Combination of Phage Display and Molecular Grafting Generates Highly Specific Tumor‐Targeting Miniproteins

Zoller, Frederic; Markert, Annette; Barthe, Philippe; Zhao, Wenye; Weichert, Wilko; Askoxylakis, Vasileios; Altmann, Annette; Mier, Walter; Haberkorn, Uwe

doi:10.1002/ange.201203857

Cited by 21 publications

(19 citation statements)

References 21 publications

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“…As expected from stability analysis of the natural occurring SFTI-I (10) and the SFTI-I derivate-targeting DLL4 (26,33) SFITGv6 demonstrated an outstanding stability in human serum over a period of 24 hours and high affinity (K D ¼14.8nmol/L) for ITGa v b 6 . In correlation to the ITGa v b 6 expression of different cell lines as measured by FACS analysis the 125 I-labeled peptide displayed specific binding to HNO97 cells and other HNSCC cells as well as to further carcinoma cell lines of different origin including lung, bladder and colon with an average of 7% which was competed to more than 90% by addition of 10 À6 mol/L unlabeled analog.…”

Section: Discussionmentioning

confidence: 55%

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Altmann

Sauter

Roesch

et al. 2017

Clinical Cancer Research

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Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients. For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified by using peptide histochemistry, using flow cytometry, and by positron emissions tomography (PET/CT). We identified a novel ITGαβ binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity ( = 14.8 nmol/L) for ITGαβ In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of Ga- andLu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions. Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαβ-positive carcinoma. .

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Section: Discussionmentioning

confidence: 55%

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Altmann

Sauter

Roesch

et al. 2017

Clinical Cancer Research

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“…SFTI1 has already been proven to be a suitable scaffold for the development of extremely stable peptides targeting tumor-associated receptors, including the a v b 6 integrin-specific peptide SFITGv6 (12,20). a v b 6 integrin has been shown to be expressed in up to 99.9% of HNSCC (9,14), as well as in lung (4), colon (7), breast (21), and pancreas carcinoma (6), and is often associated with a poor prognosis (9).…”

Section: Discussionmentioning

confidence: 99%

Comparison of the RGD Motif–Containing α_vβ₆Integrin–Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC

et al. 2018

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αβ integrin is overexpressed by several carcinomas and thus considered a target for diagnostic imaging and anticancer therapies. Recently, we presented the αβ integrin-binding peptide SFITGv6 as a novel potential tracer for imaging and targeted therapy of αβ integrin-positive carcinomas. Here, we analyzed the affinity and specificity of 5 native αβ integrin-specific binders in comparison to SFITGv6. Sunflower trypsin inhibitor 1 (SFTI1)-based peptides containing arginine-glycine-aspartic acid (RGD) motif-spanning octamers of fibronectin (SFFN1), tenascin C (SFTNC), vitronectin (SFVTN), and latency-associated peptides (LAP) 1 (SFLAP1) and 3 (SFLAP3) were synthesized, and their binding potential to αβ integrin-expressing head and neck squamous cell carcinoma (HNSCC) cell lines was evaluated. Subsequently, stability, affinity, and specificity were assessed in vitro using radio-high-pressure liquid chromatography, surface plasmon resonance assay, and binding experiments including competition, kinetics, internalization, and efflux. αβ integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal PET imaging and biodistribution experiments in HNSCC-bearing mice, andGa-DOTA-SFLAP3 was applied for diagnostic PET/CT of an HNSCC patient. When the newly designed peptides were compared, significant binding (>20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% were observed for SFLAP3 (GRGDLGRL) and SFITGv6 (FRGDLMQL). In contrast, the other peptides displayed binding that was moderate (SFLAP1, 4.1%-12.1%) to marginal (SFFN1, SFTNC, and SFVTN, <1%) and were therefore excluded from further analysis. Notably, SFLAP3 exhibited improved affinity for αβ integrin (mean half-maximal inhibitory concentration, 3.5 nM; dissociation constant, 7.4). Moreover, small-animal PET imaging and biodistribution studies of HNSCC xenograft mice revealed an increased tumor-specific accumulation 30-60 min after injection of Ga-labeled orLu-labeled DOTA-SFLAP3. Peptide staining further demonstrated binding specificity for SFLAP3 to HNSCC tumor cells. Finally, PET/CT scanning of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (SUV, 5.1) and in corresponding lymph node metastases (SUV, 4.1). SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging, and possibly targeted therapy of αβ integrin-expressing tumors.

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“…Many of the scaffolds that have been used for molecular grafting to date are derived from nature; for example, the peptide MCoTI-II (Momordica cochinchinensis trypsin inhibitor-II) has been re-engineered to antagonize intracellular p53 degradation [16] and inhibit serine proteases involved in inflammatory disease [17], and kalata B1 from Oldenlandia affinis has been reengineered to become an orally-active bradykinin B1 receptor antagonist [18]. One of the smallest disulfiderich scaffolds that has been used for molecular grafting is SFTI-1 (sunflower trypsin inhibitor-1) [19][20][21][22][23], which has been re-engineered for the treatment of cancer [20][21][22][23][24][25]. Compared to scaffolds containing unnatural amino acids, these nature-derived scaffolds have the benefit of being easily amenable to both chemical and recombinant methods for engineering and production [26][27][28].…”

Section: Introductionmentioning

confidence: 99%

Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold

Wang

Northfield

Huang

et al. 2016

European Journal of Medicinal Chemistry

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Disulfide-rich macrocyclic peptides are emerging as versatile scaffolds for the development of stable biochemical tools. This potential is due to the combination of their structural stability and range of bioactivities. Here, we explored the activity of these peptides on fibril growth of the hexapeptide Ac-VQIVYK-NH2 (AcPHF6), which is a tau-derived peptide that has been widely used to understand the pathological mechanism of numerous tauopathies, including Alzheimer's disease. Of the cyclic peptides tested, SFTI-1 and kB1 showed an inherent ability to inhibit AcPHF6 fibril formation. Using an end-capping strategy and combining it with a molecular grafting approach, we demonstrated that SFTI-1 could be used as a starting point to design more potent fibril inhibitors. We further identified chemical and structural features of SFTI-1 and its analogues that underpin their inhibitory activity. The ability to inhibit fibril growth using the strategy employed herein supports the 'steric zipper' model of AcPHF6 fibril formation and shows that naturally-occurring cyclic peptides have potential as drug leads or molecular probes for understanding fibril formation.

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Combination of Phage Display and Molecular Grafting Generates Highly Specific Tumor‐Targeting Miniproteins

Cited by 21 publications

References 21 publications

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Comparison of the RGD Motif–Containing α_vβ₆Integrin–Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC

Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold

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Combination of Phage Display and Molecular Grafting Generates Highly Specific Tumor‐Targeting Miniproteins

Cited by 21 publications

References 21 publications

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Identification of a Novel ITGαvβ6-Binding Peptide Using Protein Separation and Phage Display

Comparison of the RGD Motif–Containing αvβ6Integrin–Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC

Inhibition of tau aggregation using a naturally-occurring cyclic peptide scaffold

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Comparison of the RGD Motif–Containing α_vβ₆Integrin–Binding Peptides SFLAP3 and SFITGv6 for Diagnostic Application in HNSCC