Affinity maturation: highlights in the application of <i>in vitro</i> strategies for the directed evolution of antibodies

Chan, Denice T. Y.; Groves, Maria

doi:10.1042/etls20200331

Cited by 8 publications

(5 citation statements)

References 61 publications

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“…The resulting antibodies can be highly mutated compared to their germline-encoded counterparts, with the affinity for antigens being significantly increased compared to the corresponding naïve B cell receptors [ 55 , 56 ]. This makes affinity maturation a key technique in protein engineering to improve affinity and binding interactions in vitro in order to optimize the therapeutic potential of antibodies [ 57 ]. Adler et al observed that in vivo affinity-matured antibodies predominantly accumulate mutations within the CDR3 regions, with a total of 6–7 amino acid changes in the heavy and light chain [ 58 ].…”

Section: Discussionmentioning

confidence: 99%

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1

Harwardt,

Geyer,

Schoenfeld

et al. 2024

Antibodies

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The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens.

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Section: Discussionmentioning

confidence: 99%

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1

Harwardt,

Geyer,

Schoenfeld

et al. 2024

Antibodies

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show abstract

“…Antibodies undergo affinity optimization before they can be considered potential therapeutic drugs [ 8 ]. Techniques such as site-directed mutagenesis, chain shuffling, and error-prone PCR are commonly utilized for antibody affinity maturation [ 9 , 10 ]. However, this process is often time-consuming, spanning several months.…”

Section: Introductionmentioning

confidence: 99%

A dual computational and experimental strategy to enhance TSLP antibody affinity for improved asthma treatment

Lv,

Gong,

Liu

et al. 2024

PLoS Comput Biol

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Thymic stromal lymphopoietin is a key cytokine involved in the pathogenesis of asthma and other allergic diseases. Targeting TSLP and its signaling pathways is increasingly recognized as an effective strategy for asthma treatment. This study focused on enhancing the affinity of the T6 antibody, which specifically targets TSLP, by integrating computational and experimental methods. The initial affinity of the T6 antibody for TSLP was lower than the benchmark antibody AMG157. To improve this, we utilized alanine scanning, molecular docking, and computational tools including mCSM-PPI2 and GEO-PPI to identify critical amino acid residues for site-directed mutagenesis. Subsequent mutations and experimental validations resulted in an antibody with significantly enhanced blocking capacity against TSLP. Our findings demonstrate the potential of computer-assisted techniques in expediting antibody affinity maturation, thereby reducing both the time and cost of experiments. The integration of computational methods with experimental approaches holds great promise for the development of targeted therapeutic antibodies for TSLP-related diseases.

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“…However, this daunting task of enhancing the affinity using in vitro methods is rather complicated, expensive, and time-consuming. Structure-based computational engineering offers an attractive alternative strategy for the development of biotherapeutic antibodies [10][11][12] .…”

Section: Introductionmentioning

confidence: 99%

Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs

Singh,

Bhutkar,

Choudhary

et al. 2024

Preprint

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The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for development of the next generation therapeutics. This study delves into the refinement and engineering of CDRs through in silico affinity maturation followed by binding validation using ITC and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of RBD of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Subsequent biophysical studies using ITC provided insights into binding affinity and key thermodynamic parameters. Consistent with in silico findings, seven single mutations resulted in enhanced affinity. The mutants were further tested for neutralization activity against SARS-CoV-2 pseudovirus. L106T, L106Q, S107R, and S107Q generated mutants were more effective in virus-neutralizing with IC50 values of ~0.03 uM , ~0.13 uM , ~0.14 uM , and ~0.14 uM , respectively as compared to the native nanobody (IC50 ~0.77 uM ). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

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Affinity maturation: highlights in the application of in vitro strategies for the directed evolution of antibodies

Cited by 8 publications

References 61 publications

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1

A dual computational and experimental strategy to enhance TSLP antibody affinity for improved asthma treatment

Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs

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