Anti-PD-L1 nano antibody and application thereof

A PD-L1 and nanobody technology, applied in the field of biomedicine and molecular biology, can solve the problems of harsh antigen requirements, singleness, long immunization time, etc., and achieve the effect of excellent stability

Active Publication Date: 2020-11-10
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the antibody target specificity of the immune library is high, it has limitations
For example, not only the immunization time is too long, only for a single immune antigen, but also the requirements for the antigen are harsh
The natural library is rich in diversity, but the antibody binding ability of its library is weak

Method used

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  • Anti-PD-L1 nano antibody and application thereof
  • Anti-PD-L1 nano antibody and application thereof
  • Anti-PD-L1 nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: the design of novel V-NAR frame

[0035]Access 2YWZ_A, AAP86762, 4HGK_C, AAN75852, AAM33845, Lep-12E1, ABY64741, Tom70, etc. through PDB (https: / / www.rcsb.org) and NCBI (https: / / www.ncbi.nlm.nih.gov) databases V-NAR Nanobody amino acid sequence, and the V-NAR amino acid sequence was analyzed using clustalw (https: / / www.ebi.ac.uk) and WebLogo (http: / / weblogo.berkeley.edu / logo.cgi). The amino acid sequences of the four FR regions of the V-NAR framework were determined according to the frequency of amino acids at the corresponding positions in the framework region on the sequence alignment results. For the amino acid sequences of the CDR1, HV2 and HV4 regions of the V-NAR framework, not only the sequence comparison results, but also the amino acids at specific sites in the V-NAR that are conducive to antibody stability should be referred to, and finally the CDR1 of the V-NAR framework should be determined. , the amino acid sequences of the HV2 and HV4 region...

Embodiment 2

[0036] Example 2: Construction and evaluation of V-NAR phage library

[0037] The full-length gene fragment of V-NAR was amplified by overlap extension PCR, a total of 3 rounds of PCR. The first round of PCR amplification obtained DNA fragments in the FR1-FR3 region. The DNA fragment of the CDR3-FR4 region was obtained in the second round of PCR amplification, and the complete V-NAR full antibody gene fragment was obtained in the third round of PCR amplification ( figure 2 ). Subsequently, it and the pCANTAB5E phagemid vector were digested with Sfi I and Not I endonucleases, ligated and electrotransformed into Escherichia coli TG1. Then use 2×TY medium to dilute the V-NAR synthetic library according to 10 -1 、10 -2 、10 -3 to 10 -8 (10-fold) gradient dilution, take 100 μL of each concentration of bacterial solution and spread it on a 2×TY plate containing Amp, and detect the storage capacity of V-NAR according to the dilution factor and the number of single colonies on t...

Embodiment 3

[0040] Example 3: Screening and preliminary identification of PD-L1-specific nanobodies

[0041] Dilute PD-L1 protein and BSA with NaHCO 3 The buffer was diluted to a concentration of 100 μg / mL, PD-L1 protein was used as the experimental group, and BSA was used as the control group. Add 150 μL to each well, set up 3 replicate wells, and incubate overnight at 4°C. Then the reaction wells were washed with TBST (0.1%) buffer and blocked with 3% skimmed milk at 4° C. for 2 h. After washing, 100 μL of phage library solution was added and incubated at room temperature for 60 min. Then, after washing 10 times with TBST buffer, 200 μL of eluate was added to the wells, and incubated at room temperature for 10 min. After the incubation, 15 μL of neutralization buffer was added to each well, which was the positive phage obtained in the first round of screening, and the titer was determined. Dilute it with 2×TY medium, infect the logarithmic phase TG1 glycerolbacterium, and incubate f...

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Abstract

The invention provides an anti-PD-L1 nano antibody and application thereof. The sequence of the antibody comprises an FR region, a CDR region and an HV region, wherein the FR region comprises an FR1 region with an amino acid sequence as shown in SEQ ID NO.1, an FR2 region with an amino acid sequence as shown in SEQ ID NO.2, an FR3 region with an amino acid sequence as shown in SEQ ID NO.3 and an FR4 region with an amino acid sequence as shown in SEQ ID NO.4; the CDR region comprises a CDR1 region with an amino acid sequence shown as SEQ ID NO.5 and a CDR3 region with an amino acid sequence shown as any one of SEQ ID NO.8 to SEQ ID NO.12; and the HV region comprises an HV2 region with an amino acid sequence as shown in SEQ ID NO.6 and an HV4 region with an amino acid sequence as shown in SEQ ID NO.7. The anti-PD-L1 nano antibody screened on the basis of the novel shark V-NAR framework sequence has excellent stability and can provide a new variety for research, development and acquisition of novel antitumor drugs.

Description

technical field [0001] The present invention relates to the fields of biomedicine and molecular biology, in particular to the anti-PD-L1 nanobody and its application based on the screening of a constructed shark antibody variable region (V-NAR) phage synthetic peptide library. Background technique [0002] There is a class of heavy chain-only antibody IgNAR in sharks, and its variable region V-NAR is the antibody fragment with the smallest molecular weight known so far, known as nanobodies. V-NAR has the advantages of high affinity, strong stability, good solubility, easy coupling transformation and good tissue penetration ability, so it has broad application prospects in the biomedical industry. [0003] Phage display library is currently a widely used method for library construction. Although the antibody target specificity of the immune library is high, it has limitations. For example, not only the immunization time is too long, only for a single immune antigen, but als...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2827C07K16/005A61P35/00C07K2317/569C07K2317/92C07K2317/76C07K2317/94
Inventor 郑文云刘秋丽马兴元
Owner EAST CHINA UNIV OF SCI & TECH
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