Phage display technology and its impact in the discovery of novel protein-based drugs

Expert Opinion on Drug Discovery, Journal Year: 2024, Volume and Issue: 19(8), P. 887 - 915

Published: June 18, 2024

Introduction Phage display technology is a well-established versatile in vitro that has been used for over 35 years to identify peptides and antibodies use as reagents therapeutics, well exploring the diversity of alternative scaffolds another option conventional therapeutic antibody discovery. Such successes have responsible spawning range biotechnology companies, many complementary technologies devised expedite drug discovery process resolve bottlenecks workflow.

Language: Английский

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Benchmarking antibody clustering methods using sequence, structural, and machine learning similarity measures for antibody discovery applications

Frontiers in Molecular Biosciences, Journal Year: 2024, Volume and Issue: 11

Published: March 28, 2024

Antibodies are proteins produced by our immune system that have been harnessed as biotherapeutics. The discovery of antibody-based therapeutics relies on analyzing large volumes diverse sequences coming from phage display or animal immunizations. Identification suitable therapeutic candidates is achieved grouping the their similarity and subsequent selection a set antibodies for further tests. Such groupings typically created using sequence-similarity measures alone. Maximizing diversity in selected crucial to reducing number tests molecules with near-identical properties. With advances structural modeling machine learning, can now be grouped across other dimensions, such predicted paratopes three-dimensional structures. Here we benchmarked antibody methods clonotype, sequence, paratope prediction, structure embedding information. results were two tasks: binder detection epitope mapping. We demonstrate no method appears outperform others, while mapping, paratope, clusterings top performers. Most importantly, all propose orthogonal groupings, offering more pools when multiple than any single To facilitate exploring different methods, an online tool-CLAP-available at ( clap.naturalantibody.com ) allows users group, contrast, visualize methods.

Language: Английский

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An approach to produce thousands of single-chain antibody variants on a SPR biosensor chip for measuring target binding kinetics and for deep characterization of antibody paratopes

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 14, 2025

Drug discovery continues to face a staggering 90% failure rate, with many setbacks occurring during late-stage clinical trials. To address this challenge, there is an increasing focus on developing and evaluating new technologies enhance the "design" "test" phases of antibody-based drugs (e.g., monoclonal antibodies, bispecifics, CAR-T therapies, ADCs) biologics early preclinical development, goal identifying lead molecules higher likelihood success. Artificial intelligence (AI) becoming indispensable tool in domain, both for improving identified through traditional approaches de novo design novel therapeutics. However, critical bottlenecks persist "build" AI-designed antibodies protein binders, impeding evaluation. While AI models can rapidly generate thousands millions putative drug designs, technological cost limitations mean that only few dozen candidates are typically produced tested. developers often tradeoff between ultra-high-throughput wet lab methods provide binary yes/no binding data biophysical offer detailed characterization limited number drug-target pairs. these bottlenecks, we previously reported development Sensor-integrated Proteome On Chip (SPOC®) platform, which enables production capture-purification 1,000 - 2,400 folded proteins directly onto surface plasmon resonance (SPR) biosensor chip measuring kinetic rates picomolar affinity resolution. In study, extend SPOC technology expression single-chain (sc-antibodies), specifically scFv VHH constructs. We demonstrate constructs capture-purified at high levels SPR biosensors retain functionality as shown by specificity their respective target antigens, affinities comparable those literature. outputs comprehensive including quantitative (R max ), on-rate ( k off-rate d K D half-life t 1/2 each on-chip sc-antibodies. Additionally, present case study showcasing single amino acid mutational scan complementarity-determining regions (CDRs) HER2 (nanobody) paratope. Using 92 unique mutated variants from four different substitutions, pinpoint residues within paratope could further affinity. This serves demonstration high-throughput approach screening hundreds chain antibody sequences assay, generating resolution support AI-enabled pipelines.

Language: Английский

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Development of FAP-targeted theranostics discovered by next-generation sequencing-augmented mining of a novel immunized VNAR library

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 14, 2025

Cancer-associated fibroblasts (CAFs) in the stroma of solid tumors promote an immunosuppressive tumor microenvironment (TME) that drives resistance to therapies. The expression protease fibroblast activation protein (FAP) on surface CAFs has made FAP a target for development therapies dampen immunosuppression. Relatively few biologics have been developed and none exploit unique engagement properties Variable New Antigen Receptors (VNARs) from shark antibodies. As smallest binding domain nature, VNARs cleverage geometries recognize epitopes conventional antibodies cannot. By directly immunizing nurse with FAP, we created large anti-FAP VNAR phage display library. This library allowed us identify suite through traditional biopanning also by silico approach did not require any prior affinity-based enrichment vitro . We investigated four VNAR-Fc fusion proteins theranostic found all recognized high affinity were rapidly internalized FAP-positive cells. result, constructs effective antibody-drug conjugates able localize xenografts vivo Our findings establish as versatile platform could yield innovative cancer targeting TME.

Language: Английский

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Identification of polyreactive antibodies by high throughput enzyme-linked immunosorbent assay and surface Plasmon resonance

Journal of Immunological Methods, Journal Year: 2025, Volume and Issue: unknown, P. 113855 - 113855

Published: March 1, 2025

Language: Английский

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Can the molecular and transgenic breeding of crops be an alternative and sustainable technology to meet food demand?

Functional & Integrative Genomics, Journal Year: 2025, Volume and Issue: 25(1)

Published: April 9, 2025

Language: Английский

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Deep mining of antibody phage-display selections using Oxford Nanopore Technologies and Dual Unique Molecular Identifiers

New Biotechnology, Journal Year: 2024, Volume and Issue: 80, P. 56 - 68

Published: Feb. 12, 2024

Antibody phage-display technology identifies antibody-antigen interactions through multiple panning rounds, but traditional screening gives no information on enrichment or diversity throughout the process. This results in loss of valuable binders. Next Generation Sequencing can overcome this problem. We introduce a high accuracy long-read sequencing method based recent Oxford Nanopore Technologies (ONT) Q20+ chemistry combination with dual unique molecular identifiers (UMIs) and an optimized bioinformatic analysis pipeline to monitor selections. identified binders from two single-domain antibody libraries selected against model protein. Traditional colony-picking was compared our ONT-UMI method. enabled monitoring before after each selection round. By combining phage selections ONT-UMIs, deep mining output is possible. The approach provides alternative screening, enabling quantification round rare binder recovery, even when dominating >99% abundant. Moreover, it give insights binding motifs for further affinity maturation specificity optimizations. Our demonstrate platform future data guided strategies.

Language: Английский

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Large-scale data mining of four billion human antibody variable regions reveals convergence between therapeutic and natural antibodies that constrains search space for biologics drug discovery

mAbs, Journal Year: 2024, Volume and Issue: 16(1)

Published: June 6, 2024

The naïve human antibody repertoire has theoretical access to an estimated > 10

Language: Английский

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Determining the affinities of high-affinity antibodies using KinExA and surface plasmon resonance

mAbs, Journal Year: 2023, Volume and Issue: 15(1)

Published: Dec. 13, 2023

Accurate and efficient affinity measurement techniques are essential for the biophysical characterization of therapeutic monoclonal antibodies, one fastest growing drug classes. Surface plasmon resonance (SPR) is widely used determining antibody affinity, but does not perform well with extremely high (low picomolar to femtomolar range) molecules. In this study, we compare SPR-based Carterra LSA kinetic exclusion assay (KinExA) measuring affinities 48 antibodies generated against SARS-CoV-2 receptor-binding domain. These data reveal that high-affinity can be straight from selections using high-quality in vitro library platforms 54% correspondence between measured KinExA. Generally, where there was a 2-fold or greater difference KinExA, KinExA reported were tighter. We highlight differences identifying benefits pitfalls each terms dynamic range throughput. Furthermore, demonstrate first time single-point screening significantly improve throughput while maintaining strong correlation full binding curve equilibrium measurements, enabling accurate rank-ordering clones exceptionally tight properties.

Language: Английский

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AIntibody: an experimentally validated in silico antibody discovery design challenge

Nature Biotechnology, Journal Year: 2024, Volume and Issue: 42(11), P. 1637 - 1642

Published: Nov. 1, 2024

Language: Английский

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