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PD-L1 nanobody as well as preparation method and application thereof

A PD-L1 and nanobody technology, applied in the field of antibodies, can solve the problems of easy pollution, difficulty in large-scale production, and low yield

Active Publication Date: 2020-05-08
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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
[0007] (2) The monoclonal antibody is relatively large in size and cannot enter the tumor tissue well
[0008] (3) The monoclonal antibody development cycle is long, the production cost is high, and the output is low
[0009] (4) Monoclonal antibody drugs are expensive, complex in research and development, complex in humanization, and limited in success rate
[0010] (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
[0011] (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

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  • PD-L1 nanobody as well as preparation method and application thereof
  • PD-L1 nanobody as well as preparation method and application thereof
  • PD-L1 nanobody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0166] Example 1. The carrier construction provided by the embodiment of the present invention is specifically:

[0167] 1. Target fragment amplification is specifically:

[0168] (1) 34 primers were designed and synthesized, and a sufficient amount of the target product was amplified by PCR.

[0169] (2) The PCR reaction uses pfu high-temperature polymerase.

[0170] The amount of each component of PCR: (primer concentration is 1OD dissolved in 400ul ddH2O).

[0171] 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.

[0172] The method amplification specific steps of target fragment PCR are:

[0173] (1) The first round of PCR procedure:

[0174]

[0175] 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.

[0176] (2) The second round of...

Embodiment 2

[0196] The construction of the nanobody library provided by the embodiments of the present invention is specifically:

[0197] 1. Experimental design

[0198] 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

[0220] The identification and expression of proteins provided in the embodiments of the present invention are specifically:

[0221] (1) Construction of mammalian cell expression vector

[0222] 1. Amplify and extract the vector plasmid containing the target gene.

[0223] 2. Subcloning into eukaryotic expression vector pcDNA3.1.

[0224] 3. Sequencing to verify the accuracy of the constructed plasmid.

[0225] 4. Obtain the recombinant plasmid pcDNA3.1 by pumping.

[0226] (2) Mammalian cell culture, protein expression and purification small test

[0227] 1. Cell lines and materials

[0228] Cell line: HEK293 cells.

[0229] Medium: DMEM (10% serum), DMEM (serum-free).

[0230] Petri dish: 10cm dish or 15cm dish

[0231] 2. HEK293 cell transfection (10cm dish)

[0232] (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 ...

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Abstract

The invention belongs to the technical field of antibodies, and discloses a PD-L1 nanobody as well as a preparation method and application thereof. The method comprises the following steps: designingand synthesizing two primers, amplifying a sufficient amount of a target product by a PCR method, performing enzyme digestion, linking a target fragment and a vector, performing transformation, and performing screening and cloning; performing protein identification and expression; and constructing an antibody library: selecting an M13 phage display system to display the VHH antibody library, wherein the system is composed of a pMECS phagemid vector, E.coli TG1 and M13KO7 helper phage. In the phagemid vector pMECS provided by the invention, a sequence before a Pst I enzyme digestion site is a coding sequence of a pelB secretion signal peptide and a part of the amino acids in a first framework region of the antibody, and the pelB signal peptide can guide secretion of subsequent peptides to aperiplasm; and a sequence after a Not I enzyme digestion site is a coding sequence of HA and 6xHis tags, and can be used for purification or detection of fusion protein.

Description

technical field [0001] The invention belongs to the technical field of antibodies, and in particular relates to a PD-L1 nanobody, a preparation method and an application thereof. Background technique [0002] Currently, 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 PD1 and inhibit tumor development, the development of immunotherapy antibody drugs is also very rapid, and well-known drugs such as opdivo, keytruda, and atezolizumab also have significant effects. Therefore, it is very necessary to perform PD...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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
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