Antibody-guided design and identification of CD25-binding small antibody mimetics using mammalian cell surface display

AbstractSmall antibody mimetics that contain high-affinity target-binding peptides can be lower cost alternatives to monoclonal antibodies (mAbs). We have recently developed a method to create small antibody mimetics called FLuctuation-regulated Affinity Proteins (FLAPs), which consist of a small protein scaffold with a structurally immobilized target-binding peptide. In this study, to further develop this method, we established a novel screening system for FLAPs called monoclonal antibody-guided peptide identification and engineering (MAGPIE), in which a mAb guides selection in two manners. First, antibody-guided design allows construction of a peptide library that is relatively small in size, but sufficient to identify high-affinity binders in a single selection round. Second, in antibody-guided screening, the fluorescently labeled mAb is used to select mammalian cells that display FLAP candidates with high affinity for the target using fluorescence-activated cell sorting. We demonstrate the reliability and efficacy of MAGPIE using daclizumab, a mAb against human interleukin-2 receptor alpha chain (CD25). Three FLAPs identified by MAGPIE bound CD25 with dissociation constants of approximately 30 nM as measured by biolayer interferometry without undergoing affinity maturation. MAGPIE can be broadly adapted to any mAb to develop small antibody mimetics.

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Surface Display Technology for Biosensor Applications: A Review

Sensors ◽

10.3390/s20102775 ◽

2020 ◽

Vol 20 (10) ◽

pp. 2775 ◽

Cited By ~ 3

Author(s):

Min Park

Keyword(s):

Mammalian Cells ◽

Surface Display ◽

Cell Surface Display ◽

Bacterial Surface ◽

Yeast Cell Surface ◽

Bacterial Surface Display ◽

Display Technology ◽

Almost All ◽

Display Systems ◽

Biosensor Applications

Surface display is a recombinant technology that expresses target proteins on cell membranes and can be applied to almost all types of biological entities from viruses to mammalian cells. This technique has been used for various biotechnical and biomedical applications such as drug screening, biocatalysts, library screening, quantitative assays, and biosensors. In this review, the use of surface display technology in biosensor applications is discussed. In detail, phage display, bacterial surface display of Gram-negative and Gram-positive bacteria, and eukaryotic yeast cell surface display systems are presented. The review describes the advantages of surface display systems for biosensor applications and summarizes the applications of surface displays to biosensors.

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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 ◽

10.1155/2019/6051870 ◽

2019 ◽

Vol 2019 ◽

pp. 1-22 ◽

Cited By ~ 1

Author(s):

Biancamaria Cembrola ◽

Valentino Ruzza ◽

Fulvia Troise ◽

Maria Luisa Esposito ◽

Emanuele Sasso ◽

...

Keyword(s):

High Throughput Sequencing ◽

Surface Display ◽

Affinity Maturation ◽

Yeast Surface Display ◽

Antigen Concentration ◽

Selection Scheme ◽

High Affinity ◽

Novel Approach ◽

Yeast Surface

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.

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A Chimera of Interleukin 2 and a Binding Variant of Aerolysin Is Selectively Toxic to Cells Displaying the Interleukin 2 Receptor

Journal of Biological Chemistry ◽

10.1074/jbc.m706424200 ◽

2007 ◽

Vol 283 (3) ◽

pp. 1572-1579 ◽

Cited By ~ 7

Author(s):

Milan Osusky ◽

Lisa Teschke ◽

Xiaoying Wang ◽

Kevin Wong ◽

J. Thomas Buckley

Keyword(s):

Cell Death ◽

Mammalian Cells ◽

Therapeutic Agent ◽

Interleukin 2 ◽

Bacterial Toxin ◽

Hybrid Protein ◽

High Affinity ◽

Interleukin 2 Receptor ◽

Hybrid Molecules ◽

N Terminus

Aerolysin is a bacterial toxin that binds to glycosylphosphatidylinositol-anchored proteins (GPI-AP) on mammalian cells and oligomerizes, inserting into the target membranes and forming channels that cause cell death. We have made a variant of aerolysin, R336A, that has greatly reduced the ability to bind to GPI-AP, and as a result it is only very weakly active. Fusion of interleukin 2 (IL2) to the N terminus of R336A-aerolysin results in a hybrid that has little or no activity against cells that do not have an IL2 receptor because it cannot bind to the GPI-AP on the cells. Strikingly, the presence of the IL2 moiety allows this hybrid to bind to cells displaying high affinity IL2 receptors. Once bound, the hybrid molecules form insertion-competent oligomers. Cell death occurs at picomolar concentrations of the hybrid, whereas the same cells are insensitive to much higher concentrations of R336A-aerolysin lacking the IL2 domain. The targeted channel-forming hybrid protein may have important advantages as a therapeutic agent.

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Designing high affinity target-binding peptides to HLA-E: a key membrane antigen of multiple myeloma

Aging ◽

10.18632/aging.103858 ◽

2020 ◽

Vol 12 (20) ◽

pp. 20457-20470

Author(s):

Ying Yang ◽

Mingli Sun ◽

Zhaojin Yu ◽

Jinwei Liu ◽

Wei Yan ◽

...

Keyword(s):

Multiple Myeloma ◽

High Affinity ◽

Binding Peptides ◽

Target Binding

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A Highly Efficient Indirect P. pastoris Surface Display Method Based on the CL7/Im7 Ultra-High-Affinity System

Molecules ◽

10.3390/molecules24081483 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1483 ◽

Cited By ~ 2

Author(s):

Li ◽

Qiao ◽

Lin ◽

Liu ◽

Keyword(s):

High Efficiency ◽

Surface Display ◽

Cell Surface Display ◽

Yeast Surface Display ◽

High Affinity ◽

Highly Efficient ◽

Yeast Surface ◽

Affinity Interaction ◽

Display Systems ◽

Arginase I

Cell surface display systems for immobilization of peptides and proteins on the surface of cells have various applications, such as vaccine generation, protein engineering, bio-conversion and bio-adsorption. Though plenty of methods have been established in terms of traditional yeast surface display systems, the development of a universal display method with high efficiency remains a challenge. Here we report an indirect yeast surface display method by anchoring Im7 proteins on the surface of P. pastoris, achieving highly efficient display of target proteins, including fluorescence proteins (sfGFP and mCherry) or enzymes (human Arginase I), with a CL7 fusion tag through the ultra-high-affinity interaction between Im7 and CL7. This indirect P. pastoris surface display approach is highly efficient and provides a robust platform for displaying biomolecules.

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