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Immunity enhancing reagent

A technology of immune enhancement and reagents, applied in the field of biomedicine, can solve the problems of insurmountable immunosuppression and low curative effect

Active Publication Date: 2015-09-09
ICARTAB BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the immunosuppressive microenvironment in tumor patients, these activated immune cells in vitro are still unable to overcome the immunosuppressive effect in vivo, so adoptive immunotherapy has shown its very low efficacy in a large number of clinical trials and applications. (Trajanoski Z, Maccalli C, Mennonna D, Casorati G, Parmiani G, Dellabona P (2015) Somatically mutated tumor antigens in the quest for a more efficacious patient-oriented immunotherapy of cancer. Cancer immunology, immunotherapy : CII 64: 99- 104)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction and preparation of immunoenhancing reagents

[0049] (1) According to the corresponding relationship in Table 1, synthesize 10 full-length DNA sequences that can encode the heavy chain and light chain of the immune checkpoint monoclonal antibody. The full-length heavy chain DNA sequence is composed of signal peptide DNA sequence, heavy chain variable region DNA sequence, heavy chain constant region DNA sequence; the light chain DNA full-length sequence is composed of signal peptide DNA sequence, light chain variable region DNA sequence, light chain The DNA sequence of the chain constant region is composed, and the specific sequence is shown in the sequence listing.

[0050]Table 1 The amino acid sequence and nucleotide base sequence corresponding to the variable region of the antibody molecule encoding the immune checkpoint and the code of the virus constructed in the present invention

[0051]

[0052] A total of 5 heavy chain full-length DNA...

Embodiment 2

[0067] Example 2 Experimental verification of cytokine release in vitro

[0068] Peripheral blood mononuclear cells (PBMC cells) were isolated from human peripheral blood using Lymphoprep reagent, cultured with RPMI1640 and 10% FBS, and divided into blank group, control group, and experimental group.

[0069] The first group added PHA to the culture medium to activate peripheral blood T cells, which was the blank group; the second group was the co-culture group of PHA-activated T cells and tumor cells (lung cancer cells HCC829). Tumor cells were treated for 24 hours to increase the expression level of immune checkpoint molecular ligands (such as PD-L1 molecules, etc.) The cells were co-cultured and served as the control group; the experimental group was based on the second group of co-culture system, respectively adding different immunoenhancing reagents obtained from Example 1 (use normal saline to adjust the virus titer to the same concentration of 5× 10 13 vg / mL); 48 hou...

Embodiment 3

[0075] Example 3 Induction of apoptosis of tumor cells by immune cells secreting immune checkpoint antibodies

[0076] Peripheral blood mononuclear cell (PBMC) cells were isolated from human peripheral blood using Lymphoprep reagent, and cultured with RPMI1640 and 10% FBS. Add PHA to the medium to activate peripheral blood T cells as effector cells; treat tumor cells with γ-interferon for 24 hours to increase the expression level of immune checkpoint ligand molecules on the surface of tumor cells, such as PD-L1 molecules, on the tumor surface , and then washed three times with PBS to remove interferon-gamma in the medium, and tumor cells (hepatoma cells HepG2) treated with interferon-gamma were used as target cells. The activated effector cells were divided into two groups, and one of them was added to an immune enhancement reagent in Example 1 (immune checkpoint adeno-associated virus combination I-2:II-3:III-4=1:3:1 ), to infect the effector cells so that they can secrete a...

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Abstract

The invention discloses an immunity enhancing reagent comprising AAVs carrying molecule encoding genes of antibodies at immunodetection points, wherein the immunodetection points are one or several from PD-1, PD-L1 and CTLA-4. The AAVs carrying the molecule encoding genes of the antibodies at different immunodetection points are combined with each other, T cells, natural killer cells, cytotoxic T lymphocytes or cell factor induced killer cells and other immune cells are infected in vitro, and the antibodies at the immunodetection points are expressed and secreted after the infected immune cells are fed back to bodies of patients suffering from tumors, so that the combination of receptors at the immunodetection points and ligands of the receptors is blocked, the transfer of an immunosuppression signal is effectively blocked, the immune tumor suppression microenviroment is destroyed, the immune cell killing capacity is improved, and furthermore the curative effect of adoptive immune therapy is improved.

Description

Technical field [0001] The invention is a biomedical field, which involves a kind of immune enhanced reagent that can improve the ability of immune cells to activate, proliferation and cytokine release capabilities in vitro. Background technique [0002] During the activation of immune cells, many surface molecules are precisely adjusted, and some promote immune cell activation; some are inhibitory receptor molecules that are responsible for transmitting inhibitory signals to prevent excessive activation of the immune system.These stimulus or inhibitors of immune cell activation are usually called immune examination points, or molecules of immune examination point, immunohistos and molecules. [0003] Immunomorphic examination points include cytotoxic T lymphocyte-related antigens 4 (Cytotoxic T Lymphocyte-Associated Antigen-4, CTLA-4) and programmed death receptor 1 and its ligand (PD-1 / PD-L1)Signal channel molecules and PD-L2, IDO-1, IDO-2, KIR, OX40, CD70, LAG-3, TIM3, etc.CTL...

Claims

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Application Information

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IPC IPC(8): A61K35/76A61K39/395A61P35/00C12N5/10
Inventor 季平李凡池于继彬
Owner ICARTAB BIOMEDICAL
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