CDR-H3 Diversity Is Not Required for Antigen Recognition by Synthetic Antibodies

Persson, Helena; Ye, Wei; Wernimont, A.K.; Adams, Jarrett; Koide, Akiko; Koide, Shohei; Lam, Robert; Sidhu, Sachdev S.

doi:10.1016/j.jmb.2012.11.037

Cited by 147 publications

(221 citation statements)

References 40 publications

(43 reference statements)

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“…20 The Fv region of NB0268 was modeled and its van der Waals surface was analyzed for hydrophobicity and electrostatics (Figure 1). The log P hydrophobicity surface revealed hydrophobic regions on the complementarity-determining regions (CDRs) and polar regions on the framework surface.…”

Section: Resultsmentioning

confidence: 99%

“…Fab-phage from Library F 20 were cycled through four rounds of binding selection using a parental cancer associated fibroblast (CAF) cell line as the background depleting step and an overexpressing CAF cell line as the target selection step. 10x10^6 cells were used for selections and were incubated for 2 hrs at 4C in 1 ml growth culture medium with a library of 3 × 1013 Fab-phage.…”

Section: Methodsmentioning

confidence: 99%

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Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Jetha

Thorsteinson²,

Jmeian

et al. 2018

mAbs

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Monoclonal antibody (mAb) candidates from high-throughput screening or binding affinity optimization often contain mutations leading to liabilities for further development of the antibody, such as aggregation-prone regions and lack of solubility. In this work, we optimized a candidate integrin α11-binding mAb for developability using molecular modeling, rational design, and hydrophobic interaction chromatography (HIC). A homology model of the parental mAb Fv region was built, and this revealed hydrophobic patches on the surface of the complementarity-determining region loops. A series of 97 variants of the residues primarily responsible for the hydrophobic patches were expressed and their HIC retention times (RT) were measured. As intended, many of the computationally designed variants reduced the HIC RT compared to the parental mAb, and mutating residues that contributed most to hydrophobic patches had the greatest effect on HIC RT. A retrospective analysis was then performed where 3-dimentional protein property descriptors were evaluated for their ability to predict HIC RT using the current series of mAbs. The same descriptors were used to train a simple multi-parameter protein quantitative structure-property relationship model on this data, producing an improved correlation. We also extended this analysis to recently published HIC data for 137 clinical mAb candidates as well as 31 adnectin variants, and found that the surface area of hydrophobic patches averaged over a molecular dynamics sample consistently correlated to the experimental data across a diverse set of biotherapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Jetha

Thorsteinson²,

Jmeian

et al. 2018

mAbs

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“…1A) determined structurally to be critical for KIT activation (7) were used as an antigen to isolate binding Fabs from a naive phage-displayed library (library F; Fig. S1) containing more than 10 10 unique clones (18,19). The binding properties of the phage-derived Fabs were compared with the binding properties of a murine monoclonal anti-KIT antibody designated KTN37 that was obtained by immunization of mice with the same antigen.…”

Section: Resultsmentioning

confidence: 99%

“…Indeed, it was shown previously (25) that monoclonal antibodies against D4 domain of KIT can inhibit receptor activation. Furthermore, the advent of fully human synthetic antibody libraries allows rapid selection of binders specific to their target, coupled with an ability to affinity-mature initially isolated variants for desired attributes such as increased affinity and specificity (18). Here we describe the isolation and maturation of an antibody directed against KIT D4 that strongly impairs receptor activation and cell proliferation to a level that could be potentially used in cancer therapy.…”

Section: Discussionmentioning

confidence: 99%

“…Phage pools consisting of a phage-displayed synthetic antibody library (library F) were cycled through five rounds of selections by using KIT D4-5 immobilized on 96-well MaxiSorp immunoplates (Thermo Scientific) as antigen, as described previously (18). Culture supernatants of 96 clones from each of rounds four and five grown in 96-well format were used directly in phage ELISAs to identify clones binding KIT specifically (using BSA as a negative control).…”

Section: Methodsmentioning

confidence: 99%

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Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region

Reshetnyak

Nelson

Shi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression of disease eventually occurs in virtually all patients. In this report, we describe inhibitory KIT antibodies that bind to the membrane-proximal Ig-like D4 of KIT with significant overlap with an epitope in D4 that mediates homotypic interactions essential for KIT activation. Crystal structures of the anti-KIT antibody in complex with KIT D4 and D5 allowed design of affinity-matured libraries that were used to isolate variants with increased affinity and efficacy. Isolated antibodies showed KIT inhibition together with suppression of cell proliferation driven by ligand-stimulated WT or constitutively activated oncogenic KIT mutant. These antibodies represent a unique therapeutic approach and a step toward the development of "naked" or toxin-conjugated KIT antibodies for the treatment of KIT-driven cancers.phosphorylation | therapeutic antibodies | cancer therapy | cell signaling | protein kinase

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Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

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Kadonosono

Ota

et al. 2020

Biotechnology Journal

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Target-binding small proteins are promising alternatives to conventional monoclonal antibodies (mAbs), offering advantages in terms of tissue penetration and manufacturing costs. Recently, a design strategy to create small proteins called fluctuation-regulated affinity proteins (FLAPs) consisting of a structurally immobilized peptide from the complementarity-determining region (CDR) loops of mAbs (CDR-derived peptide) and a protein scaffold was developed. Because mAb paratopes are usually composed of multiple CDRs, FLAPs with multiple binding peptides may have an enhanced target-binding capability. Here, a strategy to create FLAPs bearing dual CDR-derived peptides (D-FLAPs) using the anti-human epithelial growth factor receptor type 2 (HER2) mAb trastuzumab as a basis is developed. Computationally selected CDR-derived peptides are first grafted onto two adjacent loops of the fibronectin type III domain (FN3) scaffold, yielding 80 D-FLAP candidates. After computational screening based on their similarity to the parental mAb with regard to the conformation of paratope residues, two candidates are selected. After further evaluation with ELISA, one D-FLAP with HYTTPP and GDGFYA peptides from CDR-L3 and CDR-H3 of the parental mAb, respectively, is found to bind HER2 with a dissociation constant of 58 nm. This method applies to various mAb drugs and allows the rational design of small protein alternatives.

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CDR-H3 Diversity Is Not Required for Antigen Recognition by Synthetic Antibodies

Cited by 147 publications

References 40 publications

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Homology modeling and structure-based design improve hydrophobic interaction chromatography behavior of integrin binding antibodies

Structural basis for KIT receptor tyrosine kinase inhibition by antibodies targeting the D4 membrane-proximal region

Reconstitution of an Anti‐HER2 Antibody Paratope by Grafting Dual CDR‐Derived Peptides onto a Small Protein Scaffold

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