Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof

A GM-CSF, anti-tumor cell technology, applied in anti-tumor drugs, medical preparations containing active ingredients, antibodies, etc., to achieve good anti-tumor efficacy, good development value, and enhanced anti-tumor immune response

Inactive Publication Date: 2015-11-11
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there are no reports on the combination of the two for tumor treatment.

Method used

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  • Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof
  • Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof
  • Tumor cell vaccine simultaneously secreting PD-1 neutralizing antibody and GM-CSF factor and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Acquisition of PD-1 neutralizing antibody and GM-CSF gene and construction of LV-anti-PD-1, LV-GM-CSF lentiviral vector

[0041] Anti-PD- 1 gene (the nucleotide sequence is shown in SEQIDNO.7, the amino acid sequence of the encoded protein is shown in SEQIDNO.8), mGM-CSF gene (the nucleotide sequence is shown in SEQIDNO.9, the amino acid sequence of the encoded protein shown in SEQ ID NO.10), hGM-CSF gene (the nucleotide sequence is shown in SEQ ID NO.11, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.12). And construct pcDNA3.1-anti-PD-1, pcDNA3.1-mGM-CSF and pcDNA3.1-hGM-CSF plasmids (synthesized by Nanjing GenScript). Then PCR method was used to amplify the target genes of PD-1 neutralizing antibody, mGM-CSF and hGM-CSF respectively, and the target vector of Ubi-MCS-3FLAG-IRES-puro was digested with BamHI / AgeI, and the digested products were recovered by electrophoresis and carried out Exchange, and its product transforms bacteri...

Embodiment 2

[0056] Example 2 Preparation of C26, 4T1, B16-F10 tumor cell vaccines of the present invention and detection of expression levels of PD-1 neutralizing antibody and GM-CSF before and after irradiation.

[0057] Use the lentivirus obtained above to infect colon cancer cell C26 (irradiation dose 100Gy), breast cancer cell 4T1 (irradiation dose 100Gy), melanoma cell line B16-F10 (irradiation dose 60Gy), and then use X-ray irradiation loss of proliferative activity. The cell vaccines of human tumor cell lung cancer cell line A549, breast cancer cell line MCF-7, melanoma cell line A375 and colon cancer HCT116 were also prepared by the same method.

[0058] The colon cancer cell C26, breast cancer cell 4T1, and melanoma cell B16-F10 cell lines stably expressing PD-1 neutralizing antibody and GM-CSF were cultured, and the cell supernatant was collected after 48 hours. At the same time, the supernatant of cells cultured for 48 h after irradiation was collected. The expression levels ...

Embodiment 3

[0059] Embodiment three Therapeutic animal experiment of product C26 vaccine of the present invention

[0060] 20 Balb / c mice were inoculated subcutaneously on the right side of each mouse with 5×10 5 For C26 cells, the tumor volume was measured every 3 days, and 1 × 10 6 The irradiated tumor cell vaccine was treated continuously for 3 times. Treatment groups include: C26 cell group after irradiation, C26 cell irradiation group infected with LV, irradiation group of C26 cell infected with LV-GM-CSF, irradiation group of C26 cell infected with LV-anti-PD-1, simultaneously The cells infected with LV-GM-CSF and LV-anti-PD-1 were irradiated group, and the untreated group was set up as blank control. Observe the tumor growth and the survival period of mice in each group of mice, each experiment is repeated 3 times, the results can be found in image 3 . The results showed that compared with the control group, the tumor inhibition rate of the co-expression of PD-1 neutralizing a...

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Abstract

The invention discloses the field of tumor cellular immunotherapy and particularly relates to a tumor cell vaccine which is modified by a PD-1 neutralizing antibody with combination of a GM-CSF factor and a preparation method thereof. In the invention, tumor cellular vaccine therapy is creatively combined with antibody therapy which relieves tumor immune-suppression, so that the prepared tumor cell vaccine simultaneously secreting the PD-1 neutralizing antibody and the GM-CSF factor can not only relieve the immune-suppression status of a tumor micro-environment but also enhance an antitumor immune reaction. The tumor cell vaccine is good in antitumor treatment effect, is excellent in development value and provides a new approach of tumor cellular immunotherapy.

Description

technical field [0001] The invention belongs to the field of tumor cell immunotherapy, and in particular relates to a tumor cell vaccine that simultaneously secretes PD-1 neutralizing antibodies and GM-CSF factors and a preparation method thereof. Background technique [0002] Tumor vaccines can activate the body's own immune system, induce specific immune responses, and improve autoimmunity. It is an active immunotherapy method. It has mild adverse reactions, good tolerance, and has both preventive and therapeutic effects, and has become one of the hotspots in global anti-tumor research. Programmed death receptor 1 (PD-1) is a costimulatory molecule that conducts inhibitory signals in the CD28 family of T cell regulatory receptors, mediates negative regulatory signals of immune responses, and is involved in tumorigenesis, viral infection and autoimmune diseases. All play a specific immunomodulatory role. PD-1 is widely expressed on the surface of activated T cells, memory...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00A61K39/395A61P35/00A61P37/04
Inventor 邓洪新田红卫魏于全
Owner SICHUAN UNIV
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