Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors

Andrey Krokhotin, Hongwei Du, Koichi Hirabayashi, Konstantin Popov, Tomohiro Kurokawa, Xinhui Wan, Soldano Ferrone, Gianpietro Dotti, Nikolay V. Dokholyan

Research output: Contribution to journalArticle

Abstract

Chimeric antigen receptor (CAR)-T cell-based immunotherapy of malignant disease relies on the specificity and association constant of single-chain variable fragments (scFvs). The latter are synthesized from parent antibodies by fusing their light (VL) and heavy (VH)-chain variable domains into a single chain using a flexible linker peptide. The fusion of VL and VH domains can distort their relative orientation, thereby compromising specificity and association constant of scFv, and reducing the lytic efficacy of CAR-T cells. Here, we circumvent the complications of domains’ fusion by designing scFv mutants that stabilize interaction between scFv and its target, thereby rescuing scFv efficacy. We employ an iterative approach, based on structural modeling and mutagenesis driven by computational protein design. To demonstrate the power of this approach, we use the scFv derived from an antibody specific to a human leukocyte antigen A2 (HLA-A2)-HER2-derived peptide complex. Whereas the parental antibody is highly specific to its target, the scFv showed reduced specificity. Using our approach, we design mutations into scFvs that restore specificity of the original antibody.

Original languageEnglish (US)
Pages (from-to)30-37
Number of pages8
JournalMolecular Therapy - Oncolytics
Volume15
DOIs
StatePublished - Dec 20 2019

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Single-Chain Antibodies
Antigen Receptors
T-Cell Antigen Receptor
Peptides
Antibody Specificity
Antibodies
HLA Antigens
varespladib methyl
Mutagenesis
Immunotherapy
Light
Mutation
Proteins

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Oncology
  • Cancer Research
  • Pharmacology (medical)

Cite this

Krokhotin, Andrey ; Du, Hongwei ; Hirabayashi, Koichi ; Popov, Konstantin ; Kurokawa, Tomohiro ; Wan, Xinhui ; Ferrone, Soldano ; Dotti, Gianpietro ; Dokholyan, Nikolay V. / Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors. In: Molecular Therapy - Oncolytics. 2019 ; Vol. 15. pp. 30-37.
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Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors. / Krokhotin, Andrey; Du, Hongwei; Hirabayashi, Koichi; Popov, Konstantin; Kurokawa, Tomohiro; Wan, Xinhui; Ferrone, Soldano; Dotti, Gianpietro; Dokholyan, Nikolay V.

In: Molecular Therapy - Oncolytics, Vol. 15, 20.12.2019, p. 30-37.

Research output: Contribution to journalArticle

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T1 - Computationally Guided Design of Single-Chain Variable Fragment Improves Specificity of Chimeric Antigen Receptors

AU - Krokhotin, Andrey

AU - Du, Hongwei

AU - Hirabayashi, Koichi

AU - Popov, Konstantin

AU - Kurokawa, Tomohiro

AU - Wan, Xinhui

AU - Ferrone, Soldano

AU - Dotti, Gianpietro

AU - Dokholyan, Nikolay V.

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