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Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo


Gut, Melanie; Dreier, Birgit; Furler, Sven; Sobek, Jens; Plückthun, Andreas; Holland, Jason P (2023). Designed ankyrin repeat proteins for detecting prostate-specific antigen expression in vivo. RSC Chemical Biology, 4(7):494-505.

Abstract

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide $^{68}$Ga. [$^{68}$Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [$^{68}$Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [$^{68}$Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g$^{-1}$ (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g$^{-1}$; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

Abstract

Late-stage prostate cancer often acquires resistance to conventional chemotherapies and transforms into a hormone-refractory, drug-resistant, and non-curative disease. Developing non-invasive tools to detect the biochemical changes that correlate with drug efficacy and reveal the onset of drug resistance would have important ramifications in managing the treatment regimen for individual patients. Here, we report the selection of new Designed Ankyrin Repeat Proteins (DARPins) that show high affinity toward prostate-specific antigen (PSA), a biomarker used in clinical monitoring of prostate cancer. Ribosome display and in vitro screening tools were used to select PSA-binding DARPins based on their binding affinity, selectivity, and chemical constitution. Surface plasmon resonance measurements demonstrated that the four lead candidates bind to PSA with nanomolar affinity. DARPins were site-specifically functionalised at a unique C-terminal cysteine with a hexadentate aza-nonamacrocyclic chelate (NODAGA) for subsequent radiolabelling with the positron-emitting radionuclide $^{68}$Ga. [$^{68}$Ga]GaNODAGA-DARPins showed high stability toward transchelation and were stable in human serum for >2 h. Radioactive binding assays using streptavidin-loaded magnetic beads confirmed that the functionalisation and radiolabelling did not compromise the specificity of [$^{68}$Ga]GaNODAGA-DARPins toward PSA. Biodistribution experiments in athymic nude mice bearing subcutaneous prostate cancer xenografts derived from the LNCaP cell line revealed that three of the four [$^{68}$Ga]GaNODAGA-DARPins displayed specific tumour-binding in vivo. For DARPin-6, tumour-uptake in the normal group reached 4.16 ± 0.58% ID g$^{-1}$ (n = 3; 2 h post-administration) and was reduced by ∼50% by competitive binding with a low molar activity formulation (blocking group: 2.47 ± 0.42% ID g$^{-1}$; n = 3; P value = 0.018). Collectively, the experimental results support the future development of new PSA-specific imaging agents for potential use in monitoring the efficacy of androgen receptor (AR)-targeted therapies.

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Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry

04 Faculty of Medicine > Functional Genomics Center Zurich
07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:610 Medicine & health
570 Life sciences; biology
Scopus Subject Areas:Physical Sciences > Chemistry (miscellaneous)
Life Sciences > Biochemistry
Life Sciences > Molecular Biology
Life Sciences > Biochemistry, Genetics and Molecular Biology (miscellaneous)
Language:English
Date:17 May 2023
Deposited On:24 Nov 2023 12:51
Last Modified:29 Jun 2024 01:40
Publisher:Royal Society of Chemistry
ISSN:2633-0679
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1039/d3cb00010a
PubMed ID:37415866
Other Identification Number:PMCID: PMC10320840
Project Information:
  • : FunderSNF
  • : Grant IDPP00P2_163683
  • : Project Title
  • : FunderSNF
  • : Grant IDPP00P2_190093
  • : Project Title
  • : FunderSwiss Cancer Research Foundation
  • : Grant IDKFS-4257-08-2017
  • : Project Title
  • Content: Published Version
  • Language: English
  • Licence: Creative Commons: Attribution 4.0 International (CC BY 4.0)