CD47 nanobody and application thereof

A single-domain antibody, expression vector technology, applied in the field of biomedicine or biopharmaceuticals, can solve the problems of harsh storage conditions and low stability

Active Publication Date: 2019-08-20
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to their special structural properties, single-domain antibodies have the advantages of both traditional antibodies and small molecule drugs, and almost perfectly overcome the shortcomings of traditional antibodies such as long development cycle, low stability, and harsh storage conditions.

Method used

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  • CD47 nanobody and application thereof
  • CD47 nanobody and application thereof
  • CD47 nanobody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0132] Example 1: Expression and purification of human CD47 protein

[0133] (1) The nucleotide sequence of human CD47 was synthesized on the pCDNA3.1(-) vector, and then its extracellular segment sequence was subcloned into the pFUSE-IgG1 vector; (2) The Omega plasmid extraction kit was used to extract and construct pFUSE-IgG1-hCD47 (ECD) plasmid; (3) Culture HEK293F cells to an OD of 2.0×10 6 cells / mL; (4) Mix the plasmid and transfection reagent PEI 1:3 evenly, let it stand for 20min, and then add it to HEK293F cells at 37°C, 6% CO 2 Cultivate in a shaker incubator for 5-6 days; (5) collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; (6) wash the beads with phosphate buffer solution pH 7.0, and then use 0.1M pH 3.0 Glycine Elution protein; (7) ultrafilter the eluted protein into PBS, take a sample after measuring yield and carry out SDS-PAGE detection (detection result such as figure 1 shown), and the rest of the protein was store...

Embodiment 2

[0134] Example 2: Construction and screening of CD47 single domain antibody library

[0135] Library construction: Briefly, (1) mix 1 mg hCD47(ECD)-Fc antigen with Freund's adjuvant in equal volume, immunize a Xinjiang Bactrian camel once a week, immunize 7 times in total, and stimulate B cells to express antigen-specific (2) After 7 times of immunization, extract 100mL camel peripheral blood lymphocytes and extract total RNA; (3) Synthesize cDNA and amplify VHH by nested PCR; (4) Use restriction endonuclease 20 μg pMECS phage display vector (supplied by Biovector) and 10 μg VHH were digested with Pst I and Not I enzymes, and the two fragments were ligated; (5) The ligated product was transformed into electroporation-competent cells TG1 to construct a CD47 single-domain antibody library and measure the library capacity, Storage capacity is 2.5×10 9 CFU (results such as figure 2 shown). At the same time, 24 clones were randomly selected for colony PCR detection, and the res...

Embodiment 3

[0137] Example 3: Expression and purification of CD47 single domain antibody in eukaryotic cells HEK293 and detection of blocking function of single domain antibody by flow cytometry

[0138]Eukaryotic cell HEK293F expresses CD47Nb-Fc fusion protein: (1) Cloning the CD47Nb sequence with correct sequencing results into pFUSE-IgG4 vector (purchased from Invivogen), and extracting pFUSE-IgG4-Nb plasmid with Omega plasmid extraction kit; (2) ) to culture HEK293F cells to OD of 2.0×10 6 cells / mL; (3) Mix the plasmid and the transfection reagent PEI at a ratio of 1:3, let it stand for 20 minutes, and then add it to HEK293F cells at 37°C, 6% CO 2 Cultivate in a shaker incubator for 5-6 days; (4) collect the cell supernatant and combine with Protein A beads at room temperature for 1 hour; (5) wash the beads with phosphate buffer solution pH 7.0, and then use 0.1M pH 3.0 Glycine Elution protein; (6) the protein ultrafiltration of elution is entered in PBS, after measuring output, take...

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Abstract

The invention relates to the field of biomedicine, discloses a CD47 nanobody and an application thereof, and particularly discloses a blocking type nanobody for integrin-associated protein (CD47) andderived protein thereof. Particularly, the invention discloses an integrin-associated protein (CD47) binding molecule and an application thereof, particularly in treatment and / or prevention or diagnosis of CD47 associated diseases such as tumor. The related CD47 nanobody can effectively block interaction between the CD47 and a ligand SIRPa thereof, and the antibody cannot cause human erythrocyte agglutination.

Description

technical field [0001] The invention belongs to the technical field of biomedicine or biopharmaceuticals, and relates to a blocking single-domain antibody and its derivative protein for the extracellular segment of an integrin-related protein (CD47) molecule. It also discloses its coding sequence, related preparation methods and uses thereof, especially the use in treating and / or preventing or diagnosing CD47-related diseases such as tumors. Background technique [0002] In recent years, antibody drugs have become a research hotspot in the field of global biomedicine in the 21st century due to their unique advantages, and more and more antibody drugs have entered clinical practice. Blocking the interaction between CD47 and SIRPα by using anti-CD47 antibodies has the effect of targeted therapy. The three drugs currently in Phase I are Forty Seven's Hu5F9-G4, Celgene's CC-90002 and Trillium's TTI-621. Trillium's CD47 antibody project is a SIRPa-Fc fusion protein, which has s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2803A61K47/6849C07K2317/565C07K2317/56C07K2317/569A61K2039/505A61P35/00C07K2317/52C07K2317/76C07K2317/24C07K2317/92C07K2317/33C07K2317/73C07K2317/94
Inventor 万亚坤朱敏沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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