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Nanobody and its encoding gene, expression vector, host cell and application

A technology of nano-antibody and coding gene, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, application, etc. It can solve the problems of rapid and uniform dispersion, high production cost, large molecular weight, etc. Problems, achieving remarkable application results, low production costs, and strong tissue penetration

Active Publication Date: 2022-08-02
沈阳荣欣生物制药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Siglec-15 monoclonal antibody reported earlier is a complete antibody molecule. Due to its large molecular weight, it is difficult to quickly and evenly disperse in the internal tumor tissue and tumor stroma, and its structure is complex, suitable for cell expression, and the production cost is high.

Method used

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  • Nanobody and its encoding gene, expression vector, host cell and application
  • Nanobody and its encoding gene, expression vector, host cell and application
  • Nanobody and its encoding gene, expression vector, host cell and application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] This example provides an anti-Siglec-15 nanobody, which is a nanobody against a specific extracellular region of Siglec-15, the amino acid sequence of which is such as SEQ ID NO.8, SEQ ID NO.13, SEQ ID NO.18, SEQ ID NO. 23, and the nucleotide sequences encoding them are shown in SEQ ID NO. 7, SEQ ID NO. 12, SEQ ID NO. 17, and SEQ ID NO. 22, respectively. Specifically, the nucleotide sequences encoding them consist of 3 framework regions and 3 CDRs, wherein the nucleotide sequences of the 3 framework regions are respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 As shown, the nucleotide sequences of the three CDRs are respectively SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6; SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.11; SEQ ID NO.11; NO.14, SEQ ID NO.15 and SEQ ID NO.16, SEQ ID NO.19, SEQ ID NO.20 and SEQID NO.21 are shown; the nucleotide sequences of the three framework regions are shown as SEQ ID NO. 24 (corresponding to SEQ ID NO.1), SEQID NO.25 (corresponding to SEQ I...

Embodiment 2

[0081] This embodiment provides a screening method for Siglec-15 nanobody, which includes:

[0082] Using the human phage nanobody library, Siglec-15 was used as the antigen, and 3 rounds of screening were performed from the antibody library, and each round of screening was carried out according to the steps of adsorption-elution-amplification.

[0083] (1) Amplification of VH nanobody library: Thaw the frozen phage antibody library on ice, pipette 400 μL into pre-warmed 200 mL of 2×TY, add 1% Glu and 1% Amp. Incubate at 37°C and 250 rpm to OD600=0.4. Take 50 mL of bacterial solution and add 2 × 10 11 KM13 helper phage was incubated in a water bath at 37°C for 30 min. Centrifuge at 3000 g for 10 min to discard the supernatant, resuspend the pellet with 100 mL of 2×TY, add 100 μg / mL Amp and 50 μg / mL Kan, and incubate overnight at 30°C and 250 rpm. The next day, the overnight bacterial solution was centrifuged at 3300 g for 30 min, the precipitate was discarded, PEG / NaCl wit...

Embodiment 3

[0088] This embodiment provides a yeast expression and purification method of an anti-Siglec-15 Nanobody, comprising the following steps:

[0089] S1. Sequence analysis of 3 positive clones, clone into pPICzα A yeast expression plasmid, and transform into Pichia pastoris GS115 for soluble expression. The nucleotide and amino acid sequences are SEQ ID: NO.7 and SEQ ID: NO.8; SEQ ID NO.12 and SEQ ID: NO.13; SEQ ID: NO.17 and SEQ ID: NO.18; NO .22 and SEQ ID: NO.23.

[0090] S2. Extract the PIT2-Siglec-15-VH recombinant plasmid and pPICZα A plasmid respectively, and use PIT2-Siglec-15-VH as the template for PCR to amplify the VH of the target fragment. After recovering the target fragment and the enzyme-cut vector, use T4 DNA ligase to connect the target gene fragment and the vector. Ethanol-precipitated DNA; 5-20 µg of linearized DNA was electroporated into Pichia GS115. Transformants were selected for gene sequencing, positive clones were picked and cultured, and methanol wa...

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Abstract

The present invention is applicable to the field of biological gene technology, and provides a nanobody and its encoding gene, expression vector, host cell and application. The nanobody is a nanobody directed against the Siglec-15 extracellular specific region, which comprises at least one amino acid Nanobodies whose sequences are shown in SEQ ID NO.8, SEQ ID NO.13, SEQ ID NO.18, and SEQ ID NO.23. In the present invention, a nanobody capable of specifically binding to the extracellular region of Siglec-15 is obtained, which has great potential in the research and development of Siglec-15 therapeutic antibody, preferably an anti-tumor antibody treatment. The nanobody has strong and fast tissue penetration ability, and can enter dense solid tumor tissue to play a role; its molecular structure is relatively simple, and can be fermented and produced by Escherichia coli or yeast expression system, which greatly reduces the production cost.

Description

technical field [0001] The invention belongs to the technical field of biological genes, and in particular relates to a nanobody and its encoding gene, expression vector, host cell and application. Background technique [0002] Cancer is the main cause of death worldwide. According to the International Union Against Cancer, about 9.6 million people die from cancer every year, far exceeding the sum of deaths from diseases such as AIDS, tuberculosis, and malaria. Experts estimate that by 2030, the number of people dying from cancer worldwide will reach 13 million each year. Immunotherapy has become the fourth largest cancer treatment after conventional treatments such as surgery, radiotherapy, and chemotherapy because of its small side effects, high antitumor activity, and wide indications. PD1 / PDL1 immunotherapy is currently a high-profile anti-cancer immunotherapy. PDL1 molecules are highly expressed on the surface of tumor cells, which can bind to PD1 molecules expressed o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00C12N15/70C12N15/81G01N33/574G01N33/68C12R1/84C12R1/19
CPCC07K16/2803C07K16/005C12N15/815C12N15/70G01N33/574G01N33/57484G01N33/6854A61P35/00A61K2039/505C07K2317/569C07K2317/565C07K2317/567G01N2333/70503C07K2317/94C07K2317/14
Inventor 王天怡姜海涛
Owner 沈阳荣欣生物制药科技有限公司
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