Co-expression cell of secretory anti-immune checkpoint antibody and tEGFR molecule and application thereof

An immune checkpoint, secreted technology, applied in the fields of transgenic cells, expression vectors, therapeutic compositions for cancer treatment, secreted antibodies, and lentivirus

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, a...

Method used

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  • Co-expression cell of secretory anti-immune checkpoint antibody and tEGFR molecule and application thereof
  • Co-expression cell of secretory anti-immune checkpoint antibody and tEGFR molecule and application thereof
  • Co-expression cell of secretory anti-immune checkpoint antibody and tEGFR molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1 Construction of vectors for co-expression of secreted anti-immune checkpoint antibodies and non-functional EGFR receptors

[0128] 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 enzyme digestion, and NotI and XhoI double enzyme digestion. Through enzyme...

Embodiment 2

[0129] Example 2 PD-1 isolated from tumor patients + CD8 + T cells have killing activity against autologous tumor cells.

[0130] 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], 1mg / ml collagenase IV [Sigma-Aldrich ], 30U / ml DNase and antibiotics), the digestion conditions were overnight at room temperature or several hours at 37°C, and mechanical separation was performed intermittently using gentleMACS (Miltenyi Biotech). 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 ++ ...

Embodiment 3

[0132] Example 3 Lentiviral vector LV-αPDL1 / M transduction enhances PD-1 + CD8 + Tumor killing activity of T cells.

[0133] PD1 isolated from patients with metastatic melanoma + CD8 + T cells, PD1 - CD8 + T cells and unisolated CD8 + T cells were seeded on cell culture dishes lined with recombinant fibronectin fragments (FN ch–296; Retronectin) and transduced with lentiviruses, respectively LV-αPDL1 / M or empty LV-M transduction. Transduced T cells were expanded for cell killing assay. Melanoma target cells were used as target cells from the same patient. The target cells were treated with 100μCi (1Ci=37GBq) 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 incubated at 37°C for 4 hours . 51 The amount of Cr released was calculated by gamma counting, and a dissolution rate was calculated for three samples. imag...

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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 first nucleic acid molecule, which is used for encoding a secretory immune checkpoint inhibiting molecule fragment; optionally, a second nucleic acid molecule is further carried, wherein the second nucleic acid molecule is used for encoding a non-functional EGFR. The expression vector of the embodiment of the invention is introduced into receptor cells, a secretory anti-immunecheckpoint antibody is efficiently expressed in the receptor cells and secreted outside the cells, 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 specific killing of tumor cells by lymphocytes is strong and 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 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 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, it is of great clinical value to find efficient and specific cancer treatment methods. [0003] Traditional tumor treatment methods mainly include surgery, radiotherapy and chem...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N7/01C12N5/10C07K19/00A61P35/00C12R1/93
Inventor 黄雪芬陈思毅
Owner ILIFESEQ LTD CORP
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