of 1,000 units / mL, placed in a 5% CO2 incubator at 37°C for 24 hours, and rhIL-1α was added at a final concentration of 1000 U / ml, and the final concentration of mouse anti-human CD3 McAb was 50ng / ml, the final concentration of rhIL-2 was 1000U / ml, add rhIL-2 every 2 days, on the 14th day of culture, check the CD3, CD4, CD8 and CD56 phenotype of cultured cells by flow cytometry, and harvest CIK cells.

[0070] 2. Detection of CIK cell phenotype

[0071] Adjust the concentration of CIK ce...

Embodiment 3

[0077] Example 3: Detecting the Cytotoxicity of CIK Cells Equipped with Bispecific Antibodies to SW1990

[0078] CIK cells were used as effector cells, and the human pancreatic cancer cell line SW1990 was used as target cells to detect the killing effect of CIK cells equipped with anti-CD3 / anti-CD133 bispecific antibodies.

[0079] Take 10 samples of normal people and 10 samples of tumor patients' peripheral venous blood to prepare CIK cells, and load each case of CIK cells with anti-CD3 / anti-CD133 bispecific antibody, and compare and detect CIK cells and those loaded with anti-CD3 / anti-CD133 bispecific antibody, respectively. The killing experiment of SW1990 cells by CIK cells with specific antibodies.

[0080] The experimental procedure is as follows: take tumor cells in the logarithmic growth phase, spread 5000 cells / well on a 96-well cell culture plate, and add CIK cells and CIK cells equipped with anti-CD3 / anti-CD133 bispecific antibody prepared in Example 2 respectively ...

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Abstract

The invention relates to an application of a CIK (cytokine induced killer) cell loaded by an anti-CD3 / anti-CD133 bispecific antibody, and particularly relates to an application of the CIK cell loaded by the anti-CD3 / anti-CD133 bispecific antibody in the forming process of a preparation for treating cancer cells of targeting killing highly-expressed CD133, an application of the CIK cell loaded by the anti-CD3 / anti-CD133 bispecific antibody in the forming process of a drug for reducing S100P gene expression in the cancer cell, and an application of the CIK cell loaded by the anti-CD3 / anti-CD133 bispecific antibody in the forming process of a drug for increasing STAT1 gene expression in the cancer cell.

Description

technical field [0001] The invention relates to the application of CIK cells loaded with anti-CD3 / anti-CD133 bispecific antibodies. Background technique [0002] More and more evidence shows that tumor stem cells play a vital role in tumor immune escape, resistance to radiotherapy, chemotherapy, and recurrence and metastasis. Therefore, it is necessary to create new strategies to effectively target and kill tumor stem cells in order to cure tumors. Immunotherapy targeting cancer stem cells has been actively explored in the research of monoclonal antibodies and stem cell vaccines. So far, the tumor stem cell-specific markers found are very rare, and CD133 is one of the most common markers found in tumor stem cells, in brain tumors, leukemia, melanoma, kidney cancer, colorectal cancer, pancreatic cancer It exists in various tumor stem cells such as lung cancer, liver cancer and ovarian cancer, and its high expression is closely related to the low survival rate of tumor patien...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/395A61K35/12A61K47/48A61P35/00A61K35/15
Inventor 黄建华
Owner 黄 建华
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