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Responsible editor | Enzyme's PD-1/PD-L1 immune checkpoint antagonist antibody has made breakthrough progress in the field of tumor immunotherapy [1].
In recent years, bispecific antibody T Cell Engager and targeted costimulation The receptor's agonistic antibodies ushered in a wave of development [2-4]
.
However, the development of immunostimulatory antibodies poses great challenges, requiring comprehensive considerations such as antibody affinity, epitope, titer, cluster formation, Fcγ receptor interaction, and receptor occupancy [5]
.
The pillar technology of biotechnology phage display technology cleverly realizes the coupling of protein phenotype and genotype, so that hundreds of millions of molecules can be efficiently screened based on affinity [6]
.
The screening of activated antibodies can only rely on the purification, expression and detection of thousands of antibodies one by one.
Therefore, there is an urgent need to develop a high-throughput screening method for activated antibodies in the field of drug research and development
.
Recently, the Zhang Hongkai research group of Nankai University, in cooperation with Gaocheng Biomedical Co.
, Ltd.
, Shanghai University of Science and Technology and Shanghai Jiaotong University, published an article entitled High throughput functional screening for next generation cancer immunotherapy using droplet-based microfluidics in Science Advances
.
The research team developed a high-throughput functional antibody screening technology based on a droplet microfluidic system (Figure 1).
First, a lentiviral antibody library was constructed and the library was used to infect cells.
Each cell was infected by only one virus and each cell secreted An antibody; afterwards, a single antibody-secreting cell and a single reporter cell are co-wrapped in the same microdroplet through a droplet microfluidic system.
Each droplet is equivalent to an independent microreactor and does not affect each other; After culturing in the droplets, the fluorescence activated droplet sorting (FADS) system is used to sort out the droplets containing activated reporter cells.
The droplet microfluidic system can generate and generate millions of droplets within a few hours.
Sorting; subsequently, the antibody-secreting cells are recovered from the droplets, amplified and subjected to the next round of screening; after multiple rounds of screening, antibody genes are extracted from the enriched cells and sequenced to determine the sequence of the active antibody
.
Figure 1 Flow chart of high-throughput functional antibody screening technology based on droplet microfluidic system Researchers use high-throughput functional antibody screening technology based on droplet microfluidic system to screen CD40 receptor agonistic antibodies
.
After two rounds of screening, the researchers performed three-generation sequencing and bioinformatics analysis on the initial library and the antibody library after two rounds of screening, and found that some antibodies were enriched round by round, and the five antibodies with the highest enrichment were expressed.
Purification and verification found that the 5 antibodies are all CD40 strongly agonistic antibodies
.
Researchers used this method to screen out active antibodies with an initial frequency of only 0.
02%, while a high proportion of inactive antibodies in the initial library were eliminated in the screening process
.
It is worth mentioning that the CD40 agonist antibody C04 screened based on this platform can effectively activate the immune system and inhibit tumor growth in mouse models.
The safety and effectiveness of the C04 antibody without any optimization are comparable to those in clinical trials.
The CD40 agonistic antibody CP870893
.
In addition, the researchers used this method to obtain multiple anti-Her2×anti-CD3 bispecific antibody T cell adaptors after one round of screening
.
This paper reports a high-throughput functional antibody screening method based on a droplet microfluidic system, which effectively realizes the coupling of genotype and active phenotype, allowing rapid screening of active antibodies from millions of diverse antibodies It becomes possible and saves manpower and capital investment, which will promote the development of next-generation tumor immunotherapy drugs
.
At the same time, the research paradigm will be able to be applied to researches in many aspects such as polypeptide and cytokine modification, cell-to-cell communication, and TCR and neoantigen pairing
.
Assistant researcher Dr.
Wang Yuan of Nankai University and doctoral student Jin Ruina are the co-first authors of the paper.
Dr.
Shen Bingqing from Gaocheng Biosciences and Dr.
Wei Wang from Shanghai University of Science and Technology made key contributions
.
Original link: https://advances.
sciencemag.
org/lookup/doi/10.
1126/sciadv.
eabe3839 Platemaker: Eleven References [1] Andrews LP, Yano H, Vignali DAA.
Inhibitory receptors and ligands beyond PD-1, PD-L1 and CTLA-4: breakthroughs or backups.
Nat Immunol.
2019 Nov;20(11):1425-1434.
doi: 10.
1038/s41590-019-0512-0.
Epub 2019 Oct 14.
PMID: 31611702.
[2 ] Mayes PA, Hance KW, Hoos A.
The promise and challenges of immune agonist antibody development in cancer.
Nat Rev Drug Discov.
2018 Jul;17(7):509-527.
doi: 10.
1038/nrd.
2018.
75.
Epub 2018 Jun 15.
PMID: 29904196.
[3] Vonderheide RH.
CD40 Agonist Antibodies in Cancer Immunotherapy.
Annu Rev Med.
2020 Jan 27;71:47-58.
doi: 10.
1146/annurev-med-062518-045435.
Epub 2019 Aug 14.
PMID: 31412220.
[4] Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI.
Bispecific antibodies: a mechanistic review of the pipeline.
Nat Rev Drug Discov.
2019 Aug;18(8):585-608.
doi: 10.
1038/s41573-019-0028-1.
PMID: 31175342.
[5] Garber K.
Immune agonist antibodies face critical test.
Nat Rev Drug Discov.
2020 Jan;19(1):3-5.
doi: 10.
1038/d41573-019-00214-5.
PMID: 31907434.
[6] Lerner RA.
Manufacturing immunity to disease in a test tube: the magic bullet realized.
Angew Chem Int Ed Engl.
2006 Dec 11;45(48) :8106-25.
doi: 10.
1002/anie.
200603381.
PMID: 17120282.
Reprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author owns all legal rights.
Violators Must be investigatedReprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights, and offenders must be investigated.
Reprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights, and offenders must be investigated. .
In recent years, bispecific antibody T Cell Engager and targeted costimulation The receptor's agonistic antibodies ushered in a wave of development [2-4]
.
However, the development of immunostimulatory antibodies poses great challenges, requiring comprehensive considerations such as antibody affinity, epitope, titer, cluster formation, Fcγ receptor interaction, and receptor occupancy [5]
.
The pillar technology of biotechnology phage display technology cleverly realizes the coupling of protein phenotype and genotype, so that hundreds of millions of molecules can be efficiently screened based on affinity [6]
.
The screening of activated antibodies can only rely on the purification, expression and detection of thousands of antibodies one by one.
Therefore, there is an urgent need to develop a high-throughput screening method for activated antibodies in the field of drug research and development
.
Recently, the Zhang Hongkai research group of Nankai University, in cooperation with Gaocheng Biomedical Co.
, Ltd.
, Shanghai University of Science and Technology and Shanghai Jiaotong University, published an article entitled High throughput functional screening for next generation cancer immunotherapy using droplet-based microfluidics in Science Advances
.
The research team developed a high-throughput functional antibody screening technology based on a droplet microfluidic system (Figure 1).
First, a lentiviral antibody library was constructed and the library was used to infect cells.
Each cell was infected by only one virus and each cell secreted An antibody; afterwards, a single antibody-secreting cell and a single reporter cell are co-wrapped in the same microdroplet through a droplet microfluidic system.
Each droplet is equivalent to an independent microreactor and does not affect each other; After culturing in the droplets, the fluorescence activated droplet sorting (FADS) system is used to sort out the droplets containing activated reporter cells.
The droplet microfluidic system can generate and generate millions of droplets within a few hours.
Sorting; subsequently, the antibody-secreting cells are recovered from the droplets, amplified and subjected to the next round of screening; after multiple rounds of screening, antibody genes are extracted from the enriched cells and sequenced to determine the sequence of the active antibody
.
Figure 1 Flow chart of high-throughput functional antibody screening technology based on droplet microfluidic system Researchers use high-throughput functional antibody screening technology based on droplet microfluidic system to screen CD40 receptor agonistic antibodies
.
After two rounds of screening, the researchers performed three-generation sequencing and bioinformatics analysis on the initial library and the antibody library after two rounds of screening, and found that some antibodies were enriched round by round, and the five antibodies with the highest enrichment were expressed.
Purification and verification found that the 5 antibodies are all CD40 strongly agonistic antibodies
.
Researchers used this method to screen out active antibodies with an initial frequency of only 0.
02%, while a high proportion of inactive antibodies in the initial library were eliminated in the screening process
.
It is worth mentioning that the CD40 agonist antibody C04 screened based on this platform can effectively activate the immune system and inhibit tumor growth in mouse models.
The safety and effectiveness of the C04 antibody without any optimization are comparable to those in clinical trials.
The CD40 agonistic antibody CP870893
.
In addition, the researchers used this method to obtain multiple anti-Her2×anti-CD3 bispecific antibody T cell adaptors after one round of screening
.
This paper reports a high-throughput functional antibody screening method based on a droplet microfluidic system, which effectively realizes the coupling of genotype and active phenotype, allowing rapid screening of active antibodies from millions of diverse antibodies It becomes possible and saves manpower and capital investment, which will promote the development of next-generation tumor immunotherapy drugs
.
At the same time, the research paradigm will be able to be applied to researches in many aspects such as polypeptide and cytokine modification, cell-to-cell communication, and TCR and neoantigen pairing
.
Assistant researcher Dr.
Wang Yuan of Nankai University and doctoral student Jin Ruina are the co-first authors of the paper.
Dr.
Shen Bingqing from Gaocheng Biosciences and Dr.
Wei Wang from Shanghai University of Science and Technology made key contributions
.
Original link: https://advances.
sciencemag.
org/lookup/doi/10.
1126/sciadv.
eabe3839 Platemaker: Eleven References [1] Andrews LP, Yano H, Vignali DAA.
Inhibitory receptors and ligands beyond PD-1, PD-L1 and CTLA-4: breakthroughs or backups.
Nat Immunol.
2019 Nov;20(11):1425-1434.
doi: 10.
1038/s41590-019-0512-0.
Epub 2019 Oct 14.
PMID: 31611702.
[2 ] Mayes PA, Hance KW, Hoos A.
The promise and challenges of immune agonist antibody development in cancer.
Nat Rev Drug Discov.
2018 Jul;17(7):509-527.
doi: 10.
1038/nrd.
2018.
75.
Epub 2018 Jun 15.
PMID: 29904196.
[3] Vonderheide RH.
CD40 Agonist Antibodies in Cancer Immunotherapy.
Annu Rev Med.
2020 Jan 27;71:47-58.
doi: 10.
1146/annurev-med-062518-045435.
Epub 2019 Aug 14.
PMID: 31412220.
[4] Labrijn AF, Janmaat ML, Reichert JM, Parren PWHI.
Bispecific antibodies: a mechanistic review of the pipeline.
Nat Rev Drug Discov.
2019 Aug;18(8):585-608.
doi: 10.
1038/s41573-019-0028-1.
PMID: 31175342.
[5] Garber K.
Immune agonist antibodies face critical test.
Nat Rev Drug Discov.
2020 Jan;19(1):3-5.
doi: 10.
1038/d41573-019-00214-5.
PMID: 31907434.
[6] Lerner RA.
Manufacturing immunity to disease in a test tube: the magic bullet realized.
Angew Chem Int Ed Engl.
2006 Dec 11;45(48) :8106-25.
doi: 10.
1002/anie.
200603381.
PMID: 17120282.
Reprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author owns all legal rights.
Violators Must be investigatedReprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights, and offenders must be investigated.
Reprinting instructions [Non-original articles] The copyright of this article belongs to the author of the article.
Personal forwarding and sharing are welcome.
Reprinting is prohibited without permission.
The author has all legal rights, and offenders must be investigated. .
