Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold

Yimchuen, Wanaporn; Kadonosono, Tetsuya; Ota, Yumi; Sato, Shinichi; Kitazawa, Maika; Shiozawa, Tadashi; Kuchimaru, Takahiro; Taki, Masumi; Ito, Yuji; Nakamura, Hiroyuki; Kizaka‐Kondoh, Shinae

doi:10.1039/d0ra00427h

Cited by 6 publications

(7 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is supported by findings from our recent study involving the grafting of single CDR peptides onto several scaffold proteins, which showed that the FN3 scaffold produced more target‐binding mimetics than other scaffold proteins. [ 15 ] The current study builds on our previous work and improves upon them by demonstrating the following: 1) Computational design and selection of FN3 mutants bearing grafted CDR peptides can generate a target‐binding mimetic on par with that obtained through conventional screening methods, [ 18 ] and 2) the binding affinity of FN3 mutants bearing single CDR‐derived peptides [ 15 ] can be improved using multiple CDR peptides (Figure 4A).…”

Section: Discussionmentioning

confidence: 81%

“…[24,25] Our group has also succeeded in creating HER2-binding small proteins based on the FN3 protein scaffold through conventional phage display library screening and subsequent affinity maturation. [18] In this study, an FN3-based antibody mimetic was developed using an alternative approach that does not involve designing and creating libraries in vitro. The FN3 protein has structural similarity to the mAb immunoglobulin domain, making it an attractive candidate scaffold for mAb-derived CDR grafting because CDR peptides grafted onto the FN3 scaffold would be more likely to mimic their original conformation on the parental mAb.…”

Section: Discussionmentioning

confidence: 99%

“…Recently, we reported a computational design strategy to develop target‐binding small proteins called fluctuation‐regulated affinity proteins (FLAPs). These FLAPs have a structurally immobilized single CDR‐derived peptide [ 15 ] or target‐binding peptide [ 18 ] to reduce entropic energy loss upon target binding. Based on the same concept, in this study, we describe the computational design of FLAPs bearing dual CDR‐derived peptides (D‐FLAPs).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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

See

Kadonosono

Ota

et al. 2020

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

See

Kadonosono

Ota

et al. 2020

Biotechnology Journal

Self Cite

View full text Add to dashboard Cite

show abstract

“…The FN3 domain has a stable structure and has been used as a protein scaffold for monobodies [ 25 , 75 ]. Yimchuen W. et.al demonstrated that monobodies grafted with HER2-targeting peptide sequences showed improved resistance to proteolysis and specific binding to HER2 [ 76 ]. In this study, we successfully developed CRT-binding monobodies by grafting CRT-binding peptide sequences (Int-α and Hep-I) onto the FN3 domain.…”

Section: Discussionmentioning

confidence: 99%

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Zhang

Thangam

You

et al. 2021

Cancers

View full text Add to dashboard Cite

Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

show abstract

“…The MRI contrast agent is based on the amphiphilic Gd(III)-complex Gd-DOTAMA(C18)2, which is inserted into the phospholipid membrane. By this strategy, a single nanosystem can bring multiple copies of the contrast agents on site, thus circumventing the inherent low sensitivity of MRI in molecular imaging applications. − To achieve active targeting for HER2 positive tumors, the external surface of the liposomes could be decorated with the A9 targeting sequence. The A9 targeting sequence will be linked to the liposome through a polyethylene glycol spacer to avoid sterical hindrance that could decrease binding affinity.…”

Section: Perspectivesmentioning

confidence: 99%

Peptide Ligands Specifically Targeting HER2 Receptor and the Role Played by a Synthetic Model System of the Receptor Extracellular Domain: Hypothesized Future Perspectives

Luca

Verdoliva

2020

J. Med. Chem.

View full text Add to dashboard Cite

A short (Fab)trastuzumab-derived peptide specific for HER2 receptor was identified. Its affinity for the model system HER2-DIVMP was found in a nanomolar range. The structural determinants responsible for the interaction between this ligand (A9) and HER2-DIVMP were investigated by both computational and NMR analysis. Next, the possibility of using A9 as HER2- specific probe for the nuclear medicine imaging was evaluated by conjugating A9 with the DTPA chelator and radiolabeling it with 111In. The developed probe retained a nanomolar affinity to HER2-overexpressing cancer cells, however, some unspecific binding also occurred. The peptide internalization into cells by receptor-mediated endocytosis was also studied. Future perspectives are aimed at using A9 as a probe for molecular imaging diagnostics as well as active targeting of anticancer drugs. Lead structure optimization is needed to minimize the percentage of A9 unspecific binding and to increase the binding affinity to the receptor.

show abstract

Strategic design to create HER2-targeting proteins with target-binding peptides immobilized on a fibronectin type III domain scaffold

Abstract: A HER2-binding protein, HBP-FLAP, developed by peptide immobilization specifically binds to HER2 and has improved resistance to proteases.

Cited by 6 publications

References 27 publications

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

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

Engineering Calreticulin-Targeting Monobodies to Detect Immunogenic Cell Death in Cancer Chemotherapy

Peptide Ligands Specifically Targeting HER2 Receptor and the Role Played by a Synthetic Model System of the Receptor Extracellular Domain: Hypothesized Future Perspectives

Contact Info

Product

Resources

About