MC (Minicircle) DNA (Deoxyribonucleic Acid) for expressing Her2-BTEC [Bridge between Her2 (Human Epidermal Growth Factor Receptor-2)-Positive Cells and Effector Cells] and application thereof

A technology of positive cells and effector cells, which is applied in the field of biomedicine to achieve the effect of improving the killing effect

Inactive Publication Date: 2019-03-01
SYNO MINICIRCLE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no relevant report on the generation of Her2 bispecific antibody in vivo by means of gene delivery

Method used

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  • MC (Minicircle) DNA (Deoxyribonucleic Acid) for expressing Her2-BTEC [Bridge between Her2 (Human Epidermal Growth Factor Receptor-2)-Positive Cells and Effector Cells] and application thereof
  • MC (Minicircle) DNA (Deoxyribonucleic Acid) for expressing Her2-BTEC [Bridge between Her2 (Human Epidermal Growth Factor Receptor-2)-Positive Cells and Effector Cells] and application thereof
  • MC (Minicircle) DNA (Deoxyribonucleic Acid) for expressing Her2-BTEC [Bridge between Her2 (Human Epidermal Growth Factor Receptor-2)-Positive Cells and Effector Cells] and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1, Construction of Her2-positive cells and effector cell bridging molecule (Her2-BTEC)

[0048] Design Her2-BTEC and construct the corresponding minicircle DNA expression vector. The Her2-BTEC expression cassette includes a variety of structures, which are listed as follows:

[0049] (1) scFv-scFv structure:

[0050]

[0051] Wherein, SP is a signal peptide (Signal Peptide); Linker is a connecting sequence. V L stands for light chain variable region, V H Represents the heavy chain variable region. The positions of anti-Her2 scFv and antiCD3 (or antiCD16) scFv are interchangeable.

[0052] Among them, antiHer2.V H The amino acid sequence of the gene is shown in SEQ ID NO: 1, and the nucleotide sequence of the coding gene is shown in SEQ ID NO: 2; antiHer2.V L The amino acid sequence of the gene is shown in SEQ ID NO: 3, and the nucleotide sequence of the coding gene is shown in SEQ ID NO: 4; antiCD3.V H The amino acid sequence of the gene is shown in SE...

Embodiment 2

[0063] Example 2, Construction of Her2-BTEC minicircle DNA carrier and preparation of minicircle (MC)

[0064] 1. Experimental method

[0065] 1. The gene encoding Her2-BTEC was synthesized. To facilitate the purification and detection of BTEC protein, a 6xHis tag was inserted at the C-terminus.

[0066] 2. Select an appropriate site to double-enzyme digest the linearized minicircle DNA empty vector pMC.BESPX.

[0067] 3. Insert the gene encoding Her2-BTEC into the linearized empty vector pMC.BESPX by using seamless cloning or traditional cloning methods (eg, restriction enzyme digestion-ligation) to construct a minicircle DNA vector pMC.Her2-BTEC.

[0068] 4. The microcircle DNA carrier pMC.Her2-BTEC was transformed into Escherichia coli E coli.ZYCY10P3S2T, and the microcircle MC.Her2-BTEC was obtained according to the standard microcircle preparation method.

[0069] Among them, the microcircle DNA empty vector pMC.BESPX, the engineering bacteria E coli.ZYCY10P3S2T, and th...

Embodiment 3

[0072] Embodiment 3, expression and purification of Her2-BTEC

[0073]1. Expression of Her2-BTEC in 293T cells

[0074] The superfect plasmid transfection kit (Invitrogen Company) was used to transfect 293T cells with the above-mentioned minicircle DNA, and culture supernatants of 293T cells were collected after culturing in serum-free medium for three days.

[0075] 2. Purification of Her2-BTEC

[0076] 1. Collect the cell culture supernatant for low-temperature ultracentrifugation, and take the supernatant.

[0077] 2. Her2-BTEC was purified by His-Tag affinity resin (cOmplete His-Tag Purification Resin, Roche).

[0078] 3. The purified protein was qualitatively detected by PAGE or Western Blot, and the protein concentration was quantitatively detected by Bradford method.

[0079] 4. Store the purified product at -20°C or -80°C for long-term storage.

[0080] 3. Experimental results

[0081] figure 2 (Right) shows the results of SDS-PAGE detection of Her2-BTEC express...

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Abstract

The invention relates to MC (Minicircle) DNA (Deoxyribonucleic Acid) for expressing Her2-BTEC [Bridge between Her2 (Human Epidermal Growth Factor Receptor-2)-Positive Cells and Effector Cells] and application thereof, in particular to an MC DNA carrier for expressing an anti-Her2 bispecific antibody in vivo. Her2 positive cancer cells (target cells) are killed by bispecific antibody mediated effector cells (T cells or NK cells). The invention firstly discloses an MC DNA carrier design scheme of MC.Her2-BTEC, and the MC DNA carrier design scheme is suitable for treating cancer related to Her2.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a minicircle (Minicircle, MC) DNA carrier, more specifically a minicircle DNA carrier for expressing a bridging molecule connecting Her2 positive cells and effector cells in vivo. Background technique [0002] Her2 (human epidermal growth factor receptor-2, human epidermal growth factor receptor 2), also known as Her2 / neu or ErbB2, is a member of the type I transmembrane tyrosine kinase receptor family and belongs to a type of transmembrane protein. When it is overexpressed or mutated, it often induces the homodimerization of Her2 or the heterodimerization between Her2 and other family members, and then activates the downstream RAS-MAPK and the AKT signaling pathway targeting mTOR, resulting in abnormal cell proliferation and survive. It has been found that Her2 is highly expressed on the surface of various cancer cells, including breast cancer, non-small cell lung cancer,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/13C07K16/46A61K39/395A61P35/00
CPCA61K2039/505A61K2039/53A61P35/00C07K16/2809C07K16/283C07K16/32C07K2317/31C07K2317/33C07K2317/622C12N15/85C12N2800/107
Inventor 谌平谢亦武陈志英
Owner SYNO MINICIRCLE BIOTECH CO LTD
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