CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody

Camps, Estel Collado; Lith, Sanne A. M. van; Frielink, Cathelijne; Lankhof, Jordi; Dijkgraaf, Ingrid; Gotthardt, Martin; Brock, Roland

doi:10.3390/ph14070602

Cited by 13 publications

(9 citation statements)

References 57 publications

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“…This shows that efficient uptake only occurs when interaction of the ligand with the receptor is present. We have previously observed similar combined effects for penetratin and the nanobody 7D12 [22].…”

Section: Discussionsupporting

confidence: 69%

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Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Camps

Lith

Kip

et al. 2022

Eur J Nucl Med Mol Imaging

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Purpose Exendin, an analogue of the glucagon-like peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9-39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9-39) could turn it into a useful alternative tracer with less side-effects than exendin-4. Methods We conjugated exendin-4 and exendin(9-39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro and their biodistribution in vivo. Results Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9-39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9-39). Conclusion We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9-39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

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Section: Discussionsupporting

confidence: 69%

“…Importantly, we had previously established that conjugation of various CPPs to a nanobody binding the epidermal growth factor receptor (EGFR) increased endocytosis of the compound without increasing EGFR activation. This creates the rationale for combining penetratin and exendin variants for the GLP1R-directed approach [21,22].…”

Section: Introductionmentioning

confidence: 99%

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Camps

Lith

Kip

et al. 2022

Eur J Nucl Med Mol Imaging

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show abstract

“…This shows that e cient uptake only occurs when interaction of the ligand with the receptor is present. We have previously observed similar combined effects for penetratin and the nanobody 7D12 [22].…”

Section: Discussionsupporting

confidence: 69%

“…Importantly, we had previously established that conjugation of various CPPs to a nanobody binding the epidermal growth factor receptor (EGFR) increased endocytosis of the compound without increasing EGFR activation creating a strong rationale for the GLP1R-directed approach [21,22].…”

Section: Introductionmentioning

confidence: 99%

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Camps

Lith

Kip

et al. 2022

Preprint

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Purpose Exendin, an analogue of the Glucagon-Like Peptide 1 (GLP1), is an excellent tracer for molecular imaging of pancreatic beta cells and beta cell-derived tumours. The commonly used form, exendin-4, activates the GLP1 receptor and causes internalisation of the peptide-receptor complex. As a consequence, injection of exendin-4 can lead to adverse effects such as nausea, vomiting and hypoglycaemia and thus requires close monitoring during application. By comparison, the antagonist exendin(9–39) does not activate the receptor, but its lack of internalisation has precluded its use as a tracer. Improving the cellular uptake of exendin(9–39) could turn it into a useful alternative tracer with less side-effects than exendin-4. Methods We conjugated exendin-4 and exendin(9–39) to the well-known cell-penetrating peptide (CPP) penetratin. We evaluated cell binding and internalisation of the radiolabelled peptides in vitro, and their biodistribution in vivo. Results Exendin-4 showed internalisation irrespective of the presence of the CPP, whereas for exendin(9–39) only the penetratin conjugate internalised. Conjugation to the CPP also enhanced the in vivo tumour uptake and retention of exendin(9–39). Conclusion We demonstrate that penetratin robustly improves internalisation and tumour retention of exendin(9–39), opening new avenues for antagonist-based in vivo imaging of GLP1R.

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“…Recently, a peptide ligation method using sortase and transglutaminase has attracted attention as a method for specifically ligating proteins under mild conditions 24 . Recently, a VHH and CPP bioconjugate formed using sortase has been reportedly taken-up by cells 25 , 26 . Microbial transglutaminase (MTG; EC 2.3.2.13), belonging to the class of a protein γ-glutamyl transferases, is the most widely used enzyme because of its high activity, Ca + independence, and low reverse reaction 27 .…”

Section: Introductionmentioning

confidence: 99%

Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery

Ando

Nakazawa

Miura

et al. 2021

Sci Rep

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A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

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CPPs to the Test: Effects on Binding, Uptake and Biodistribution of a Tumor Targeting Nanobody

Cited by 13 publications

References 57 publications

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Conjugation to a cell-penetrating peptide drives the tumour accumulation of the GLP1R antagonist exendin(9-39)

Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery

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