Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries
The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface dis...
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doaj-94bdb67c9cfb471a9f97586c3b7bc0e72020-11-24T21:21:16ZengHindawi LimitedBioMed Research International2314-61332314-61412019-01-01201910.1155/2019/60518706051870Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast LibrariesBiancamaria Cembrola0Valentino Ruzza1Fulvia Troise2Maria Luisa Esposito3Emanuele Sasso4Valeria Cafaro5Margherita Passariello6Feliciano Visconte7Maddalena Raia8Luigi Del Vecchio9Anna Morena D’Alise10Riccardo Cortese11Elisa Scarselli12Nicola Zambrano13Claudia De Lorenzo14Alfredo Nicosia15Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyNouscom s.r.l., Via di Castel Romano 100, 00128 Rome, ItalyCEINGE–Biotecnologie Avanzate s.c.a r.l., Via G. Salvatore 486, 80145 Naples, ItalyReithera s.r.l., Via di Castel Romano 100, 00128 Rome, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyDepartment of Biology, University of Naples Federico II, Cupa Nuova Cintia 21, 80126 Naples, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyCEINGE–Biotecnologie Avanzate s.c.a r.l., Via G. Salvatore 486, 80145 Naples, ItalyCEINGE–Biotecnologie Avanzate s.c.a r.l., Via G. Salvatore 486, 80145 Naples, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyNouscom s.r.l., Via di Castel Romano 100, 00128 Rome, ItalyNouscom s.r.l., Via di Castel Romano 100, 00128 Rome, ItalyNouscom s.r.l., Via di Castel Romano 100, 00128 Rome, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, ItalyThe affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.http://dx.doi.org/10.1155/2019/6051870 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Biancamaria Cembrola Valentino Ruzza Fulvia Troise Maria Luisa Esposito Emanuele Sasso Valeria Cafaro Margherita Passariello Feliciano Visconte Maddalena Raia Luigi Del Vecchio Anna Morena D’Alise Riccardo Cortese Elisa Scarselli Nicola Zambrano Claudia De Lorenzo Alfredo Nicosia |
spellingShingle |
Biancamaria Cembrola Valentino Ruzza Fulvia Troise Maria Luisa Esposito Emanuele Sasso Valeria Cafaro Margherita Passariello Feliciano Visconte Maddalena Raia Luigi Del Vecchio Anna Morena D’Alise Riccardo Cortese Elisa Scarselli Nicola Zambrano Claudia De Lorenzo Alfredo Nicosia Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries BioMed Research International |
author_facet |
Biancamaria Cembrola Valentino Ruzza Fulvia Troise Maria Luisa Esposito Emanuele Sasso Valeria Cafaro Margherita Passariello Feliciano Visconte Maddalena Raia Luigi Del Vecchio Anna Morena D’Alise Riccardo Cortese Elisa Scarselli Nicola Zambrano Claudia De Lorenzo Alfredo Nicosia |
author_sort |
Biancamaria Cembrola |
title |
Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries |
title_short |
Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries |
title_full |
Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries |
title_fullStr |
Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries |
title_full_unstemmed |
Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries |
title_sort |
rapid affinity maturation of novel anti-pd-l1 antibodies by a fast drop of the antigen concentration and facs selection of yeast libraries |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2019-01-01 |
description |
The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders. |
url |
http://dx.doi.org/10.1155/2019/6051870 |
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