Composition for targeted or immune therapy of cancers and application of composition

A therapeutic composition and composition technology, applied in the field of biomedicine, can solve the problems of far low CAR-T cell reports, off-target effects, and little research

Inactive Publication Date: 2019-09-20
BEIJING OBSTETRICS & GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, CAR-T cells have a significant effect on the treatment of circulating lymphocyte tumors, especially for the treatment of B cell malignancies, but the reports of CAR-T cells applied to solid tumors are far lower than those in the blood system
Analyzing the reasons, there are the following situations: ① In addition to the tumor tissue itself, solid tumors are surrounded by mesenchymal components, normal tissues and necrotic tissues, forming a solid barrier. Immune cells need to cross these barriers to reach the target tumor tissue. It is difficult for cells to enter the tumor body, which limits the killing effect
②For solid tumors, there is no perfect tumor-specific antigen. Sometimes the target antigen is also expressed on the surface of normal cells. Any antigen expressed on normal cells has the risk of being accidentally injured, resulting in off-target effects and toxic reactions on normal cells.
③ In the transfection technology, the transfection efficiency of different vectors is limited, and it is extremely difficult to obtain high-efficiency CAR-T cells
At present, the clinical application of CAR-T cells in cervical cancer has not been reported at home and abroad, which may be related to the following factors: ① lack of cervical cancer-specific tumor-associated antigens; ② in developed countries, such as the United States, due to the complete screening of cervical cancer According to the screening system, most of the patients are found in the early stage, and there are few patients with advanced cervical cancer. Therefore, there are few studies in developed countries.

Method used

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  • Composition for targeted or immune therapy of cancers and application of composition
  • Composition for targeted or immune therapy of cancers and application of composition
  • Composition for targeted or immune therapy of cancers and application of composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Screening of specific tumor-associated antigens in cervical cancer

[0036] According to domestic and foreign literature and existing clinical trial reports, four membrane proteins, MESO, MUC1, CD133 and GD2, were selected as tumor-associated specific antigens (TAAs) for cervical cancer. Collect 8 cases of cancer tissues, adjacent cancer tissues and normal cervical tissues of uterine fibroids from patients with "cervical squamous cell carcinoma". According to the expression in tissues, the most specific and highly expressed protein in cancer tissues was selected as cervical cancer TAA.

[0037] Western blot experiments found that GD2 protein was not expressed in cervical squamous cell carcinoma, adjacent cancer and cervical inflammatory tissues, while MESO and MUC1 were highly expressed in cervical squamous cell carcinoma and adjacent cancer tissues. The positive expression rate of MUC1 in cervical squamous cell carcinoma and adjacent tissues is similar to th...

Embodiment 2

[0047] Example 2 Functional detection of MESO protein gene (MSLN)

[0048] A lentivirus with low expression of MESO was constructed, and FACS flow cytometry was used to detect cell apoptosis, MTT method was used to detect cell proliferation, and cell scratch experiment was used to understand cell metastasis.

[0049] Lentivirus interference technology knocked down the expression of MESO in SiHa cells, and RT-PCR showed that the knockdown efficiency was 78.3%. The MESO low expression group (Knock-down, KD) was compared with the SiHa cell group (MOCK group) and SiHa cells infected with empty virus (Normal control, NC group), and the results are as follows.

[0050] 1. FACS flow cytometry cell apoptosis detection

[0051] ShRNA lentivirus was used to infect SiHa cells. After 3 days of culture, the apoptosis rate was compared. The experiment was repeated three times. Compared with 4.77% in NC group, the apoptosis rate in KD group was significantly increased to 5.81%, the differe...

Embodiment 3

[0067] Example 3 Construction of MESO-CAR-T cells

[0068] experimental method:

[0069] 3.1 Construction of MESO-overexpression lentivirus (for the test procedure, see figure 2 )

[0070] 3.1.1 Clone build

[0071] (1) Target gene and tool carrier information (see image 3 )

[0072] Carrier name: GV401

[0073] Cloning site: BamHI / BamHI

[0074] Component sequence: EF1a-ScFv-second generation CarT-2A-EGFP

[0075] (2) Carrier digestion

[0076] According to Table 7 below, prepare 50 μl enzyme digestion system. Add various reagents sequentially according to the order in Table 7, gently blow and mix with a pipette, briefly centrifuge, and place at 37°C for 3 hours or overnight, perform agarose gel electrophoresis on the digested product of the carrier, and recover the target band.

[0077] Table 7. Enzyme digestion system configuration

[0078]

[0079] (3) Acquisition of target gene fragments

[0080] Primers:

[0081] Table 8. Primer sequences

[0082] ...

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Abstract

The invention provides a cervical cancer treatment composition and application thereof. The composition comprises one or various anticancer antibodies which are selected from an MESO antibody, a MUC 1 antibody, a CD 133 antibody and a GD2 antibody. The composition can be used for preparing medicines for cervical cancer treatment, especially cervical cancer immune or targeted therapy medicines.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a composition for cancer targeting or immunotherapy and its application. Background technique [0002] A chimeric antigen receptor (CAR) is a fusion of an antigen-specific CAR and a T cell signaling region, and the antigen specificity is determined by the targeting region in the CAR. At present, CAR-T cells have a significant effect on the treatment of circulating lymphocyte tumors, especially for the treatment of B cell malignancies, but the reports of CAR-T cells applied to solid tumors are far lower than those in the blood system. Analyzing the reasons, there are the following situations: ① In addition to the tumor tissue itself, solid tumors are surrounded by mesenchymal components, normal tissues and necrotic tissues, forming a solid barrier. Immune cells need to cross these barriers to reach the target tumor tissue. It is difficult for cells to enter the tumor body, which limits...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): A61K39/00A61K39/395A61P35/00
CPCA61K39/39558A61P35/00A61K39/4611A61K39/464468A61K39/4631C07K14/7051C07K2319/03
Inventor吴玉梅何玥
OwnerBEIJING OBSTETRICS & GYNECOLOGY HOSPITAL CAPITAL MEDICAL UNIV