Co-expression of secretory anti-immune checkpoint antibody, intracellular immune checkpoint inhibiting molecule and tEGFR molecule and application thereof

An immune checkpoint, secreted technology, applied in the direction of antibodies, applications, anti-tumor drugs, etc.

Inactive Publication Date: 2019-05-14
ILIFESEQ LTD CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, tumor immunotherapy is only effective for some patients, and further research and development are still needed to enhance clinical efficacy

Method used

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  • Co-expression of secretory anti-immune checkpoint antibody, intracellular immune checkpoint inhibiting molecule and tEGFR molecule and application thereof
  • Co-expression of secretory anti-immune checkpoint antibody, intracellular immune checkpoint inhibiting molecule and tEGFR molecule and application thereof
  • Co-expression of secretory anti-immune checkpoint antibody, intracellular immune checkpoint inhibiting molecule and tEGFR molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0275] Example 1 Construction of co-expression of secreted anti-immune checkpoint antibody, shRNA for intracellular silencing of immune checkpoint, and non-functional EGFR receptor vector

[0276]In this example, the inventors cloned the fusion gene encoding anti-human PD-L1 single-chain antibody and human IgG1 Fc into a lentiviral vector containing an EF-1 promoter. During the cloning process, the selected restriction enzymes are XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion. Through enzyme digestion, ligation, screening and amplification of the target plasmid, the expression of secreted anti-immune checkpoint antibody is generated. Lentiviral plasmid (LV-αPDL1 scFv-Fc (LV-αPDL1). The inventors cloned the fusion gene encoding anti-human PD-1 single-chain antibody and human IgG4Fc into a lentiviral vector containing EF-1 promoter. During the cloning process, the selected restriction enzymes are XbaI and NotI double-digestion, and NotI and Xho...

Embodiment 2

[0277] Example 2 PD-1 isolated from tumor patients + CD8 + T cells have killing activity against autologous tumor cells.

[0278] from HLA-A2 + PBLs (peripheral blood) and fresh tumor samples were obtained from patients with metastatic melanoma. PBLs were removed by venipuncture, subjected to gradient centrifugation (LSM; ICN Biomedicals Inc.), and stored frozen until analysis. Fresh tumor samples were minced under sterile conditions and digested with enzymes (the composition of the digestion solution was: RPMI-1640 medium containing L-glutamine [Lonza], 1 mg / ml collagenase IV [Sigma-Aldrich ], 30U / ml DNase and antibiotics), the digestion conditions were digested overnight at room temperature or several hours at 37°C, and mechanically separated using MACS (Miltenyi Biotech) intermittently. Tumor single-cell suspensions were used to obtain primary tumor cell lines and to isolate and amplify PD-1 + CD8 + T cells. PD-1 from tumor single cell suspension + CD8 ++ T cells w...

Embodiment 3

[0280] Example 3 Lentiviral vector LV-αPDL1 / iCbl / M transduction enhances PD-1 + CD8 + Tumor killing activity of T cells.

[0281] PD1 isolated from patients with metastatic melanoma + CD8 + T cells, PD1 - CD8 + T cells and unisolated CD8 + T cells were planted on cell culture dishes with recombinant fibronectin fragments (FN ch-296; Retronectin) and transduced with lentiviruses, the transduction lentiviruses were LV-αPDL1 / M / iCbl or empty LV-M transduction guide. Transduced T cells were expanded and used for cell killing experiments. Melanoma target cells were used as target cells from the same patient. The target cells were treated with 100μCi (1Ci=37 GBq) 51 Cr (PerkinElmer) labeled, after elution twice, matched effector T cells from the same patient were plated in a 96-well U-bottom culture dish at the specified effector / target ratio in the culture dish, and cultured at 37°C for 4 Hour. 51 The amount of Cr released was calculated by gamma counting, and a dissolution...

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Abstract

The invention provides an expression vector, a lentivirus, a transgenic cell, a secretory antibody and a therapeutic composition for treating cancer. The expression vector carries the following nucleic acid molecules: (a) a first nucleic acid molecule, which is used for encoding a secretory immune checkpoint inhibiting molecule fragment; (b) a second nucleic acid molecule, which is used for silencing an intracellular immune checkpoint and optionally further carrying a third nucleic acid molecule, wherein the third nucleic acid molecule is used for encoding a non-functional EGFR. The expressionvector of the embodiment of the invention is introduced into receptor cells, a secretory anti-immune checkpoint antibody is efficiently expressed in the receptor cells and secreted outside the cells,meanwhile, the immune checkpoint molecules of the receptor cells are specifically silenced, and an immune escape mechanism mediated by the immune checkpoint is repressed. When the expression vector of the embodiment of the invention is introduced into the receptor cells, such as lymphocytes, the proliferation capacity and the effect function of the lymphocytes are enhanced, and the specific killing of tumor cells is stronger and more effective.

Description

technical field [0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to cells co-expressing secreted anti-immune checkpoint antibodies, intracellular immune checkpoint inhibitory molecules and tEGFR surface markers and their applications. More specifically, the present invention relates to expression vectors, Lentiviruses, transgenic cells, secreted antibodies, and therapeutic compositions for treating cancer. Background technique [0002] Cancer is a disease in which cells proliferate uncontrollably due to genetic mutations in cells. It has become a major threat to human health and is one of the main causes of human death. The World Health Organization (WHO) pointed out in the "Global Cancer Report 2014" that in 2012, the number of cancer patients and deaths worldwide increased rapidly. Therefore, finding efficient and specific cancer treatment methods has great clinical value. [0003] Traditional tumor treatment ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N7/01C12N5/10C07K19/00A61K39/395A61P35/00
Inventor 黄雪芬陈思毅
Owner ILIFESEQ LTD CORP
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