Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen

A PD-L1 and nanobody technology, applied in the field of antibodies, can solve the problems of high storage cost, instability, and non-repeatable application

Inactive Publication Date: 2019-09-20
SHIHEZI UNIVERSITY
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the monoclonal antibody is used for mouse monoclonal antibody, the application to the human body will produce anti-mouse monoclonal antibody, which cannot be used repeatedly, which will affect its curative effect
[0008] (2) The monoclonal antibody is relatively large in size and cannot enter the tumor tissue well
[0009] (3) The monoclonal antibody development cycle is long, the production cost is high, and the output is low
[0010] (4) Monoclonal antibody drugs are expensive, complex in research and development, complex in humanization, and limited in success rate
[0011] (5) It is difficult to produce on a large scale, and monoclonal antibody drugs consume a huge amount of money in the process of building factories and producing them
[0012] (6) Unstable, easy to degrade, high storage cost; easy to pollute, high maintenance cost
It has the same structure as the VH of human antibodies, and sequencing shows that it has a high homology with VH3, but the CDR1 (Complementarity-determining region-1) and CDR3 (Complementarity-determining region-3) of Nanobodies are relatively long

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen
  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen
  • Method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with PD-L1 antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0158] Example 1. The carrier construction provided by the embodiment of the present invention is specifically:

[0159] 1. Target fragment amplification is specifically:

[0160] (1) 34 primers were designed and synthesized, and a sufficient amount of the target product was amplified by PCR.

[0161] (2) The PCR reaction uses pfu high-temperature polymerase.

[0162] The amount of each component of PCR: (primer concentration is 1OD dissolved in 400ul ddH2O).

[0163] Reaction system: 50ul. Primer mix, (1 / 34) 0.4ul x 13.6 total 8ul. 10X pfu Buffer, 5ul. Each upstream and downstream primers, each 2ul. Pfu, 0.4ul (5u / ul). ddH2O, add water to 50ul respectively.

[0164] The method amplification specific steps of target fragment PCR are:

[0165] (1) The first round of PCR procedure:

[0166]

[0167] The above is the first-round PCR reaction system, and the second-round PCR is performed with the first-round PCR product as a template.

[0168] (2) The second round of...

Embodiment 2

[0187] The construction of the nanobody library provided by the embodiments of the present invention is specifically:

[0188] 1. Experimental design

[0189] The M13 phage display system was selected to display the VHH antibody library, which consists of pMECS phagemid vector, E.coliTG1 and M13KO7 helper phage. In the phagemid vector pMECS, the sequence before the Pst I restriction site is the coding sequence of the pelB secretion signal peptide and some amino acids in the first framework region of the antibody. The pelB signal peptide can guide the secretion of subsequent polypeptides into the periplasmic cavity. The Not I restriction site is followed by the coding sequence of HA and 6×His tag, which can be used for purification or detection of fusion protein. The sequence immediately following it encodes the phage PIII capsid protein ( Figure 4 shown). There is an amber stop codon between the 6×His tag and the gene III sequence, and 10% to 20% of the amber stop codon ca...

Embodiment 3

[0211] The protein identification and expression provided by the embodiments of the present invention are specifically:

[0212] (1) Construction of mammalian cell expression vector

[0213] 1. Amplify and extract the vector plasmid containing the target gene.

[0214] 2. Subcloning into eukaryotic expression vector pcDNA3.1.

[0215] 3. Sequencing to verify the accuracy of the constructed plasmid.

[0216] 4. Obtain the recombinant plasmid pcDNA3.1 by pumping.

[0217] (2) Mammalian cell culture, protein expression and purification experiments

[0218] 1. Cell lines and materials

[0219] Cell line: HEK293 cells.

[0220] Medium: DMEM (10% serum), DMEM (serum-free).

[0221] Petri dish: 10cm dish or 15cm dish

[0222] 2. HEK293 cell transfection (10cm dish)

[0223] (1) 24 hours before transfection, the total amount of cells in a 4-5X106 / 10cm culture dish is plated, and the transfection can be carried out when the cells are growing well and the density of adherent cells...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
Molecular weightaaaaaaaaaa
Molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention belongs to the technical field of antibodies and discloses a method for preparing PD-L1 (programmed death-ligand 1) nanoantibody by immunizing alpacas with a PD-L1 antigen. The method comprises: designing two synthetic primers, amplifying by PCR (polymerase chain reaction) to obtain sufficient target products for enzyme digestion, linking target fragments to a vector, converting, and carrying out screening and cloning; carrying out protein verifying and expressing; constructing an antibody library; displaying a VHH antibody library through an M13 bacteriophage display system which is composed of a pMECS phagemid vector, E. coli TG1 and an M13KO7 helper phage. In the phasmid vector pMECS, a sequence before a Pst I enzyme digestion site is a coding sequence for part of amino acids in first frame regions of pelB secretory signal peptides and antibodies; the pelB signal peptides can guide subsequent polypeptides secreted to periplasmic cavities; a sequence after a Not I enzyme digestion site is a coding sequence for HA and 6*His tags; the method is suitable for purification or detection of fusion proteins.

Description

technical field [0001] The invention belongs to the technical field of antibodies, and in particular relates to a method for preparing PD-L1 nanobody by immunizing alpaca with PD-L1 antigen. Specifically, it relates to a method for preparing PD-L1 nanobody by immunizing alpaca with mammalian cells expressing PD-L1 antigen. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] At present, some anti-PD-L1 monoclonal antibodies have been approved for the treatment of cancer. For example, the PD-L1 inhibitor Tecentriq (atezolizumab or MDL3280a), the approved indication is bladder cancer (urothelial carcinoma). The expression of PD-L1 is increased in most tumor tissues, such as breast cancer, non-small cell lung cancer, and melanoma. The expression of PD-L1 in tumor cells is also considered to be a prognostic factor for many types of malignant tumors. In order to block the combination of PD-L1 and receptor PD...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/28C12N15/13C12N15/85A61K47/68
CPCC07K16/2827C12N15/85A61K47/6849C07K2317/22C07K2317/569C12N2800/107C12N15/1037C07K2317/70C12Q1/686
Inventor 陈创夫吴鹏
Owner SHIHEZI UNIVERSITY
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com