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Minicircle DNA expression bridging molecules for connecting human and animal target cells with effector cells, and application of minicircle DNA expression bridging molecules

A technology of effector cells and target cells, applied in the field of biomedicine, can solve the problems of evaluating safety and effectiveness of monkeys or mice, and achieve the effect of promoting the development process

Pending Publication Date: 2019-05-03
SYNO MINICIRCLE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the bispecific antibodies currently under development are humanized or humanized. They are only specific for human-derived antigens and have no cross-reaction with monkey or mouse antigens. It is difficult to use monkeys or mice to evaluate their safety. sex and effectiveness

Method used

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  • Minicircle DNA expression bridging molecules for connecting human and animal target cells with effector cells, and application of minicircle DNA expression bridging molecules
  • Minicircle DNA expression bridging molecules for connecting human and animal target cells with effector cells, and application of minicircle DNA expression bridging molecules
  • Minicircle DNA expression bridging molecules for connecting human and animal target cells with effector cells, and application of minicircle DNA expression bridging molecules

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the construction of human-animal cross-reactive bridging molecule (crBTEC)

[0044] Design crBTEC and construct the corresponding microcircle DNA expression vector. The crBTEC expression cassette includes a variety of structures, as follows:

[0045] (1) scFv-scFv structure:

[0046]

[0047] Wherein, SP is a signal peptide (Signal Peptide); Linker is a connecting sequence. Tar represents the target cell (Target) antigen, such as CD19, CD20, EpCAM, Her2 and so on. anti-Tar.V H The amino acid sequence is shown in SEQ ID NO: 1, 5 or 9, anti-Tar.V L The amino acid sequence is shown in SEQ ID NO: 3, 7 or 11; anti-CD3.V H The amino acid sequence is shown in SEQ ID NO: 13, anti-CD3.V L The amino acid sequence is shown in SEQ ID NO: 15;

[0048] anti-CD16.V H The amino acid sequence is shown in SEQ ID NO: 17, anti-CD16.V L The amino acid sequence of is shown in SEQ ID NO:19. It also includes the coding genes of the above elements; the coding genes of ...

Embodiment 2

[0057] Embodiment 2, the construction of microcircle DNA carrier and the preparation of microcircle (MC)

[0058] (a) Synthesis of the gene encoding crBTEC.

[0059] (b) Select an appropriate site to double-enzyme digest the empty minicircle DNA vector pMC.BESPX.

[0060] (c) Insert the gene encoding crBTEC into the empty vector pMC.BESPX after double digestion by using seamless cloning or traditional cloning methods (eg, enzyme digestion-ligation) to construct a minicircle DNA vector (pMC.crBTEC).

[0061] (d) Escherichia coli E coli.ZYCY10P3S2T was transformed with the vector pMC.crBTEC, and the microcircle (MC.crBTEC) was obtained according to the standard microcircle preparation method.

[0062] Among them, the microcircle DNA empty vector pMC.BESPX, the engineering bacteria E coli.ZYCY10P3S2T, and the preparation method of the microcircle refer to the reference Nat Biotechnol.2010,28:1287-1289.

Embodiment 3

[0063] Expression and purification of embodiment 3, crBTEC

[0064] 1. Expression of crBTEC in 293T cells

[0065] The superfect plasmid transfection kit (Invitrogen) was used to transfect 293T cells with the above-mentioned minicircle DNA, and the supernatants of 293T cells were collected after culturing in serum-free medium for three days.

[0066] 2. Purification of crBTEC

[0067] Collect the cell culture supernatant by low-temperature ultracentrifugation, take the supernatant, and then use His-Tag affinity resin to purify (cOmplete His-Tag Purification Resin, Roche). The purified protein is qualitatively detected by PAGE or Western Blot, and quantitatively detected by Bradford method protein concentration. Store the purified product at -20 or -80°C for long-term storage.

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Abstract

The application relates to a minicircle DNA expression bridging molecules for connecting human and animal target cells with effector cells, and an application of the minicircle DNA expression bridgingmolecules, in particular to a minicircle DNA vector for in vivo expression human-animal cross reaction bispecific antibody(crBsAb). The human-animal bispecific antibody(crBsAb) can combine human or animal target cells, and can also combine human or animal effector cells (T cells or natural killer cells) so as to treat cancer, infection and other diseases. Before clinical testing with medicines, the human-animal cross reaction bispecific antibody is favorable for evaluation of the safety and the validity of bispecific antibody drugs with an animal model, so as to promote development process ofthe medicines. The bridging molecules are completely the same in large animal experimentation and clinical minicircle DNA-gene drugs for human, and the condition that in the prior art, two differenteditions of biology drugs for common-used animal and human can cause difficult result interpretation, is avoided.

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 kind of expression in vivo of human-animal cross-reactive bispecific antibody (Bispecific antibody cross-reactive between human and animal, crBsAb) minicircle DNA vector. Background technique [0002] Bispecific antibody (Bispecific antibody, referred to as BsAb) is a kind of genetically engineered antibody, which is a type of antibody hybrid molecule with dual functions. The variable domains (or Fab segments) in bivalent antibodies have different specificities. , capable of binding to two different ligands. When designing BsAb molecules, researchers consider that BsAb can bind to antigen molecules on target cells and to functional molecules on effector cells. In this way, BsAb molecules build bridges between target cells (antigen molecules) and effector cells (functional molecules) to stimulate a directional ...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/13A61K48/00A61P35/00A61P31/00
Inventor 谢亦武
Owner SYNO MINICIRCLE BIOTECH CO LTD
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