Somatic Hypermutation and Framework Mutations of Variable Region Contribute to Anti-Zika Virus-Specific Monoclonal Antibody Binding and Function

Tsuji, Isamu; Vang, Fue; Domínguez, David Ernesto Marón; Karwal, Lovkesh; Sanjali, Ankita; Livengood, Jill A.; Davidson, Edgar; Fouch, Mallorie E.; Doranz, Benjamin J.; Das, Subash C.; Dean, Hansi

doi:10.1128/jvi.00071-22

Cited by 4 publications

(3 citation statements)

References 73 publications

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“…Importantly though, as they do not directly alter the contact surface, mutations in these positions should also be able to alter or even improve TCR specificity. Indeed, naturally selected as well as engineered framework mutations are associated with the generation of more potent and specific antibodies ( 20 – 23 ).…”

Section: Discussionmentioning

confidence: 99%

“…Such mutations may be advantageous in that they avoid directly altering the chemical makeup of the binding site, thus reducing the risk of introducing new reactivities. Indeed, although the targets of antibodies and TCRs are very different, framework sites have long been known to influence antibody loop conformational and dynamic properties ( 17 – 19 ), and mutations in framework regions above CDR loops have been associated with the generation of more potent and specific antibodies ( 20 – 23 ).…”

Section: Introductionmentioning

confidence: 99%

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Enhanced T cell receptor specificity through framework engineering

Rosenberg,

Ayres,

Medina-Cucurella

et al. 2024

Front. Immunol.

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Development of T cell receptors (TCRs) as immunotherapeutics is hindered by inherent TCR cross-reactivity. Engineering more specific TCRs has proven challenging, as unlike antibodies, improving TCR affinity does not usually improve specificity. Although various protein design approaches have been explored to surmount this, mutations in TCR binding interfaces risk broadening specificity or introducing new reactivities. Here we explored if TCR specificity could alternatively be tuned through framework mutations distant from the interface. Studying the 868 TCR specific for the HIV SL9 epitope presented by HLA-A2, we used deep mutational scanning to identify a framework mutation above the mobile CDR3β loop. This glycine to proline mutation had no discernable impact on binding affinity or functional avidity towards the SL9 epitope but weakened recognition of SL9 escape variants and led to fewer responses in a SL9-derived positional scanning library. In contrast, an interfacial mutation near the tip of CDR3α that also did not impact affinity or functional avidity towards SL9 weakened specificity. Simulations indicated that the specificity-enhancing mutation functions by reducing the range of loop motions, limiting the ability of the TCR to adjust to different ligands. Although our results are likely to be TCR dependent, using framework engineering to control TCR loop motions may be a viable strategy for improving the specificity of TCR-based immunotherapies.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhanced T cell receptor specificity through framework engineering

Rosenberg,

Ayres,

Medina-Cucurella

et al. 2024

Front. Immunol.

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“…During antibody affinity maturation, CDRs undergo a high degree of somatic mutation. In mammals, the generation of high-affinity antibodies occurs in activated B cells by the mutagenic diversification of the variable regions of immunoglobulin (Ig) genes, a process called somatic hypermutation (SHM) [ 3 , 4 ]. As a result of an alternating process between the stochastic SHM of Ig genes and the selection and clonal expansion of B cells that contain affinity-enhanced mutations, the affinity of antibodies increases massively during an immune response [ 5 ].…”

Section: Introductionmentioning

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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