Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C. Ronald; Sidhu, Sachdev S.

doi:10.1021/acschembio.6b00990

Cited by 17 publications

(15 citation statements)

References 49 publications

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“…Anti-EGFR Fabs, 8708 and 8709, were previously developed and characterized 16. We cloned the (scFv) 2 , scFv-Fc, and IgG probes based on CDRs of 8708 and 8709 Fabs (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…CDRs from 8708 and 8709 Fabs 16 were subcloned as (scFv) 2 , scFv-Fc, and IgG as described previously 17.…”

Section: Methodsmentioning

confidence: 99%

“…8 x 10 5 HEK293T cells were plated 24 hours before transfection in complete media. Plasmids (2 µg) expressing either wild-type EGFR-GFP (Addgene, Cambridge MA) or mutant EGFR vIII -GFP 16 were mixed with 100 µL of Opti-Eagles minimum essential media (Opti-MEM) (Thermo Fisher Scientific, Hampton, NH) to a final concentration of 30 µg/mL. Polyethylenimine (PEI) (Sigma, Aldrich, St. Louis, MI) was added to 100 µL of Opti-MEM to a final concentration of 120 µg/mL.…”

Section: Methodsmentioning

confidence: 99%

“…Here, we evaluated imaging properties of antibody fragments that recognize domains I/II of EGFR. We previously isolated two anti-EGFR Fabs, 8708 and 8709, which bind domains I/II of EGFR 16. We used the complementarity determining regions (CDRs) of these Fabs to construct IRDye800CW-labeled (scFv) 2 , scFv-Fc, and IgG imaging probes.…”

Section: Introductionmentioning

confidence: 99%

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Near infrared imaging of epidermal growth factor receptor positive xenografts in mice with domain I/II specific antibody fragments

Bernhard¹,

El-Sayed²,

Barreto³

et al. 2019

Theranostics

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Epidermal growth factor receptor (EGFR) is a transmembrane cell surface receptor that is frequently overexpressed and/or mutated in many cancers. Therapies targeting EGFR have poor outcomes due to the lack of reliable diagnostic tests to monitor EGFR. Current in vitro EGFR diagnostic methods are invasive, requiring biopsies, which limits tumor sampling and availability. EGFR molecular imaging provides non-invasive whole-body images capable of detecting primary tumors and metastases, which can be used to diagnose and monitor response to therapy. Methods : We evaluated properties of two anti-EGFR fragments, 8708 and 8709, as molecular-targeted imaging probes. 8708 and 8709 are anti-EGFR antigen binding fragments (Fabs) that recognize domain I/II of EGFR, which is distinct from epitopes recognized by current anti-EGFR therapeutic antibodies. We used complementarity determining region sequences from 8708 and 8709 Fabs to generate an anti-EGFR IgG and (scFv) 2 and scFv-Fc antibody fragments. We expressed, purified, and labeled the IgG and fragments with IRDye800CW and used them to image EGFR-positive and -negative xenografts in CD-1 nude mice. 8709 scFv-Fc was also tested for competitive binding with the therapeutic anti-EGFR antibody nimotuzumab and for quantifying ratios of EGFR and EGFR vIII deletion mutant. Results : IRDye800CW-labeled 8708 (scFv) 2 and 8709 scFv-Fc imaging probes showed high levels of accumulation and good retention in EGFR-positive xenografts, with peak accumulation occurring at 24 and 48 hours post injection, respectively. IRDye680RD-labeled 8709 scFv-Fc did not compete with IRDye800CW-labeled nimotuzumab for EGFR binding as assayed by flow cytometry using an EGFR-positive cell line. IRDye680RD-labeled 8709 scFv-Fc and IRDye800CW-labeled nimotuzumab used in combination were able to determine the ratio of cells expressing EGFR and a deletion mutant EGFR vIII . Conclusion : IRDye800CW-labeled 8708 (scFv) 2 and 8709 scFv-Fc had desirable binding affinities, clearance times, and tumor accumulation to be used for imaging in combination with current EGFR targeted therapies. This study highlights the potential for using 8708 (scFv) 2 and 8709 scFv-Fc as EGFR diagnostic and therapy monitoring tools.

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“…Anti-EGFR Fabs, 8708 and 8709, were previously developed and characterized 16. We cloned the (scFv) 2 , scFv-Fc, and IgG probes based on CDRs of 8708 and 8709 Fabs (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…CDRs from 8708 and 8709 Fabs 16 were subcloned as (scFv) 2 , scFv-Fc, and IgG as described previously 17.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Near infrared imaging of epidermal growth factor receptor positive xenografts in mice with domain I/II specific antibody fragments

Bernhard¹,

El-Sayed²,

Barreto³

et al. 2019

Theranostics

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“…Phage display is a popular tool for the development of therapeutic antibodies in the pharmaceutical industry. In most cases, therapeutic antibodies have biological functions; for example, antibodies bind to target proteins and act as antagonists, allosteric regulators, or agonists (10,11). The main challenge with the use of phage panning for drug discovery is that the panning is based purely on the affinity between the antibody and the antigen (SI Appendix, Fig.…”

Section: Discussionmentioning

confidence: 99%

Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems

Zheng

Xie

Yang

et al. 2018

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

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We describe a method for the rapid selection of functional antibodies. The method depends on the cocultivation of that produce phage with target eukaryotic cells in very small volumes. The antibodies on phage induce selectable phenotypes in the target cells, and the nature of the antibody is determined by gene sequencing of the phage genome. To select functional antibodies from the diverse antibody repertoire, we devised a selection platform that contains millions of picoliter-sized droplet ecosystems. In each miniecosystem, the bacteria produce phage displaying unique members of the antibody repertoire. These phage interact only with eukaryotic cells in the same miniecosystem, making phage available directly for activity-based antibody selection in biological systems.

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Epidermal growth factor receptor (EGFR) involvement in epithelial‐derived cancers and its current antibody‐based immunotherapies

London

Gallo

2020

Cell Biology International

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The epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein that is part of the family of tyrosine kinase receptors. The binding of EGFR to its cognate ligands leads to its autophosphorylation and subsequent activation of the signal transduction pathways involved in regulating cellular proliferation, differentiation, and survival. Accordingly, this receptor carries out both redundant and restricted functions in the germline development of mammals and in the maintenance of various adult tissues. Correspondingly, the loss of EGFR regulation results in many human diseases, with the most notable cancer. This receptor is overexpressed and/or mutated in multiple epithelial-derived tumors, and associated with poor prognosis and survival in cancer patients. Here, we discuss in detail the role of EGFR in specific epithelial-derived cancer pathologies; these include lung cancer, colorectal cancer, and squamous cell carcinomas. The development of multiple anticancer agents against EGFR diminished the progression and metastasis of tumors. Some of the most versatile therapeutic anti-EGFR agents include the monoclonal antibodies (mAbs), demonstrating success in clinical settings when used in combination with cytotoxic treatments, such as chemotherapy and/or radiation. We thus discuss the development and application of two of the most notable therapeutic mAbs, cetuximab, and panitumumab, currently utilized in various EGFR-related epithelial cancers.

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Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists

Cited by 17 publications

References 49 publications

Near infrared imaging of epidermal growth factor receptor positive xenografts in mice with domain I/II specific antibody fragments

Near infrared imaging of epidermal growth factor receptor positive xenografts in mice with domain I/II specific antibody fragments

Antibody selection using clonal cocultivation of Escherichia coli and eukaryotic cells in miniecosystems

Epidermal growth factor receptor (EGFR) involvement in epithelial‐derived cancers and its current antibody‐based immunotherapies

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