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Fusion protein based on single-chain antibody fragment, nano assembly and preparation method and application thereof

A nano-assembly, fusion protein technology, applied in the field of medicine, can solve the problems of destroying the antigen recognition region of the antibody, reducing the antigen recognition of the antibody, complicated reaction and purification process, etc., and achieving the effect of excellent stability

Active Publication Date: 2022-05-20
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported ways of immobilizing antibodies are mainly to use amino groups, carboxyl groups, sulfhydryl groups and other groups on antibody drug molecules to bond them to the surface of particles, but these methods have many problems, such as complex reaction and purification processes, and damage The advanced structure of the antibody or the blocking of the antigen recognition region of the therapeutic antibody significantly reduces the ability of the antibody to recognize the antigen, etc.

Method used

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  • Fusion protein based on single-chain antibody fragment, nano assembly and preparation method and application thereof
  • Fusion protein based on single-chain antibody fragment, nano assembly and preparation method and application thereof
  • Fusion protein based on single-chain antibody fragment, nano assembly and preparation method and application thereof

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preparation example Construction

[0067] In some embodiments of the present invention, it relates to a method for preparing the above-mentioned nano-assembly, comprising the following steps:

[0068] (1) mixing the fusion protein with water or an aqueous solution to obtain an aqueous phase; mixing the hydrophobic degradable polyester and its derivatives with an organic solvent to obtain an oil phase;

[0069] (2) preparing the water phase and the oil phase described in step (1) into an oil-in-water emulsion;

[0070] (3) Separating and purifying the emulsion to obtain a nano assembly.

[0071] In some of these embodiments, an oil-in-water emulsion is prepared by means of microfluidics, high-pressure homogenization, and ultrasound;

[0072] Alternatively, the emulsion is separated and purified by means of rotary evaporation, freeze-drying, centrifugation, chromatography, ultrafiltration, etc. to obtain a nano assembly.

[0073] In some of these embodiments, the concentration of the fusion protein in the aqueo...

Embodiment 1

[0139] Embodiment 1 Design of single-chain antibody fragment fusion protein gene

[0140] Enter the IMGT database, enter the antibody sequence search entry, enter the antibody target to be queried, select the antibody, enter the amino acid sequence interface, select the VH and VL Kappa or Lamda segment, and use the two segments (GGGGS) 3 Linked to obtain the corresponding single-chain antibody fragment peptide sequence.

[0141] The structures of αHER2 scFv and α4-1BB scFv are VL-L-VH, and the structures of αPDL1 scFv and αTIGIT scFv are VH-L-VL.

[0142] Pass the single-chain antibody fragment with the peptide sequence of HSA or MSA without signal peptide (GGGGS) 3 The corresponding single-chain antibody fragment-albumin recombinant fusion protein peptide sequence is obtained.

[0143] The peptide sequence is converted into a nucleotide sequence by SnapGene or other similar software, which is synthesized by Shanghai Sangon.

[0144] αHER2 scFv-HSA was constructed by the abov...

Embodiment 2

[0149] Embodiment 2 Amplifies MSA sequence from mouse cDNA library

[0150] Obtain the MSA (mouse serum albumin, Mouse Serum Albumin) cDNA that does not have signal peptide coding sequence from mouse liver fetal cDNA library by PCR method, used primer MSA F (SEQ ID NO.8) and MSA R (SEQ ID NO.9) was synthesized with an oligonucleotide synthesizer, the downstream primer was introduced into the XbaI restriction site and the protective base, and the underlined part was the endonuclease recognition sequence.

[0151] 50μL PCR reaction system: 2x Mix 25μL, DNA template 2 O is supplemented, and the reaction system can be reduced or enlarged according to the demand. Perform PCR after gentle mixing. The PCR reaction conditions are heat denaturation at 94°C for 1 min; denaturation at 94°C for 30 s; annealing at 58°C for 30 s; extension at 72°C for 1.5 min; a total of 30 cycles; and extension at 72°C for 5 min. The expected 1.6kb band was obtained by 1% agarose gel detection analysis, a...

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Abstract

The invention relates to a recombinant fusion protein, a nano assembly as well as a preparation method and application of the nano assembly. The nano assembly is constructed from at least one recombinant fusion protein, hydrophobic degradable polyester and derivatives thereof. The recombinant fusion protein comprises a single-chain antibody fragment, a connecting peptide and albumin with a hydrophobic region, wherein the single-chain antibody fragment has at least one of a competitive binding target, an inhibition signal channel, an activation signal channel and an antigen targeting effect. The invention also discloses a preparation method of the monoclonal antibody fragment-albumin recombinant fusion protein and a construction method of a nano assembly. The nano assembly provided by the invention provides a brand-new construction method of a monospecific, bispecific or multispecific antibody, and the nano assembly can be used for treating diseases such as tumors, autoimmune diseases and the like.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a recombinant fusion protein based on a single-chain antibody fragment, a nano assembly and a preparation method and application thereof. Background technique [0002] Monoclonal antibody drugs represented by immune checkpoint blocking antibodies have become a new hotspot in the global biopharmaceutical field, and the PD1 / PDL1 and CTLA4 monoclonal antibody drugs that have been marketed are profoundly changing the pattern of tumor treatment. At present, more than 10 PD1 / PDL1 monoclonal antibody drugs have been approved for the treatment of different types of tumors such as non-small cell lung cancer and melanoma in China. At the same time, the development of monoclonal antibody drugs targeting other immune checkpoint molecules of T cells (TIGIT, OX40, LAG3, TIM3, etc.) and other immune cells (including macrophages, natural killer cells, etc.) is also in full swing. [0003] Immu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62A61K47/64A61K47/65A61K39/395A61K9/14A61P35/00A61P37/02A61P29/00A61P31/00B82Y5/00B82Y40/00
CPCA61K47/643A61P35/00A61K9/146A61P29/00C07K14/715C07K14/76C07K14/47A61K38/1793A61K47/64A61K47/6873A61P37/00C07K2319/00A61K9/107A61K47/34A61P37/02C07K19/00A61K9/14A61K47/65A61P31/00B82Y40/00C12N15/62A61K47/68A61K38/17A61K47/59A61K47/62A61K47/69C07K14/35C07K17/00A61K38/00Y02A50/30C07K2317/622C07K16/32C07K16/2878C07K16/2827C07K16/2803C07K2319/31C07K2317/92C07K2317/74C07K2317/73C08L67/04C08L89/00B82Y5/00C07K16/18A61K47/6937A61K47/6849C07K14/70535
Inventor 王均沈松赵东坤
Owner SOUTH CHINA UNIV OF TECH
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