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Fusion protein of human serum albumin and interleukin 2 and application thereof

A technology of human serum albumin and human interleukin, which is applied in the field of fusion proteins and can solve problems such as side reactions

Active Publication Date: 2021-04-30
TIANJIN LINDA SINOBIOTECH CO LTD +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The real clinical efficacy of PD-1 / L1 monoclonal antibody is only 15-30%, and there are significant side effects

Method used

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  • Fusion protein of human serum albumin and interleukin 2 and application thereof
  • Fusion protein of human serum albumin and interleukin 2 and application thereof
  • Fusion protein of human serum albumin and interleukin 2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Embodiment 1: Human serum albumin (HSA) gene expression and the construction of carrier plasmid

[0108]Using the total RNA extracted from human fetal liver as a template, the HSA gene was synthesized and amplified by reverse transcription polymerase chain polymerization. 5 micrograms of total RNA were synthesized into corresponding DNA strands under the action of reverse transcriptase (purchased from GIBCO / BRL). The reaction condition was 45°C for 20 minutes, then the temperature was raised to 55°C, and the temperature was maintained for 40 minutes.

[0109] The DNA sequences of the oligonucleotide primers used are: SEQ ID No.11 and SEQ ID No.12.

[0110] Two EcoRI recognition sites were added to the two ends of the HSA gene and cloned into an expression vector. The PCR reaction conditions were 94° C. for 4 minutes, and the DNA encoding HSA was further amplified for 35 cycles: 94° C. for 30 seconds; 58° C. for 30 seconds; 72° C. for 2 minutes and 30 seconds. At the ...

Embodiment 2

[0112] Example 2: Expression of recombinant human serum albumin / interleukin-2 fusion protein and its analog fusion protein and construction of vector plasmid

[0113] According to the GeneBank registration number: NM_000586, NP_000577.2, the amino acid sequence SEQ ID No.10 of human interleukin-2 (wild type) mature peptide was obtained, the inventor designed the molecular structure of recombinant human serum albumin fusion protein, and interleukin-2 mature peptide In a "seamless connection" manner, no connecting peptides are placed between the C-terminals of human serum albumin mature peptides, SEQ ID No.1, and the corresponding nucleotide sequence is SEQ ID No.2, or interleukin- 2 The mature peptides are connected in a "seamless connection" mode, and no connecting peptides are placed between the N-terminals of the mature peptides of human serum albumin, SEQ ID No.3, and the corresponding nucleotide sequence is SEQ ID No.4.

[0114] According to the known computer analysis and...

Embodiment 3

[0117] Embodiment 3: Transformation and preparation of Pichia pastoris expression engineering bacteria

[0118] Pichia pastoris strain X33 colony was inoculated in a 50ml centrifuge tube containing 5ml YPD culture medium, and cultured overnight at 30°C at a speed of 250 rpm. The next day, take 0.2ml of the overnight culture and transfer it into 500ml of YPD culture solution, and place it in a 2-liter Erlenmeyer flask. Incubate with rotation at 30°C for 2-3 hours to bring the cell density to OD 600 =1.3-1.5. The yeasts were collected by centrifugation, resuspended in 500ml ice-cold sterile water and washed twice. Then the yeast was suspended in 20ml of ice-cold 1M sorbitol solution and washed once.

[0119] Taking the pYZ-rhIL-2v5 / SA plasmid DNA constructed in Example 2 as an example, the plasmid DNA is treated with Pme I restriction endonuclease to form a linear plasmid molecule. 5 μg of linearized plasmid DNA was mixed with 80 μl of treated yeast and placed in a 0.2 cm th...

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Abstract

The invention relates to a fusion protein of human serum albumin (especially wild-type human serum albumin) and human interleukin-2 (especially mutant human interleukin-2). The fusion protein has a significantly prolonged in vivo half-life relative to recombinant interleukin-2, so that the fusion protein can be used alone for the treatment of tumors. Moreover, the fusion protein can significantly improve the anti-tumor efficacy of an anti-PD-1 antibody or an anti-PD-L1 antibody.

Description

technical field [0001] The present invention relates to the fusion protein of human serum albumin (especially wild-type human serum albumin) and human interleukin-2 (especially mutant human interleukin-2). Compared with recombinant interleukin-2, the fusion protein has a significantly extended half-life in vivo, so it can be used alone to treat tumors. Moreover, the fusion protein can significantly improve the anti-tumor efficacy of anti-PD-1 antibody or anti-PD-L1 antibody. Background technique [0002] (1) Human serum albumin (HSA) is a soluble monomeric protein that constitutes half of the total protein in blood. As a basic carrier, albumin carries molecules such as fatty acids, steroids and hormones, and its stable and inert nature is an important factor in maintaining blood pressure. Serum albumin is a globular non-glycosylated serum protein with a molecular weight of 65 kilodaltons and 585 amino acids. This protein (precursor of albumin) is then processed by the Gol...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12P21/02A61K38/20A61K39/395A61K47/64A61P35/00A61P7/10
CPCC07K14/55A61K38/2013A61K47/643A61K39/3955A61P35/00A61P7/10C07K2319/31A61K38/00A61K2300/00C07K14/765C07K2319/00C12N15/62C07K2317/21C07K2317/76C07K16/2818A61K39/395A61K38/385
Inventor 于在林富岩杨小楠富俞淞乔国强侯琼朱涛
Owner TIANJIN LINDA SINOBIOTECH CO LTD
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