An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Identification of a high-risk immunogenic prostate cancer patient subset as candidates for T-cell engager immunotherapy and the introduction of a novel albumin-fused anti-CD3 × anti-PSMA bispecific design.

BACKGROUND: Cancer immunotherapies such as bispecific T-cell engagers have seen limited adoption in prostate cancer (PC), possibly due to differing levels of cancer receptor expression and effector T-cell infiltration between patients and inherent defects in T-cell engager design. METHODS: CD8+ T-cell infiltration and PSMA expression were determined by RNA sequencing of primary PC tissue samples from 126 patients with localised PC and 17 patients with metastatic PC. Prognostic value was assessed through clinical parameters, including CAPRA-S risk score. A panel of albumin-fused anti-CD3 × anti-PSMA T-cell engagers with different neonatal Fc receptor (FcRn) affinity were characterised by flow cytometry, Bio-Layer Interferometry and functional cellular assays. RESULTS: A subset of patients with localised (30/126 = 24%) and metastatic (10/17 = 59%) PC showed both high PSMA expression and high CD8+ T-cell enrichment. The High/High phenotype in localised PC associated with a clinically high-risk cancer subtype, confirmed in an external patient cohort (n = 550, PRAD/TCGA). The T-cell engagers exhibited tunable FcRn-driven cellular recycling, CD3 and PSMA cellular engagement, T-cell activation and PSMA level-dependent cellular cytotoxicity. CONCLUSION: This work presents an albumin-fused bispecific T-cell engager with programmable FcRn engagement and identifies a high-risk PC patient subset as candidates for treatment with the T-cell engager class of immuno-oncology biologics.

Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity.

Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy.

An albumin-angiotensin converting enzyme 2-based SARS-CoV-2 decoy with FcRn-driven half-life extension.

The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ ß = 13.5 h) than soluble ACE2 (t½ ß = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. STATEMENT OF SIGNIFICANCE: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Functionalized Acyclic (l)-Threoninol Nucleic Acid Four-Way Junction with High Stability In Vitro and In Vivo.

Oligonucleotides are increasingly being used as a programmable connection material to assemble molecules and proteins in well-defined structures. For the application of such assemblies for in vivo diagnostics or therapeutics it is crucial that the oligonucleotides form highly stable, non-toxic, and non-immunogenic structures. Only few oligonucleotide derivatives fulfil all of these requirements. Here we report on the application of acyclic l-threoninol nucleic acid (aTNA) to form a four-way junction (4WJ) that is highly stable and enables facile assembly of components for in vivo treatment and imaging. The aTNA 4WJ is serum-stable, shows no non-targeted uptake or cytotoxicity, and invokes no innate immune response. As a proof of concept, we modify the 4WJ with a cancer-targeting and a serum half-life extension moiety and show the effect of these functionalized 4WJs in vitro and in vivo, respectively.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

Targeting the IL-6-Yap-Snail signalling axis in synovial fibroblasts ameliorates inflammatory arthritis.

OBJECTIVE: We aimed to understand the role of the transcriptional co-factor Yes-associated protein (Yap) in the molecular pathway underpinning the pathogenic transformation of synovial fibroblasts (SF) in rheumatoid arthritis (RA) to become invasive and cause joint destruction. METHODS: Synovium from patients with RA and mice with antigen-induced arthritis (AIA) was analysed by immunostaining and qRT-PCR. SF were targeted using Pdgfrα-CreER and Gdf5-Cre mice, crossed with fluorescent reporters for cell tracing and Yap-flox mice for conditional Yap ablation. Fibroblast phenotypes were analysed by flow cytometry, and arthritis severity was assessed by histology. Yap activation was detected using Yap-Tead reporter cells and Yap-Snail interaction by proximity ligation assay. SF invasiveness was analysed using matrigel-coated transwells. RESULTS: Yap, its binding partner Snail and downstream target connective tissue growth factor were upregulated in hyperplastic human RA and in mouse AIA synovium, with Yap detected in SF but not macrophages. Lineage tracing showed polyclonal expansion of Pdgfrα-expressing SF during AIA, with predominant expansion of the Gdf5-lineage SF subpopulation descending from the embryonic joint interzone. Gdf5-lineage SF showed increased expression of Yap and adopted an erosive phenotype (podoplanin+Thy-1 cell surface antigen-), invading cartilage and bone. Conditional ablation of Yap in Gdf5-lineage cells or Pdgfrα-expressing fibroblasts ameliorated AIA. Interleukin (IL)-6, but not tumour necrosis factor alpha (TNF-α) or IL-1ß, Jak-dependently activated Yap and induced Yap-Snail interaction. SF invasiveness induced by IL-6 stimulation or Snail overexpression was prevented by Yap knockdown, showing a critical role for Yap in SF transformation in RA. CONCLUSIONS: Our findings uncover the IL-6-Yap-Snail signalling axis in pathogenic SF in inflammatory arthritis.

FcRn expression in cancer: Mechanistic basis and therapeutic opportunities.

There is an urgent need to identify new cellular targets to expand the repertoire, potency and safety of cancer therapeutics. Neonatal Fc Receptor (FcRn)-driven cellular recycling plays a predominant role in the prolonged serum half-life of human serum albumin (HSA) and immunoglobulin G (IgG) exploited in long-acting cancer drug designs. FcRn-mediated HSA and IgG uptake in epithelial cells and dendritic cell antigen presentation offers new therapeutic opportunities beyond half-life extension. Altered FcRn expression in solid tumours accounting for HSA catabolism or recycling supports a role for FcRn in tumour metabolism and growth. This review addresses the mechanistic basis for different FcRn expression profiles observed in cancer and exploitation for targeted drug delivery. Furthermore, the review highlights FcRn-mediated immunosurveillance and immune therapy. FcRn offers a potential attractive cancer target but in-depth understanding of role and expression profiles during cancer pathogenesis is required for tailoring targeted drug designs.

Per-glycosylation of the Surface-Accessible Lysines: One-Pot Aqueous Route to Stabilized Proteins with Native Activity.

Chemical glycosylation of proteins is a powerful tool applied widely in biomedicine and biotechnology. However, it is a challenging undertaking and typically relies on recombinant proteins and site-specific conjugations. The scope and utility of this nature-inspired methodology would be broadened tremendously by the advent of facile, scalable techniques in glycosylation, which are currently missing. In this work, we investigated a one-pot aqueous protocol to achieve indiscriminate, surface-wide glycosylation of the surface accessible amines (lysines and/or N-terminus). We reveal that this approach afforded minimal if any change in the protein activity and recognition events in biochemical and cell culture assays, but at the same time provided a significant benefit of stabilizing proteins against aggregation and fibrillation - as demonstrated on serum proteins (albumins and immunoglobulin G, IgG), an enzyme (uricase), and proteins involved in neurodegenerative disease (α-synuclein) and diabetes (insulin). Most importantly, this highly advantageous result was achieved via a one-pot aqueous protocol performed on native proteins, bypassing the use of complex chemical methodologies and recombinant proteins.

Programmable half-life and anti-tumour effects of bispecific T-cell engager-albumin fusions with tuned FcRn affinity.

Fc-less bispecific T-cell engagers have reached the immuno-oncology market but necessitate continual infusion due to rapid clearance from the circulation. This work introduces a programmable serum half-life extension platform based on fusion of human albumin sequences engineered with either null (NB), wild type (WT) or high binding (HB) FcRn affinity combined with a bispecific T-cell engager. We demonstrate in a humanised FcRn/albumin double transgenic mouse model (AlbuMus) the ability to tune half-life based on the albumin sequence fused with a BiTE-like bispecific (anti-EGFR nanobody x anti-CD3 scFv) light T-cell engager (LiTE) construct [(t½ 0.6 h (Fc-less LiTE), t½ 19 hours (Albu-LiTE-NB), t½ 26 hours (Albu-LiTE-WT), t½ 37 hours (Albu-LiTE-HB)]. We show in vitro cognate target engagement, T-cell activation and discrimination in cellular cytotoxicity dependent on EGFR expression levels. Furthermore, greater growth inhibition of EGFR-positive BRAF mutated tumours was measured following a single dose of Albu-LiTE-HB construct compared to the Fc-less LiTE format and a full-length anti-EGFR monoclonal antibody in a new AlbuMus RAG1 knockout model introduced in this work. Programmable half-life extension facilitated by this albumin platform potentially offers long-lasting effects, better patient compliance and a method to tailor pharmacokinetics to maximise therapeutic efficacy and safety of immuno-oncology targeted biologics.

Roadmap on nanomedicine.

Since the launch of the Alliance for Nanotechnology in Cancer by the National Cancer Institute in late 2004, several similar initiatives have been promoted all over the globe with the intention of advancing the diagnosis, treatment and prevention of cancer in the wake of nanoscience and nanotechnology. All this has encouraged scientists with diverse backgrounds to team up with one another, learn from each other, and generate new knowledge at the interface between engineering, physics, chemistry and biomedical sciences. Importantly, this new knowledge has been wisely channeled towards the development of novel diagnostic, imaging and therapeutic nanosystems, many of which are currently at different stages of clinical development. This roadmap collects eight brief articles elaborating on the interaction of nanomedicines with human biology; the biomedical and clinical applications of nanomedicines; and the importance of patient stratification in the development of future nanomedicines. The first article reports on the role of geometry and mechanical properties in nanomedicine rational design; the second articulates on the interaction of nanomedicines with cells of the immune system; and the third deals with exploiting endogenous molecules, such as albumin, to carry therapeutic agents. The second group of articles highlights the successful application of nanomedicines in the treatment of cancer with the optimal delivery of nucleic acids, diabetes with the sustained and controlled release of insulin, stroke by using thrombolytic particles, and atherosclerosis with the development of targeted nanoparticles. Finally, the last contribution comments on how nanomedicine and theranostics could play a pivotal role in the development of personalized medicines. As this roadmap cannot cover the massive extent of development of nanomedicine over the past 15 years, only a few major achievements are highlighted as the field progressively matures from the initial hype to the consolidation phase.

Albumin-Binding Fatty Acid-Modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics.

Prolonged circulation and modulation of the pharmacokinetic profile are important to improve the clinical potential of antisense oligonucleotides (ASOs). Gapmer ASOs demonstrate excellent nuclease stability and robust gene silencing activity without the requirement of transfection agents. A major challenge for in vivo applications, however, is the short blood circulatory half-life. This work describes utilization of the long circulation of serum albumin to increase the blood residence time of gapmer ASOs. The method introduces fatty acid modifications into the gapmer ASOs design to exploit the binding and transport property of serum albumin for endogenous ligands. The level of albumin-gapmer ASOs interaction, blood circulatory half-life and biodistribution was dependent on number, position, and fatty acid type (palmitic or myristic acid) within the gapmer ASO sequence and either phosphorothioate or phosphodiester backbone modifications. This work offers a strategy to optimize gapmer ASO pharmacokinetics by a proposed endogenous assembly process with serum albumin that can be tuned by gapmer ASO design modifications.

Albumin-based drug designs for pharmacokinetic modulation.

INTRODUCTION: A poor pharmacokinetic profile due to inadequate distribution and rapid renal clearance limits site-specific target engagement and drug efficacy. The inherent properties of human serum albumin for broad tissue distribution, prolonged circulation, and ligand transport have been engineered into albumin-based drug designs to modulate the pharmacokinetics to increase efficacy and reduce the frequency of dose. AREAS COVERED: This review highlights albumin structural features, ligand binding, and molecular interactions key to albumin-drug designs and an overview of the repertoire of albumin-drugs and approaches, with focus on pharmacokinetics of marketed products and clinical trials. EXPERT OPINION: Comparison, and advantages as well as disadvantages of the endogenous albumin-binding versus recombinant albumin construct approach, and half-life extension and intracellular drug delivery applications. The section addresses current challenges and solutions to the different drug designs, and considerations needed to progress the field such as conjugation chemistries, drug loading, and animal models. The section highlights the need for a paradigm shift in the field from 'utilizing' to 'controlling' albumin transport with recombinant human albumin variants engineered for tuned affinity to albumin cellular receptors.

FcRn overexpression in human cancer drives albumin recycling and cell growth; a mechanistic basis for exploitation in targeted albumin-drug designs.

Albumin accumulation in tumours could reflect a role of albumin in transport of endogenous nutrient cargos required for cellular growth and not just a suggested source of amino acids; a role driven by albumin engagement with its cognate cellular recycling neonatal Fc receptor. We investigate the hypothesis that albumin cellular recruitment is increased by higher human FcRn (hFcRn) expression in human cancer tissue that provides the mechanistic basis for exploitation in albumin-based drug designs engineered to optimise this process. Eight out of ten different human cancer tissue types screened for hFcRn expression by immunohistochemistry (310 samples) exhibited significantly higher hFcRn expression compared to healthy tissues. Accelerated tumour growth over 28 days in mice inoculated with hFcRn-expressing HT-29 human colorectal cancer cell xenografts, compared to CRISPR/Cas9 hFcRn-knockout HT-29, suggests a hFcRn-mediated tumour growth effect. Direct correlation between hFcRn expression and albumin recycling supports hFcRn-mediated diversion of albumin from lysosomal degradation. Two-fold increase in accumulation of fluorescent labelled high-binding hFcRn albumin, compared to wild type albumin, in luciferase MDA-MB-231-Luc-D3H2LN breast cancer xenografts was shown. This work identifies overexpression of hFcRn in several human cancer types with mechanistic data suggesting hFcRn-driven albumin recruitment for increased cellular growth that has the potential to be exploited with high hFcRn-binding albumin variants for targeted therapies.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Regulation of Gdf5 expression in joint remodelling, repair and osteoarthritis.

Growth and Differentiation Factor 5 (GDF5) is a key risk locus for osteoarthritis (OA). However, little is known regarding regulation of Gdf5 expression following joint tissue damage. Here, we employed Gdf5-LacZ reporter mouse lines to assess the spatiotemporal activity of Gdf5 regulatory sequences in experimental OA following destabilisation of the medial meniscus (DMM) and after acute cartilage injury and repair. Gdf5 expression was upregulated in articular cartilage post-DMM, and was increased in human OA cartilage as determined by immunohistochemistry and microarray analysis. Gdf5 expression was also upregulated during cartilage repair in mice and was switched on in injured synovium in prospective areas of cartilage formation, where it inversely correlated with expression of the transcriptional co-factor Yes-associated protein (Yap). Indeed, overexpression of Yap suppressed Gdf5 expression in chondroprogenitors in vitro. Gdf5 expression in both mouse injury models required regulatory sequence downstream of Gdf5 coding exons. Our findings suggest that Gdf5 upregulation in articular cartilage and synovium is a generic response to knee injury that is dependent on downstream regulatory sequence and in progenitors is associated with chondrogenic specification. We propose a role for Gdf5 in tissue remodelling and repair after injury, which may partly underpin its association with OA risk.

Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

Independent Validation of a Diagnostic Noninvasive 3-MicroRNA Ratio Model (uCaP) for Prostate Cancer in Cell-Free Urine.

BACKGROUND: Detection of prostate cancer (PC) based on serum prostate-specific antigen (PSA) testing leads to many unnecessary prostate biopsies, overdiagnosis, and overtreatment of clinically insignificant tumors. Thus, novel and more accurate molecular biomarkers are required. METHODS: Using reverse transcription quantitative PCR, we measured the concentrations of 45 preselected microRNAs (miRNAs) in extracellular vesicle-enriched cell-free urine samples from 4 independent patient cohorts from Spain and Denmark, including 758 patients with clinically localized PC, 289 noncancer controls with benign prostatic hyperplasia (BPH), and 233 patients undergoing initial transrectal ultrasound (TRUS)-guided prostate biopsy owing to PC suspicion (101 with benign and 132 with malignant outcome). Diagnostic potential was assessed by ROC and decision curve analysis. RESULTS: We identified and successfully validated 8 upregulated and 21 downregulated miRNAs in urine from PC patients. Furthermore, we validated a previously identified 3-miRNA diagnostic ratio model, uCaP (miR-222-3p*miR-24-3p/miR-30c-5p). High uCaP scores were distinctive of PC in urine samples from BPH vs PC patients in 3 independent cohorts [area under the curve (AUC) = 0.84, 0.71, 0.72]. Additionally, uCaP predicted TRUS biopsy results with greater accuracy than PSA (AUC uCaP = 0.644; AUC PSA = 0.527) for patients within the diagnostic gray zone (PSA ≤ 10 ng/mL). CONCLUSIONS: We successfully validated a urine-based diagnostic 3-miRNA signature for PC (uCaP) in 3 independent patient cohorts from 2 countries. In the future, the simple and noninvasive uCaP test may be used to help more accurately select patients for prostate biopsy. Prospective clinical validation is warranted.