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Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier

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Abstract

Delivery of therapeutic proteins into tissues and across the blood–brain barrier (BBB) is limited by the size and biochemical properties of the proteins. Efficient delivery across BBB is generally restricted to small, highly lipophilic molecules. However, in the last decades, several peptides that can pass cell membranes have been identified. It has been shown that these peptides are also capable of delivering large hydrophilic cargoes into cells and are therefore a powerful biological tool for transporting drugs across cell membranes and even into the brain. We designed and prepared a single-chain antibody fragment (scFvs), specific for the pathological form of the prion protein (PrPSc), where a cell-penetrating peptide (CPP) was used as a linker between the two variable domains of the scFv. The intravenously administered recombinant scFv-CPP was successfully targeted to and delivered into mouse brain cells. Our single-chain antibody fragments are of special interest in view of possible therapeutic reagents design not only for prion diseases but also for other neurodegenerative diseases.

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Abbreviations

ABTS:

2,2′-Azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

CNS:

Central nervous system

CPP:

Cell-penetrating peptides

DAPI:

4′,6-Diamidino-2-phenylindole

Fv:

Antibody variable domains (Vl + Vh)

HRP:

Horseradish peroxidase

IMAC:

Immobilized metal ion affinity chromatography

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LLH:

scFv in Vl-linker-Vh chain arrangement

PBS:

Phosphate-buffered saline

PrP:

Prion protein

PrPC :

Cellular form of the PrP

PrPSc :

Pathogenic form of the PrP

scFv:

Antibody single-chain fragment

TBS:

Tris-buffered saline

TMB:

3,3′,5,5′-Tetramethylbenzidine

Vh:

Variable domain of the heavy-chain of an antibody

Vl:

Variable domain of the light chain of an antibody

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Acknowledgments

This work was supported by the Slovenian Research Agency Ph.D. grant for N. Škrlj.

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Authors and Affiliations

  1. Biochemistry Chair, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia

    Nives Škrlj, Vladka Čurin Šerbec & Marko Dolinar

  2. Institute of Pharmacology and Experimental Toxicology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

    Gorazd Drevenšek

  3. Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia

    Samo Hudoklin & Rok Romih

  4. Department for Production of Diagnostic Reagents and Research, Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia

    Vladka Čurin Šerbec

Authors
  1. Nives Škrlj
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  2. Gorazd Drevenšek
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Correspondence to Marko Dolinar.

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Škrlj, N., Drevenšek, G., Hudoklin, S. et al. Recombinant Single-Chain Antibody with the Trojan Peptide Penetratin Positioned in the Linker Region Enables Cargo Transfer Across the Blood–Brain Barrier. Appl Biochem Biotechnol 169, 159–169 (2013). https://doi.org/10.1007/s12010-012-9962-7

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