Targeting of Epithelial Cell Adhesion Molecule-Expressing Malignant Tumors Using an Albumin-Binding Domain-Fused Designed Ankyrin Repeat Protein: Effect of the Molecular Architecture.

Designed ankyrin repeat protein (DARPin) Ec1, a small scaffold protein (18 kDa), binds with high affinity the epithelial cell adhesion molecule (EpCAM) that is overexpressed in several carcinomas. To enhance the targeted delivery of cytotoxic drugs using Ec1, we investigated the potential of fusing Ec1 with an albumin-binding domain (ABD) to improve its circulation time and decrease renal uptake. Two fusion proteins were created, Ec1-ABD, with the ABD at the C-terminus, and ABD-Ec1, with the ABD at the N-terminus.

Bioengineered Nanomedicines Targeting the Intestinal Fc Receptor Achieve the Improved Glucoregulatory Effect of Semaglutide in a Type 2 Diabetic Mice Model.

The oral administration of the glucagon-like peptide-1 analogue, semaglutide, remains a hurdle due to its limited bioavailability. Herein, neonatal Fc receptor (FcRn)-targeted nanoparticles (NPs) were designed to enhance the oral delivery of semaglutide. The nanocarriers were covalently linked to the FcRn-binding peptide FcBP or the affibody molecule Z that specifically binds to the human FcRn (hFcRn) in a pH-dependent manner.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2).

Nanoparticles targeting the intestinal Fc receptor enhance intestinal cellular trafficking of semaglutide.

Semaglutide is the first oral glucagon-like peptide-1 (GLP-1) analog commercially available for the treatment of type 2 diabetes. In this work, semaglutide was incorporated into poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles (NPs) to improve its delivery across the intestinal barrier. The nanocarriers were surface-decorated with either a peptide or an affibody that target the human neonatal Fc receptor (hFcRn), located on the luminal cell surface of the enterocytes.

Comparative Preclinical Evaluation of Peptide-Based Chelators for the Labeling of DARPin G3 with Tc for Radionuclide Imaging of HER2 Expression in Cancer.

Non-invasive radionuclide imaging of human epidermal growth factor receptor type 2 (HER2) expression in breast, gastroesophageal, and ovarian cancers may stratify patients for treatment using HER2-targeted therapeutics. Designed ankyrin repeat proteins (DARPins) are a promising type of targeting probe for radionuclide imaging. In clinical studies, the DARPin [Tc]Tc-(HE)-G3 labeled using a peptide-based chelator His-Glu-His-Glu-His-Glu ((HE)), provided clear imaging of HER2 expressing breast cancer 2-4 h after injection.

Experimental HER2-Targeted Therapy Using ADAPT6-ABD-mcDM1 in Mice Bearing SKOV3 Ovarian Cancer Xenografts: Efficacy and Selection of Companion Imaging Counterpart.

Overexpression of the human epidermal growth factor receptor 2 (HER2) in breast and gastric cancer is exploited for targeted therapy using monoclonal antibodies and antibody-drug conjugates. Small engineered scaffold proteins, such as the albumin binding domain (ABD) derived affinity proteins (ADAPTs), are a promising new format of targeting probes for development of drug conjugates with well-defined structure and tunable pharmacokinetics. Radiolabeled ADAPT6 has shown excellent tumor-targeting properties in clinical trials.

Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein.

Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins.

Influence of the Position and Composition of Radiometals and Radioiodine Labels on Imaging of Epcam Expression in Prostate Cancer Model Using the DARPin Ec1.

The epithelial cell adhesion molecule (EpCAM) is intensively overexpressed in 40-60% of prostate cancer (PCa) cases and can be used as a target for the delivery of drugs and toxins. The designed ankyrin repeat protein (DARPin) Ec1 has a high affinity to EpCAM (68 pM) and a small size (18 kDa). Radiolabeled Ec1 might be used as a companion diagnostic for the selection of PCa patients for therapy.

The Use of a Non-Conventional Long-Lived Gallium Radioisotope Ga Improves Imaging Contrast of EGFR Expression in Malignant Tumours Using DFO-ZEGFR:2377 Affibody Molecule.

Epidermal growth factor receptor (EGFR) is overexpressed in many malignancies. EGFR-targeted therapy extends survival of patients with disseminated cancers. Radionuclide molecular imaging of EGFR expression would make EGFR-directed treatment more personalized and therefore more efficient.

Radionuclide therapy using ABD-fused ADAPT scaffold protein: Proof of Principle.

Molecular recognition in targeted therapeutics is typically based on immunoglobulins. Development of engineered scaffold proteins (ESPs) has provided additional opportunities for the development of targeted therapies. ESPs offer inexpensive production in prokaryotic hosts, high stability and convenient approaches to modify their biodistribution.

Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies.

Investigation of a Pharmacological Approach for Reduction of Renal Uptake of Radiolabeled ADAPT Scaffold Protein.

Albumin binding domain-Derived Affinity ProTeins (ADAPTs) are small (5 kDa) engineered scaffold proteins that are promising targeting agents for radionuclide-based imaging. A recent clinical study has demonstrated that radiolabeled ADAPTs can efficiently visualize human epidermal growth factor receptor 2 (HER2) expression in breast cancer using SPECT imaging. However, the use of ADAPTs directly labeled with radiometals for targeted radionuclide therapy is limited by their high reabsorption and prolonged retention of activity in kidneys.

Phase I Study of Tc-ADAPT6, a Scaffold Protein-Based Probe for Visualization of HER2 Expression in Breast Cancer.

Radionuclide molecular imaging of human epidermal growth factor receptor type 2 (HER2) expression may help to stratify breast and gastroesophageal cancer patients for HER2-targeting therapies. Albumin-binding domain-derived affinity proteins (ADAPTs) are a new type of small (46-59 amino acids) protein useful as probes for molecular imaging. The aim of this first-in-humans study was to evaluate the biodistribution, dosimetry, and safety of the HER2-specific Tc-ADAPT6.

Imaging using radiolabelled targeted proteins: radioimmunodetection and beyond.

The use of radiolabelled antibodies was proposed in 1970s for staging of malignant tumours. Intensive research established chemistry for radiolabelling of proteins and understanding of factors determining biodistribution and targeting properties. The use of radioimmunodetection for staging of cancer was not established as common practice due to approval and widespread use of [F]-FDG, which provided a more general diagnostic use than antibodies or their fragments.

Influence of Several Compounds and Drugs on the Renal Uptake of Radiolabeled Affibody Molecules.

Affibody molecules are the most studied class of engineered scaffold proteins (ESPs) in radionuclide molecular imaging. Attempts to use affibody molecules directly labelled with radiometals for targeted radionuclide therapy were hampered by the high uptake and retention of radioactivity in kidneys. Several promising strategies have been implemented to circumvent this problem.

Feasibility of Imaging EpCAM Expression in Ovarian Cancer Using Radiolabeled DARPin Ec1.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in 55%-75% of ovarian carcinomas (OC). EpCAM might be used as a target for a treatment of disseminated OC. Designed ankyrin repeats protein (DARPin) Ec1 is a small (18 kDa) protein, which binds to EpCAM with subnanomolar affinity.

HER2-Specific Pseudomonas Exotoxin A PE25 Based Fusions: Influence of Targeting Domain on Target Binding, Toxicity, and In Vivo Biodistribution.

The human epidermal growth factor receptor 2 (HER2) is a clinically validated target for cancer therapy, and targeted therapies are often used in regimens for patients with a high HER2 expression level. Despite the success of current drugs, a number of patients succumb to their disease, which motivates development of novel drugs with other modes of action. We have previously shown that an albumin binding domain-derived affinity protein with specific affinity for HER2, ADAPT, can be used to deliver the highly cytotoxic protein domain PE25, a derivative of Pseudomonas exotoxin A, to HER2 overexpressing malignant cells, leading to potent and specific cell killing.

On the prevention of kidney uptake of radiolabeled DARPins.

Background: Designed ankyrin repeat proteins (DARPins) are small engineered scaffold proteins (14-18 kDa) that demonstrated promising tumor-targeting properties in preclinical studies. However, high renal accumulation of activity for DARPins labeled with residualizing labels is a limitation for targeted radionuclide therapy. A better understanding of the mechanisms behind the kidney uptake of DARPins could aid the development of strategies to reduce it.

Effect of a radiolabel biochemical nature on tumor-targeting properties of EpCAM-binding engineered scaffold protein DARPin Ec1.

Radionuclide-based imaging of molecular therapeutic targets might facilitate stratifying patients for specific biotherapeutics. New type of imaging probes, based on designed ankyrin repeat proteins (DARPins), have demonstrated excellent contrast of imaging of human epidermal growth factor type 2 (HER2) expression in preclinical models. We hypothesized that labeling approaches, which result in lipophilic radiometabolites (non-residualizing labels), would provide the best imaging contrast for DARPins that internalize slowly after binding to cancer cells.

CAIX-targeting radiotracers for hypoxia imaging in head and neck cancer models.

Hypoxia-induced carbonic anhydrase IX (CAIX) expression is a prognostic marker in solid tumors. In recent years many radiotracers have been developed, but a fair comparison of these compounds is not possible because of the diversity in tumor models and other experimental parameters. In this study we performed a direct in vivo comparison of three promising CAIX targeting radiotracers in xenografted head and neck cancer models.

Comparative evaluation of affibody- and antibody fragments-based CAIX imaging probes in mice bearing renal cell carcinoma xenografts.

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics.

Optimal composition and position of histidine-containing tags improves biodistribution of Tc-labeled DARPin G3.

Radionuclide molecular imaging of HER2 expression in disseminated cancer enables stratification of patients for HER2-targeted therapies. DARPin G3, a small (14 kDa) engineered scaffold protein, is a promising probe for imaging of HER2. We hypothesized that position (C- or N-terminus) and composition (hexahistidine or (HE)) of histidine-containing tags would influence the biodistribution of [Tc]Tc(CO)-labeled DARPin G3.

Potent and specific fusion toxins consisting of a HER2‑binding, ABD‑derived affinity protein, fused to truncated versions of Pseudomonas exotoxin A.

Fusion toxins consisting of an affinity protein fused to toxic polypeptides derived from Pseudomonas exotoxin A (ETA) are promising agents for targeted cancer therapy. In this study, we examined whether fusion toxins consisting of an albumin binding domain‑derived affinity protein (ADAPT) interacting with human epidermal growth factor receptor 2 (HER2), coupled to the ETA‑derived polypeptides PE38X8 or PE25, with or without an albumin binding domain (ABD) for half‑life extension, can be used for specific killing of HER2‑expressing cells. The fusion toxins could easily be expressed in a soluble form in Escherichia coli and purified to homogeneity.

Selection of the optimal macrocyclic chelators for labeling with In and Ga improves contrast of HER2 imaging using engineered scaffold protein ADAPT6.

Radionuclide molecular imaging is a promising tool that becomes increasingly important as targeted cancer therapies are developed. To ensure an effective treatment, a molecular stratification of the cancer is a necessity. To accomplish this, visualization of cancer associated molecular abnormalities in vivo by molecular imaging is the method of choice.