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

ABSTRACT: Nanobodies are well-established targeting ligands for molecular imaging and therapy. Their short circulation time enables early imaging and reduces systemic radiation exposure. However, shorter circulation time leads to lower tracer accumulation in the target tissue. Cell-penetrating peptides (CPPs) improve cellular uptake of various cargoes, including nanobodies. CPPs could enhance tissue retention without compromising rapid clearance. However, systematic investigations on how the functionalities of nanobody and CPP combine with each other at the level of 2D and 3D cell cultures and in vivo are lacking. Here, we demonstrate that conjugates of the epidermal growth factor receptor (EGFR)-binding nanobody 7D12 with different CPPs (nonaarginine, penetratin, Tat and hLF) differ with respect to cell binding and induction of endocytosis. For nonaarginine and penetratin we compared the competition of EGF binding and performance of L- and D-peptide stereoisomers, and tested the D-peptide conjugates in tumor cell spheroids and in vivo. The D-peptide conjugates showed better penetration into spheroids than the unconjugated 7D12. Both in vivo and in vitro, the behavior of the agent reflects the combination of both functionalities. Although CPPs cause promising increases in in vitro uptake and 3D penetration, the dominant effect of the CPP in the control of biodistribution warrants further investigation.

SUBMITTER: Collado Camps E

PROVIDER: S-EPMC8308549 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: The non-homogenous distribution of antibody-drug conjugates (ADCs) within solid tumors is a major limiting factor for their wide clinical application. Nanobodies have been shown to rapidly penetrate into xenografts, achieving more homogeneous tumor targeting. However, their rapid renal clearance can hamper their application as nanobody drug conjugates (NDCs). Here, we evaluate whether half-life extension via non-covalent interaction with albumin can benefit the efficacy of a HER2-targeted NDC. Methods: HER2-targeted nanobody 11A4 and the irrelevant nanobody R2 were genetically fused to an albumin-binding domain (ABD) at their C-terminus. Binding to both albumin and tumor cells was determined by ELISA-based assays. The internalization potential as well as the in vitro efficacy of NDCs were tested on HER2 expressing cells. Serum half-life of iodinated R2 and R2-ABD was studied in tumor-free mice. The distribution of fluorescently labelled 11A4 and 11A4-ABD was assessed in vitro in 3D spheroids. Subsequently, the in vivo distribution was evaluated by optical molecular imaging and ex vivo by tissue biodistribution and tumor immunohistochemical analysis after intravenous injection of IRDye800-conjugated nanobodies in mice bearing HER2-positive subcutaneous xenografts. Finally, efficacy studies were performed in HER2-positive NCI-N87 xenograft-bearing mice intravenously injected with a single dose (250 nmol/kg) of nanobodies conjugated to auristatin F (AF) either via a maleimide or the organic Pt(II)‑based linker, coined Lx ®. Results: 11A4-ABD was able to bind albumin and HER2 and was internalized by HER2 expressing cells, irrespective of albumin presence. Interaction with albumin did not alter its distribution through 3D spheroids. Fusion to ABD resulted in a 14.8-fold increase in the serum half-life, as illustrated with the irrelevant nanobody. Furthermore, ABD fusion prolonged the accumulation of 11A4-ABD in HER2-expressing xenografts without affecting the expected homogenous intratumoral distribution. Next to that, reduced kidney retention of ABD-fused nanobodies was observed. Finally, a single dose administration of either 11A4-ABD-maleimide-AF or 11A4-ABD-Lx-AF led to long-lasting tumor remission in HER2-positive NCI-N87 xenograft-bearing mice. Conclusion: Our results demonstrate that genetic fusion of a nanobody to ABD can significantly extend serum half-life, resulting in prolonged and homogenous tumor accumulation. Most importantly, as supported by the impressive anti-tumor efficacy observed after a single dose administration of 11A4-ABD-AF, our data reveal that monovalent internalizing ABD-fused nanobodies have potential for the development of highly effective NDCs.

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

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