Fusion polypeptides and uses thereof

A technology for fusing peptides and binding sites, which is applied in the direction of hybrid peptides, plasma life extension fusion, peptides, etc., can solve problems such as short half-life in vivo and safety issues

Pending Publication Date: 2022-01-11
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Those skilled in the art also know that strong immune stimulation may bring safety problems
[0007] In ad

Method used

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  • Fusion polypeptides and uses thereof
  • Fusion polypeptides and uses thereof
  • Fusion polypeptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0095] HSA can also be modified to improve its properties. For example, the free cysteine ​​in wild-type HSA can be substituted for other amino acids, such as serine. In some embodiments, the HSA comprises the amino acid substitution C58S.

[0096] In some embodiments, the carrier protein has at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 16, and has The same or similar structure as type HSA.

[0097] In some embodiments, the N-terminus and C-terminus of the polypeptide of interest are linked to the carrier protein via a linker. In some embodiments, the N-terminus of the polypeptide of interest is connected to the carrier protein through a linker, and the C-terminus of the polypeptide of interest is directly connected to the carrier protein. In some embodiments, the N-terminus of the polypeptide of interest is directly connected to the carrier protein, and the C-terminus of the polypeptide of interest is connected to the carrier protein t...

Embodiment 1

[0138] Embodiment 1, the construction of expression vector

[0139] The construction of the expression vector was completed by Nanjing GenScript Biotechnology Co., Ltd., including: synthesizing the nucleic acid encoding the amino acid sequence of SEQ ID NO: 12-15, the nucleic acid additionally comprising the nucleotide sequence encoding the signal peptide of SEQ ID NO: 18 ; Construction of the nucleic acid into the mammalian cell expression vector pcDNA3.4 by molecular cloning methods, and amplification and purification of the plasmid, etc.

Embodiment 2

[0140] Embodiment 2, the expression of fusion polypeptide

[0141] In this example, 293-6E cells were transfected with the nucleic acid encoding the fusion polypeptide for eukaryotic expression. The expression and purification of the fusion polypeptide were completed by Nanjing GenScript Biotechnology Co., Ltd. The process is briefly described as follows:

[0142] -293-6E cells in serum-free FreeStyle TM 293Expression medium (Thermo Fisher Scientific, Carlsbad, CA, USA), placed in Erlenmeyer flasks (Corning Inc., Acton, MA), cultured on a shaking incubator (VWR Scientific, Chester, PA), culture conditions at 37°C with 5% CO 2 .

[0143] - The day before transfection, dilute the cells to an appropriate density.

[0144] - On the day of transfection, the plasmid mixture (the mass ratio of the plasmid encoding the first polypeptide and the second polypeptide is 1:1) and the transfection reagent (such as polyetherimide, Polyetherimide, PEI) in an appropriate ratio (such as 1 ...

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Abstract

The present invention relates to a fusion polypeptide comprising a carrier protein and a polypeptide of interest, the carrier protein has a plurality of helical domains linked by a loop, the polypeptide of interest is inserted into the loop of the carrier protein, the carrier protein obscuring a site of interest on the polypeptide of interest, therefore, the accessibility of the site is blocked. The invention also relates to polynucleotides and carriers encoding the fusion polypeptides, as well as host cells comprising the polynucleotides and/or carriers. Further, the invention also relates to application of the fusion polypeptide of the invention in the treatment of cancer and/or activation of immune cells.

Description

technical field [0001] The present invention relates to fusion polypeptides. In particular, the invention relates to modified immunomodulatory molecules and their use in the treatment of cancer. Background technique [0002] Modulation of the immune system through cytokines, such as activation of immune cells, is a strategy for cancer immunotherapy. However, certain sites on cytokines have an impact on their function (eg, immunomodulatory effects). [0003] For example, interleukin-2 (IL-2) can regulate the function of immune cells. After IL-2 binds to the receptors IL2Rα (CD25), IL2Rβ (CD122) and IL2Rγ (CD132), it can activate downstream signaling pathways, including JAK1 and JAK3 kinases and the transcription factor STAT5, to stimulate immune cells (such as T cells and natural killer cells) (NK) cell activation and proliferation. IL-2 can not only conduct signal transduction through CD25 / CD122 / CD132 trimer, but also activate signal pathway through CD122 / CD132 dimer. A...

Claims

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Application Information

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IPC IPC(8): C07K19/00A61K38/20A61K47/64A61P35/00A61P37/04
CPCC07K14/5443C07K14/55A61K47/643A61P35/00A61P37/04C07K2319/31A61K38/00
Inventor 沈健周家宏
Owner NANJING NORMAL UNIVERSITY
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