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Recombinant immune cytokine and application thereof

A technology of immune cells and factors, applied in the biological field, can solve the problems of adverse reactions, low response rates, off-target effects, etc., and achieve the effect of good tumor suppressor activity and secretion promotion.

Inactive Publication Date: 2018-04-17
TAIZHOU MABTECH PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the biggest limitation of the widespread use of IL-2 is the severe adverse reaction of vascular leak syndrome characterized by hypertension and fever and its half-life of only 5-7 minutes, followed by a secondary clearance time of 30-120 minutes
Another shortcoming of IL-2 is that it widely binds to receptors expressed on immune cells, making regulatory T cells (Treg) expand and hindering the body's anti-cancer immunity (Boyman, The role of interleukin-2 during homeostasis and activation of the immunesystem. Nat Rev Immunol, 2012. 12(3): p. 180-90.)
[0011] Although a variety of immune agents can elicit effective immune responses against tumor cell invasion, there are still certain disadvantages in treating cancer by activating the immune system: serious adverse reactions, severe off-target effects, and upregulated immunosuppression on the surface of cancer cells low response rate due to factor

Method used

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  • Recombinant immune cytokine and application thereof
  • Recombinant immune cytokine and application thereof
  • Recombinant immune cytokine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1, Construction, expression and identification of immune cytokine BIPI

[0043] Using genetic engineering methods, the heavy chain constant region gene of PD-L1 monoclonal antibody and the IL-2 mutated gene were synthesized to obtain the full-length heavy chain fragment of BIPI, which was digested and connected to the pcDNA3.1 eukaryotic expression vector superior. The schematic diagram of the heavy chain structure is shown in figure 1 As shown, and the schematic diagram of the protein structure is shown in figure 2 As shown, the Fc end of the heavy chain of the original anti-PD-L1 antibody passed through (GGGGS) 3 The linker connects the IL-2 gene. pass EcoR I and Hind III Double enzyme digestion identification, see the results image 3 , proving that the BIPI light chain plasmid and the BIPI heavy chain plasmid have been successfully constructed. The correctness of the light chain and heavy chain sequences was verified by sequencing.

[0044] After...

experiment example 1

[0046] Experimental example 1. Detection of the binding activity of the immune cytokine BIPI to the PD-L1 antigen on the cell surface

[0047] (1) The recombinant CHO cells with high expression of PD-L1 were divided into 2×10 5 The density was resuspended in 1% FBS PBS buffer, and 100ul per tube was added to the flow tube. Drop vertically, do not drop the cell suspension on the tube wall;

[0048] (2) Dilute the antibody: Dilute the purified BIPI with PBS to a concentration of 100ug / mL, add 100uL / tube to the flow tube, and make triplicate wells. The other control groups (IgG, PD-L1 antibody and PBS) were treated as above; the solutions in the flow tubes of each group were mixed with a vortex shaker, and kept in the refrigerator at 4°C for 1 h in the dark;

[0049](3) Centrifuge the flow tube at 1000 r / min for 5 min, quickly pour off the upper layer, do not shake to avoid cell loss at the bottom of the tube, then add 1% FBS in PBS solution at 200 uL / tube, shake and mix well ...

experiment example 2

[0053] Experimental example 2. Detection of IL2 biological activity in immune cytokine BIPI

[0054] (1) Take CTLL-2 cells in good growth state, 1×10 per well 4 Cells were spread evenly in a 96-well cell culture plate;

[0055] (2) Add different concentrations of immunocytokines and recombinant human IL-2 in gradient ratio dilution to each well, and start to dilute the antibody at a concentration of 100ng / ml. By calculation, the ratio of IL-2 contained in BIPI to IL-2 2 The standard products have the same molar mass starting point, carry out 2-fold ratio dilution, a total of 15 concentrations, and make triplicate wells, at 37 ℃, 8% CO 2 cultured in an incubator for 72 h;

[0056] (3) Add 60uL of APS and MTS reagent mixed at a ratio of 1:6 to each well for color development. After 3 hours of color development, place the cell culture plate in the iMark microplate reader, set the wavelength to 450 nm and read the absorbance value of each group;

[0057] (4) Percentage of cell...

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PUM

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Abstract

The invention relates to a recombinant immune cytokine targeting PD-L1 and IL-2 receptors and an application of the recombinant immune cytokine. By means of a whole-genome synthesis technology, an Fcheavy-chain gene of an IgG1 subclass antibody of a PD-L1 monoclonal antibody is fused with a mutated IL-2 gene through linker, and the novel recombinant immune cytokine is obtained. The recombinant immune cytokine has better tumor-inhibiting activity, especially for tumor types with PD-L1 weak expression, and achieves effects of activating in-vivo lymphocytes and promoting cytokine production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically, the invention relates to a recombinant immune cytokine targeting PD-L1 and IL-2 receptors and its application. Background technique [0002] PD-1 is a type I transmembrane glycoprotein, which is composed of an IgV type extracellular domain, a transmembrane region and an intracellular tail region with 21%-22% homologous genes with the CD28 family. The intracellular tail region is composed of two phosphorylation sites on the immunoreceptor tyrosine inhibitory motif and the immunoreceptor tyrosine switch module. This structure, when phosphorylated, can negatively regulate TCR signaling through phosphorylated Src homology phosphatase 1 (SHP-1) and SHP-2. PD-1 exists as a monomer because it lacks the cysteine ​​residues adjacent to the cell membrane necessary for dimer formation. The gene encoding PD-1, PDCD1, is on human chromosome 2 and contains 5 exons (Keir, et al., PD-1 and ...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K39/395A61K38/20A61K47/68A61P35/00A61P37/02
CPCA61K38/2013A61K39/395C07K14/55C07K16/2827C07K2319/00
Inventor 郭亚军寇庚钱卫珠郭怀祖徐进
Owner TAIZHOU MABTECH PHARM CO LTD
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