CAPRI cell and preparation method thereof

A cell and nuclear cell technology, applied in the field of CAPRI cells and their preparation, can solve the problems of doubling the probability of cell culture contamination, production safety and clinical cooperation, tumor killing immune response and other issues

Active Publication Date: 2016-04-27
赛德特生物制药有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3. The tumor itself is heterogeneous
4. Tumors can secrete cytokines such as IL-6, IL-4, IL-10, etc. that can induce immunosuppression or Th2, Th17, etc. that are not conducive to cellular immunity, resulting in insufficient tumor killing immune response
Therefore, the inhibitory effect of MHC-C on non-specific killing in CIK therapy is its main defect
In addition, in CIK therapy, cells are obtained from fresh cultures and directly reinfused into patients, causing problems in preparation safety and clinical compliance
Moreover, traditional therapies require long-term (generally more than 14 days) cell culture, which not only increases the workload, but also increases the equipment occupancy rate, increases the cost of cell culture, and doubles the chance of contamination of the cell culture

Method used

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  • CAPRI cell and preparation method thereof
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  • CAPRI cell and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Preparation of Cascade Cytokine-Induced and Activated Tumor-Specific Killer T Lymphocytes (CAPRI Cells)

[0061] 1. Peripheral Blood Mononuclear Cell Collection

[0062] 1.1 Method 1: Human peripheral blood collection: use heparin anticoagulation, manually collect blood 120ml-400ml, after separation and purification, the number of monocytes is about 1.5×10 8 -5.0×10 8 .

[0063] 1.2 Method 2: Obtain peripheral blood mononuclear cells from the patient through leukapheresis technology. The volume of apheresis is 150-500ml. 8 -5.0×10 8 .

[0064] 2. Preparation of reagents and solutions

[0065] 2.1 Preparation of coating solution

[0066] 2.1.1 Prepare 1× Phosphate Buffer Saline (PBS) with pH7.0-7.4

[0067] 2.2.2 Adjust the pH of the above PBS buffer to 8.6 with 1N sodium hydroxide solution

[0068] 2.2.3 Filter with 0.22μm sterile filter

[0069] 2.2.4 Add 0.2 ml of mouse anti-human CD3 monoclonal antibody (OKT3) at a concentration of 1 mg / ml to 100 m...

Embodiment 2

[0117] Example 2 In vitro experiment of CAPRI cells on autologous breast cancer cell cytotoxicity

[0118] The method of Example 1 and the CIK technology were used to culture the patient's own CAPRI cells and CIK cells respectively, and the patient's own breast cancer cell line cells were used as target cells (marked with Cr51) for cytotoxicity experiments. The killing rate was measured by observation under a microscope and the amount of Cr51 radioactive release. The results are shown in figure 2 and image 3 .

[0119] in, figure 2 A is observation under the microscope that after the autologous tumor cells were co-incubated with CAPRI cells and CIK cells for 20 hours, the number of residual tumors in the CAPRI cell group was significantly less than that in the CIK cell group, which proves that CAPRI cells have obvious killing and inhibitory effects on autologous tumor cells in vitro ; but under the same conditions, the effect of CIK cells was not obvious ( figure 2 B)....

Embodiment 3

[0120] In vivo test of embodiment 3CAPRI cell activity

[0121] 1×10 autologous tumor cells from patients with colorectal cancer 5 Each was subcutaneously injected into nude mice at the age of 6-8 weeks. At the same time, the patient's peripheral blood mononuclear cells were used to culture CAPRI cells according to the method of Example 1. CIK cells were prepared by CIK technology, and the tail vein injection containing 1 × 10 6 0.1ml of normal saline for each cell, administered continuously for one week. After 21 days or when the mice died, the tumor volume was measured and the survival time of the mice was recorded (observed for 45 days in total). The statistical analysis results are shown in Table 2, Figure 4-Figure 5 .

[0122] Table 2 Comparison of the largest tumor diameter in nude mice

[0123] CAPRI cell group CIK cell group 3.3mm 7.1mm 2.5mm 6.3mm 3.7mm 6.8mm 2.9mm 6.9mm 3.4mm 7.5mm 2.7mm 5.9mm 3.083±0.187...

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Abstract

The invention relates to a cascade tumor specificity killing T lymphocyte (CAPRI cell) activated by induction of cell factors, and a preparation method of the CAPRI cell. The preparation of the CAPRI cell comprises the steps of in-vitro activation of peripheral blood monouclear cells and the proliferation of the CAPRI cell. According to the invention, through the combined stimulation of the in-vitro lymphocyte differentiation antigen resisting antibodies and the cell factors, a large number of T lymphocytes capable of identifying the tumor specific antigens can be obtained through two-step activation. The tumor specificity cells after activation and induction can kill the tumor cells in the bodies of the patients, so that the effect of inhibiting the growth of the primary tumors by combining with the chemoradiotherapy or independently is achieved; by killing the tiny metastatic tumor foci, and eliminating the circulating metastatic tumors or tumor stem cells, the effects of delaying the recurrence and metastasis, prolonging the life time of the patients, and improving the life quality of the patients are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to CAPRI cells and a preparation method thereof. Background technique [0002] Biological treatment of tumors is now widely used as a fourth therapy in addition to surgery, radiotherapy and chemotherapy. Currently, the more common therapies include dendritic cell (DC) therapy and cytokine-induced killer cell (CIK) therapy. [0003] The DC therapy plan is to isolate the DC cell precursors, monocytes, from the patient's peripheral blood lymphocytes, and obtain DCs with antigen-presenting function after activation by cytokines such as IL-4, GMCSF, and TNF. Tumor information molecules such as patient tumor lysate or synthetic tumor polypeptide or tumor cell line lysate or tumor antigen mRNA can be added to the drug, and the final product DC is an antigen-presenting cell carrying tumor information. In clinical application, DC can be used as a vaccine to inoculate patients to in...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0786A61K39/00A61P35/00
CPCA61K39/0011A61K2039/5158A61K2039/545C12N5/0636C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2307C12N2501/2315C12N2501/24C12N2501/25C12N2501/515C12N2506/115
Inventor 古松海赖春宁吴茂友秦森邦古筝
Owner 赛德特生物制药有限公司
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