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Cited by 44Protease and Inhibitor MechanismsPeptidase Inhibition and AnalysisBlood Coagulation and Thrombosis Mechanisms

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The Urokinase Receptor (uPAR) as a “Trojan Horse” in Targeted Cancer Therapy: Challenges and Opportunities

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Metrangolo, Virginia, Ploug, Michael, Engelholm, Lars H.·2021·Cancers
DOI 10.3390/cancers13215376PMC PMC8582577
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Abstract

One of the largest challenges to the implementation of precision oncology is identifying and validating selective tumor-driving targets to enhance the therapeutic efficacy while limiting off-target toxicity. In this context, the urokinase-type plasminogen activator receptor (uPAR) has progressively emerged as a promising therapeutic target in the management of aggressive malignancies. By focalizing the plasminogen activation cascade and subsequent extracellular proteolysis on the cell surface of migrating cells, uPAR endows malignant cells with a high proteolytic and migratory potential to dissolve the restraining extracellular matrix (ECM) barriers and metastasize to distant sites. uPAR is also assumed to choreograph multiple other neoplastic stages via a complex molecular interplay with distinct cancer-associated signaling pathways. Accordingly, high uPAR expression is observed in virtually all human cancers and is frequently associated with poor patient prognosis and survival. The promising therapeutic potential unveiled by the pleiotropic nature of this receptor has prompted the development of distinct targeted intervention strategies. The present review will focus on recently emerged cytotoxic approaches emphasizing the novel technologies and related limits hindering their application in the clinical setting. Finally, future research directions and emerging opportunities in the field of uPAR targeting are also discussed.

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Figure 3 Function and regulation of the urokinase-type plasminogen activator receptor (uPAR) in the biology of cancer. (a) Through a high binding affinity interaction (KD ~0.2 nM), the urokinase receptor binds active uPA and its zymogen pro-uPA, favoring its focused cell-surface activation. Active uPA proteolytically converts the zymogen plasminogen (plg) into active plasmin (plm), which can reciprocally activate pro-uPA, while remaining protected from its primary plasma physiological inhibitors, α2-antiplasmin (α2-AP). These mutual zymogen activation reactions start a powerful positive feedback mechanism, resulting in efficient and localized plasmin generation on the cell surface of migrating cancer cells at their leading edge. The increased cell surface concentration of the reactants involved, respectively, uPA or pro-uPA, by binding to uPAR, and plasmin and plasminogen, via multiple receptors, strongly accelerate and amplify this reciprocal activation loop. Once activated, the broad-spectrum protease plasmin mediates the non-specific proteolysis of several ECM and basement membrane (BM) components, either directly or through the activation of pro-matrix metalloproteinases (pro-MMPs), thereby promoting cancer cell migration, invasion, and metastasis. Plasmin and MMPs can also release or activate ECM-bound cancer-related growth factors (GF) contributing to tumor progression and angiogenesis. Most of these factors then feedback in an autocrine or paracrine fashion to enhance the expression of different pro-cancer genes, including urokinase plasminogen activating system (uPAS) components, such as uPA and uPAR, that further supports the proteolytic cascade and thus tumor progression. Besides α2-AP, another important physiological regulator of uPA-uPAR-induced plg-activation is the serine protease inhibitor (serpin) plasminogen activator inhibitor 1 (PAI1), which specifically inhibits uPA by forming stable ternary complexes with uPAR-bound uPA, which are subsequently internalized via the α2-macroglobulin receptor/low-density lipoprotein receptor-related protein 1 (LRP1). (b) By becoming a part of functional units involving distinct extracellular molecules and membrane co-receptors (e.g., its second main cognate ligand, the matrix protein Vn, members of the integrin adhesion receptor superfamily, G-protein-coupled receptors (GPCR), and growth factors receptors (GFR, e.g., EGFR and VEGFR-2, epidermal growth and vascular endothelial growth factor receptor 2)), uPAR is believed to indirectly choreograph—in a non-proteolytic fashion—several cancer-associated intracellular signal-transduction pathways regulating other tumor hallmarks, including, among others, proliferation, survival, migration, invasion, metastasis, angiogenesis, and epithelial–mesenchymal transition (EMT). The intracellular signaling components are indicated (focal adhesion kinase (FAK), Src, Rac, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), etc.), but the pathways remain speculative. A more comprehensive list of uPAR putative non-canonical interacting proteins and related signaling consequences is reported in [4,16,73]. (c) Cell surface uPAR may undergo two major post-translational processing events, namely proteolytic cleavage (in the DI–DII linker region) and shedding (via hydrolysis of the GPI-anchor), resulting in diverse uPAR isoforms, including suPAR D1, suPAR DI–D3, suPAR D2–D3, and GPI-anchored uPAR D2–D3. Created with BioRender.com.
Figure 3 Function and regulation of the urokinase-type plasminogen activator receptor (uPAR) in the biology of cancer. (a) Through a high binding affinity interaction (KD ~0.2 nM), the urokinase receptor binds active uPA and its zymogen pro-uPA, favoring its focused cell-surface activation. Active uPA proteolytically converts the zymogen plasminogen (plg) into active plasmin (plm), which can reciprocally activate pro-uPA, while remaining protected from its primary plasma physiological inhibitors, α2-antiplasmin (α2-AP). These mutual zymogen activation reactions start a powerful positive feedback mechanism, resulting in efficient and localized plasmin generation on the cell surface of migrating cancer cells at their leading edge. The increased cell surface concentration of the reactants involved, respectively, uPA or pro-uPA, by binding to uPAR, and plasmin and plasminogen, via multiple receptors, strongly accelerate and amplify this reciprocal activation loop. Once activated, the broad-spectrum protease plasmin mediates the non-specific proteolysis of several ECM and basement membrane (BM) components, either directly or through the activation of pro-matrix metalloproteinases (pro-MMPs), thereby promoting cancer cell migration, invasion, and metastasis. Plasmin and MMPs can also release or activate ECM-bound cancer-related growth factors (GF) contributing to tumor progression and angiogenesis. Most of these factors then feedback in an autocrine or paracrine fashion to enhance the expression of different pro-cancer genes, including urokinase plasminogen activating system (uPAS) components, such as uPA and uPAR, that further supports the proteolytic cascade and thus tumor progression. Besides α2-AP, another important physiological regulator of uPA-uPAR-induced plg-activation is the serine protease inhibitor (serpin) plasminogen activator inhibitor 1 (PAI1), which specifically inhibits uPA by forming stable ternary complexes with uPAR-bound uPA, which are subsequently internalized via the α2-macroglobulin receptor/low-density lipoprotein receptor-related protein 1 (LRP1). (b) By becoming a part of functional units involving distinct extracellular molecules and membrane co-receptors (e.g., its second main cognate ligand, the matrix protein Vn, members of the integrin adhesion receptor superfamily, G-protein-coupled receptors (GPCR), and growth factors receptors (GFR, e.g., EGFR and VEGFR-2, epidermal growth and vascular endothelial growth factor receptor 2)), uPAR is believed to indirectly choreograph—in a non-proteolytic fashion—several cancer-associated intracellular signal-transduction pathways regulating other tumor hallmarks, including, among others, proliferation, survival, migration, invasion, metastasis, angiogenesis, and epithelial–mesenchymal transition (EMT). The intracellular signaling components are indicated (focal adhesion kinase (FAK), Src, Rac, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), etc.), but the pathways remain speculative. A more comprehensive list of uPAR putative non-canonical interacting proteins and related signaling consequences is reported in [4,16,73]. (c) Cell surface uPAR may undergo two major post-translational processing events, namely proteolytic cleavage (in the DI–DII linker region) and shedding (via hydrolysis of the GPI-anchor), resulting in diverse uPAR isoforms, including suPAR D1, suPAR DI–D3, suPAR D2–D3, and GPI-anchored uPAR D2–D3. Created with BioRender.com.CC BY · PubMed Central
Figure 11 Schematic structure and putative mechanism of action of uPAR-directed immunotherapy approaches. (a) Wang et al. developed third-generation uPAR-targeted CAR T-cells using ATF as antigen recognition domain instead of the usual antibody-derived single-chain variable fragment (scFv) [251]. Besides the CD3 ζ-signaling domain, third-generation CARs incorporate endodomains from co-stimulatory molecules, in this case, CD28 and CD137 (also known as 4-1BB), to deliver the additional signals necessary to fully activate the T-cell, physiologically provided by the antigen-presenting cells (APC) [258]. (b) ARM-U2 is a uPAR-targeted antibody-recruiting small molecule comprising a derivative of the high-affinity uPAR inhibitor IPR-803 (bottom box), functioning as the target-binding molecule (TBM, shown in red), and a 2,4-dinitrophenol (DNP) moiety as the antibody-binding domain (ABM, depicted in blue). (c) Putative action mechanisms of ATF-CAR T cells (upper) and ARM-U2 (bottom). Upon uPAR-recognition and binding, mediated by the ATF domain, ATF CAR T-cells (upper) form a non-classical immune synapse (IS) with the target cancer cell, which mediates the T-cell cytolytic effector functions, including perforin and granzyme release, ultimately leading to selective tumor cell death [258]. The simultaneous binding of ARM-U2 to surface uPAR on the target cancer cell (by the TBM) and endogenous anti-DNP antibodies (recruited via the DNP moiety, ABM) results in the formation of a ternary complex, which can subsequently trigger selective immune-mediated cytotoxicity through different mechanisms. In the complement-mediated cytotoxicity, the C1q complement protein binds to the Fc part of the antibodies and activates the complement cascade, which culminates in the formation of the membrane attack complex (MAC) on the cancer cell surface and its lysis. Alternatively, the antibody’s Fc can also interact with the Fc-receptors expressed on the surface of various immune cells, such as macrophages or natural killer cells, followed by target cell phagocytosis (ADCP) or tumor-cell lysis via NK releasing potent oxidizing agents and protein toxins, such as granzyme and perforin (ADCC) [255]. Created with BioRender.com.
Figure 11 Schematic structure and putative mechanism of action of uPAR-directed immunotherapy approaches. (a) Wang et al. developed third-generation uPAR-targeted CAR T-cells using ATF as antigen recognition domain instead of the usual antibody-derived single-chain variable fragment (scFv) [251]. Besides the CD3 ζ-signaling domain, third-generation CARs incorporate endodomains from co-stimulatory molecules, in this case, CD28 and CD137 (also known as 4-1BB), to deliver the additional signals necessary to fully activate the T-cell, physiologically provided by the antigen-presenting cells (APC) [258]. (b) ARM-U2 is a uPAR-targeted antibody-recruiting small molecule comprising a derivative of the high-affinity uPAR inhibitor IPR-803 (bottom box), functioning as the target-binding molecule (TBM, shown in red), and a 2,4-dinitrophenol (DNP) moiety as the antibody-binding domain (ABM, depicted in blue). (c) Putative action mechanisms of ATF-CAR T cells (upper) and ARM-U2 (bottom). Upon uPAR-recognition and binding, mediated by the ATF domain, ATF CAR T-cells (upper) form a non-classical immune synapse (IS) with the target cancer cell, which mediates the T-cell cytolytic effector functions, including perforin and granzyme release, ultimately leading to selective tumor cell death [258]. The simultaneous binding of ARM-U2 to surface uPAR on the target cancer cell (by the TBM) and endogenous anti-DNP antibodies (recruited via the DNP moiety, ABM) results in the formation of a ternary complex, which can subsequently trigger selective immune-mediated cytotoxicity through different mechanisms. In the complement-mediated cytotoxicity, the C1q complement protein binds to the Fc part of the antibodies and activates the complement cascade, which culminates in the formation of the membrane attack complex (MAC) on the cancer cell surface and its lysis. Alternatively, the antibody’s Fc can also interact with the Fc-receptors expressed on the surface of various immune cells, such as macrophages or natural killer cells, followed by target cell phagocytosis (ADCP) or tumor-cell lysis via NK releasing potent oxidizing agents and protein toxins, such as granzyme and perforin (ADCC) [255]. Created with BioRender.com.CC BY · PubMed Central
Figure 1 Graphical representation of the structure of uPA•uPAR complexes on the cell surface. A composite molecular model representing human uPAR based on the crystal structure solved for uPAR•ATF complexes is displayed in (A) using the PDB coordinates 2FD6 [36]. The molecular shape of uPAR is visualized by a semitransparent surface, while secondary structure elements are depicted as ribbons. The assembly of the three LU-domains is evident from the color coding, yellow (DI), blue (DII), and red (DIII). A hypothetical model for the GPI-anchor, tethering uPAR to the cell surface, is shown as sticks. In (B), the bimolecular complex of uPAR with its natural binding ligand uPA is illustrated using a solid surface representation for uPAR and a ribbon diagram for the receptor-binding fragment of uPA (ATF) used to crystallize the complex. The large hydrophobic ligand-binding cavity of uPAR is highlighted by the grey area delimited by the hatched black line using the following atomic color coding: grey (C), blue (N), red (O), and yellow (S). The inset in the bottom right corner provides a more detailed illustration of the tight engagement and burial of the tip of the β-hairpin of GFD in uPA within the deepest region of the central cavity in uPAR. Adapted from [23].
Figure 1 Graphical representation of the structure of uPA•uPAR complexes on the cell surface. A composite molecular model representing human uPAR based on the crystal structure solved for uPAR•ATF complexes is displayed in (A) using the PDB coordinates 2FD6 [36]. The molecular shape of uPAR is visualized by a semitransparent surface, while secondary structure elements are depicted as ribbons. The assembly of the three LU-domains is evident from the color coding, yellow (DI), blue (DII), and red (DIII). A hypothetical model for the GPI-anchor, tethering uPAR to the cell surface, is shown as sticks. In (B), the bimolecular complex of uPAR with its natural binding ligand uPA is illustrated using a solid surface representation for uPAR and a ribbon diagram for the receptor-binding fragment of uPA (ATF) used to crystallize the complex. The large hydrophobic ligand-binding cavity of uPAR is highlighted by the grey area delimited by the hatched black line using the following atomic color coding: grey (C), blue (N), red (O), and yellow (S). The inset in the bottom right corner provides a more detailed illustration of the tight engagement and burial of the tip of the β-hairpin of GFD in uPA within the deepest region of the central cavity in uPAR. Adapted from [23].CC BY · PubMed Central
Figure 2 Graphical representation of the crystal structure of the ternary uPA•uPAR•Vn complex. (A) A composite molecular model of the ATF•uPAR•SMB complex solved by X-ray crystallography is shown in (A) using the PDB coordinates 3BTI [37]. The structure is rotated 90° in the horizontal axis compared to Figure 1, providing a “top view” of uPAR and moving the cell surface to the back of the picture. As in Figure 1, uPAR is represented in a composite semitransparent surface and cartoon representation. The bound ligands ATF and SMB (representing uPA and Vn, respectively) are depicted as ribbons. A detailed view of the molecular binding interface between uPAR and SMB in this ternary complex is provided in (B). The corresponding hot spot residues in uPAR (R91 and W32) and SMB (F13, D22, Y27, and Y28) are highlighted and shown as sticks. Adapted from [23].
Figure 2 Graphical representation of the crystal structure of the ternary uPA•uPAR•Vn complex. (A) A composite molecular model of the ATF•uPAR•SMB complex solved by X-ray crystallography is shown in (A) using the PDB coordinates 3BTI [37]. The structure is rotated 90° in the horizontal axis compared to Figure 1, providing a “top view” of uPAR and moving the cell surface to the back of the picture. As in Figure 1, uPAR is represented in a composite semitransparent surface and cartoon representation. The bound ligands ATF and SMB (representing uPA and Vn, respectively) are depicted as ribbons. A detailed view of the molecular binding interface between uPAR and SMB in this ternary complex is provided in (B). The corresponding hot spot residues in uPAR (R91 and W32) and SMB (F13, D22, Y27, and Y28) are highlighted and shown as sticks. Adapted from [23].CC BY · PubMed Central
Figure 4 uPAR gene expression profile in human cancer. Abbreviations (TCGA, The Cancer Genome Atlas): ACC, Adenoid Cystic Carcinoma; BLCA, Bladder Urothelial Carcinoma; BRCA, Breast Invasive Carcinoma; CESC, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; CHOL, Cholangiocarcinoma; COAD, Colon Adenocarcinoma; DLBC, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA, Esophageal Carcinoma; GBM, Glioblastoma Multiforme; HNSCC, Head and Neck Squamous Cell Carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney Renal Clear Cell Carcinoma; KIRP, Kidney Renal Papillary Cell Carcinoma; LAML, Acute Myeloid Leukemia; LGG, Brain Lower Grade Glioma; LIHC, Liver Hepatocellular Carcinoma; LUAD, Lung Adenocarcinoma; LUSC, Lung Squamous Cell Carcinoma; OV, Ovarian Serous Cystadenocarcinoma; PAAD, Pancreatic Adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; PRAD, Prostate Adenocarcinoma; READ, Rectum Adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; STAD, Stomach Adenocarcinoma; TGCT, Testicular Germ Cell Tumors; THCA, Thyroid Carcinoma; THYM, Thymoma; UCES, Uterine Corpus Endometrial Carcinoma; UCS, Uterine Carcinosarcoma. Adapted from http://gepia.cancer-pku.cn/ (Accessed date: 1 August 2021).
Figure 4 uPAR gene expression profile in human cancer. Abbreviations (TCGA, The Cancer Genome Atlas): ACC, Adenoid Cystic Carcinoma; BLCA, Bladder Urothelial Carcinoma; BRCA, Breast Invasive Carcinoma; CESC, Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma; CHOL, Cholangiocarcinoma; COAD, Colon Adenocarcinoma; DLBC, Lymphoid Neoplasm Diffuse Large B-cell Lymphoma; ESCA, Esophageal Carcinoma; GBM, Glioblastoma Multiforme; HNSCC, Head and Neck Squamous Cell Carcinoma; KICH, Kidney Chromophobe; KIRC, Kidney Renal Clear Cell Carcinoma; KIRP, Kidney Renal Papillary Cell Carcinoma; LAML, Acute Myeloid Leukemia; LGG, Brain Lower Grade Glioma; LIHC, Liver Hepatocellular Carcinoma; LUAD, Lung Adenocarcinoma; LUSC, Lung Squamous Cell Carcinoma; OV, Ovarian Serous Cystadenocarcinoma; PAAD, Pancreatic Adenocarcinoma; PCPG, Pheochromocytoma and Paraganglioma; PRAD, Prostate Adenocarcinoma; READ, Rectum Adenocarcinoma; SARC, Sarcoma; SKCM, Skin Cutaneous Melanoma; STAD, Stomach Adenocarcinoma; TGCT, Testicular Germ Cell Tumors; THCA, Thyroid Carcinoma; THYM, Thymoma; UCES, Uterine Corpus Endometrial Carcinoma; UCS, Uterine Carcinosarcoma. Adapted from http://gepia.cancer-pku.cn/ (Accessed date: 1 August 2021).CC BY · PubMed Central
Figure 5 Tissue expression of uPAR in human cancer. (a) Peroxidase and multi-immunofluorescence staining of primary colon adenocarcinoma (panels 1–2) and a corresponding liver metastasis with desmoplastic growth pattern (panels 3–4). uPAR is primarily expressed by tumor-infiltrating macrophages (black arrows in 1 and 3; blue arrows in 2 and 4) along with some few detached budding cancer cells at the invasive cancer front (white arrows in 2 and 4) (Ca: cancer, ST: stroma, DS: desmoplastic stroma; LP: liver parenchyma). Reproduced from [129], Copyright (2009, John Wiley & Sons, Inc., Hoboken, New Jersey, USA) with permission of John Wiley & Sons, Inc. (b) An analogous expression pattern is seen in the intestinal subtype of gastric cancer (panels 1–2), where macrophages (green arrows in 2) and neutrophils account for the principal uPAR-expressing cells, while myofibroblasts (blue arrows in 2), at a similar location, contribute to uPAR expression to a lesser extent. In the diffuse subtype (panel 3), the uPAR-positive cells are widespread within the tumor. Despite this heterogeneity, tumor cells (black arrows) constitute a relevant fraction of the uPAR-positive cell population in both gastric cancer subtypes. Modified from [131], Copyright (2011, John Wiley & Sons, Inc.) with permission of John Wiley & Sons, Inc. (c) uPAR expression in normal (panel 1) and cancerous ovarian tissues grade II, III, and IV (panels 2, 3, and 4, respectively). Intense uPAR immunoreactivity is detected in both neoplastic epithelium and juxtatumoral stroma compared to the normal tissue and is significantly associated with the increasing tumor grade. Adapted from [144], Copyright (2011, American Association for Cancer Research, AACR, Philadelphia, Pennsylvania, USA), with permission of AACR.
Figure 5 Tissue expression of uPAR in human cancer. (a) Peroxidase and multi-immunofluorescence staining of primary colon adenocarcinoma (panels 1–2) and a corresponding liver metastasis with desmoplastic growth pattern (panels 3–4). uPAR is primarily expressed by tumor-infiltrating macrophages (black arrows in 1 and 3; blue arrows in 2 and 4) along with some few detached budding cancer cells at the invasive cancer front (white arrows in 2 and 4) (Ca: cancer, ST: stroma, DS: desmoplastic stroma; LP: liver parenchyma). Reproduced from [129], Copyright (2009, John Wiley & Sons, Inc., Hoboken, New Jersey, USA) with permission of John Wiley & Sons, Inc. (b) An analogous expression pattern is seen in the intestinal subtype of gastric cancer (panels 1–2), where macrophages (green arrows in 2) and neutrophils account for the principal uPAR-expressing cells, while myofibroblasts (blue arrows in 2), at a similar location, contribute to uPAR expression to a lesser extent. In the diffuse subtype (panel 3), the uPAR-positive cells are widespread within the tumor. Despite this heterogeneity, tumor cells (black arrows) constitute a relevant fraction of the uPAR-positive cell population in both gastric cancer subtypes. Modified from [131], Copyright (2011, John Wiley & Sons, Inc.) with permission of John Wiley & Sons, Inc. (c) uPAR expression in normal (panel 1) and cancerous ovarian tissues grade II, III, and IV (panels 2, 3, and 4, respectively). Intense uPAR immunoreactivity is detected in both neoplastic epithelium and juxtatumoral stroma compared to the normal tissue and is significantly associated with the increasing tumor grade. Adapted from [144], Copyright (2011, American Association for Cancer Research, AACR, Philadelphia, Pennsylvania, USA), with permission of AACR.CC BY · PubMed Central
Figure 6 Immunoperoxidase staining of pancreatic adenocarcinomas. Metastatic pancreatic cancers show intensive staining in stromal fibroblasts (SF, a, black arrow), while the nonmetastatic tumors (b) display moderate staining in both cancer cells (black arrow) and SF. Reproduced from [145], Copyright (2007, American Association for Cancer Research, AACR), with permission from AACR.
Figure 6 Immunoperoxidase staining of pancreatic adenocarcinomas. Metastatic pancreatic cancers show intensive staining in stromal fibroblasts (SF, a, black arrow), while the nonmetastatic tumors (b) display moderate staining in both cancer cells (black arrow) and SF. Reproduced from [145], Copyright (2007, American Association for Cancer Research, AACR), with permission from AACR.CC BY · PubMed Central
Figure 7 Putative mechanisms of action of ATF-fusions toxins: DTAT and uPA-activated PrAg-U2/FP59. (A) The uPAR-targeted cytotoxin DTAT consists of the 388-amino acid portion of DTAT, comprising the translocation (T) and N-terminal catalytic A domains of DT, fused to the amino-terminal fragment of uPA; (1A) The ATF targeting moiety mediates DTAT delivery and binding to the target uPAR-expressing cells. Cell-surface furin protease or furin-like proteases activate DT by proteolytic “nicking” of the arginine-rich bridge connecting the A and T domains, which remain linked by a disulfide bond; (2A) DTAT is then internalized in clathrin-coated pits into early endosomal vesicles (EE), where (3A) proteolytic activation of toxin molecules that escaped cleavage by cell-surface proteases may occur. (4A) Upon endosome acidification, the T domain undergoes a conformational change, inserts into the endosomal membrane, and forms a pore through which the catalytic domain A translocates to the cytoplasm, after concomitant reduction of the interdomain bridging disulfide bond. (5A) Here, the A domain inactivates the translation elongation factor 2 (eEF-2) by ADP-ribosylation, causing irreversible inhibition of protein synthesis and subsequent cell death, via apoptosis. A single molecule of toxin delivered to the cytosol is sufficient to kill the target cell [172,173,179,211]. (B) PrAg-U2/FG59 is a dual-component prodrug designed to be selectively activated by uPA on the surface of uPAR-overexpressing tumor cells. (1B) In the presence of pro-uPA and plg, receptor-bound PrAg-U2 undergoes a uPA-mediated activating cleavage and, subsequently, (2B) self-associates into a barrel-shaped heptamer, which binds the recombinant cytotoxin FP59. (3B) The resulting toxin complex is then internalized into endosomes where FP59 translocates to the cytosol in a PrAg-dependent manner (4B). (5B, 6B) As for DTAT, cell death occurs via apoptosis following protein synthesis arrest induced by EF2 inactivation catalyzed by the ADP-ribosylating domain of Pseudomonas exotoxin A (PE3) included in the fusion protein FP59 [83,212]. Abbreviations: ATR, anthrax toxin receptor. Created with BioRender.com.
Figure 7 Putative mechanisms of action of ATF-fusions toxins: DTAT and uPA-activated PrAg-U2/FP59. (A) The uPAR-targeted cytotoxin DTAT consists of the 388-amino acid portion of DTAT, comprising the translocation (T) and N-terminal catalytic A domains of DT, fused to the amino-terminal fragment of uPA; (1A) The ATF targeting moiety mediates DTAT delivery and binding to the target uPAR-expressing cells. Cell-surface furin protease or furin-like proteases activate DT by proteolytic “nicking” of the arginine-rich bridge connecting the A and T domains, which remain linked by a disulfide bond; (2A) DTAT is then internalized in clathrin-coated pits into early endosomal vesicles (EE), where (3A) proteolytic activation of toxin molecules that escaped cleavage by cell-surface proteases may occur. (4A) Upon endosome acidification, the T domain undergoes a conformational change, inserts into the endosomal membrane, and forms a pore through which the catalytic domain A translocates to the cytoplasm, after concomitant reduction of the interdomain bridging disulfide bond. (5A) Here, the A domain inactivates the translation elongation factor 2 (eEF-2) by ADP-ribosylation, causing irreversible inhibition of protein synthesis and subsequent cell death, via apoptosis. A single molecule of toxin delivered to the cytosol is sufficient to kill the target cell [172,173,179,211]. (B) PrAg-U2/FG59 is a dual-component prodrug designed to be selectively activated by uPA on the surface of uPAR-overexpressing tumor cells. (1B) In the presence of pro-uPA and plg, receptor-bound PrAg-U2 undergoes a uPA-mediated activating cleavage and, subsequently, (2B) self-associates into a barrel-shaped heptamer, which binds the recombinant cytotoxin FP59. (3B) The resulting toxin complex is then internalized into endosomes where FP59 translocates to the cytosol in a PrAg-dependent manner (4B). (5B, 6B) As for DTAT, cell death occurs via apoptosis following protein synthesis arrest induced by EF2 inactivation catalyzed by the ADP-ribosylating domain of Pseudomonas exotoxin A (PE3) included in the fusion protein FP59 [83,212]. Abbreviations: ATR, anthrax toxin receptor. Created with BioRender.com.CC BY · PubMed Central
Figure 8 Design of anti-uPAR 2G10 ADCs. The parental anti-uPAR Ab 2G10 was site-specifically modified via the SMARTag® technology, which genetically incorporates an aldehyde tag to the designated location within the antibody, yielding homogeneous ADC with defined attachment sites for the payload. One or two aldehyde tags were incorporated into the IgG heavy chain (Fc portion), resulting in two or four conjugation sites. Inhibitors of tubulin polymerization (maytansine and MMAE) covalently linked to Hydrazino-iso-Pictet-Spengler (HIPS)-functionalized cleavable or non-cleavable linkers were then conjugated to the reactive 2G10 Fc sites to generate nine site-specifically conjugated 2G10 ADCs. Created with BioRender.com.
Figure 8 Design of anti-uPAR 2G10 ADCs. The parental anti-uPAR Ab 2G10 was site-specifically modified via the SMARTag® technology, which genetically incorporates an aldehyde tag to the designated location within the antibody, yielding homogeneous ADC with defined attachment sites for the payload. One or two aldehyde tags were incorporated into the IgG heavy chain (Fc portion), resulting in two or four conjugation sites. Inhibitors of tubulin polymerization (maytansine and MMAE) covalently linked to Hydrazino-iso-Pictet-Spengler (HIPS)-functionalized cleavable or non-cleavable linkers were then conjugated to the reactive 2G10 Fc sites to generate nine site-specifically conjugated 2G10 ADCs. Created with BioRender.com.CC BY · PubMed Central
Figure 9 Composition and drug loading strategies of uPAR-targeted IONPs. (A) Most of the uPAR-targeted NPs developed up to date consist of magnetic IONPs coated with amphiphilic polymers. In addition to high stability, these polymers provide active functional carboxyl groups (COOH) on the particle surface for bioconjugation of targeting moieties, such as recombinant ATF (PDB accession number: 1URK) or other uPAR-peptide antagonists (not shown in the figure). (B) Different drug encapsulation methods can be utilized based on the physicochemical properties of the cargo, as well as drug-delivery strategies. Direct adsorption of the drug on the NP surface through non-covalent interaction increases the amount of drug encapsulated and efficiency of release into the targeted cells, compared to covalent linking [242]. Using this direct encapsulation method, doxorubicin and noscapine, two hydrophobic anticancer drugs, were physically adsorbed onto the amphiphilic polymer coating of ATF-IONPs via hydrophobic interactions (top) [236,237]. Similarly, the chemotherapeutic cisplatin (middle) was conjugated on the surface of ATF-PEG-IONPs via a coordinate bond between the positive charge of platinum (Pt+) with the negative charge of the coating polymer’s carboxylate groups (O=C-O-, Lewis base) [239]. In both cases, drug release is triggered at acidic pH values, typical of the hypoxic tumor milieu or intracellular lysosomes. Alternatively, more controlled drug release may be achieved via linker-mediated adsorption [242]. Using a pH-sensitive lysosomally cleavable peptide linker, gemcitabine, a hydrophilic drug, was covalently conjugated on the polymer surface of ATF-IONPs, achieving cathepsin B-mediated intracellular drug release (bottom) [236]. Created with BioRender.com.
Figure 9 Composition and drug loading strategies of uPAR-targeted IONPs. (A) Most of the uPAR-targeted NPs developed up to date consist of magnetic IONPs coated with amphiphilic polymers. In addition to high stability, these polymers provide active functional carboxyl groups (COOH) on the particle surface for bioconjugation of targeting moieties, such as recombinant ATF (PDB accession number: 1URK) or other uPAR-peptide antagonists (not shown in the figure). (B) Different drug encapsulation methods can be utilized based on the physicochemical properties of the cargo, as well as drug-delivery strategies. Direct adsorption of the drug on the NP surface through non-covalent interaction increases the amount of drug encapsulated and efficiency of release into the targeted cells, compared to covalent linking [242]. Using this direct encapsulation method, doxorubicin and noscapine, two hydrophobic anticancer drugs, were physically adsorbed onto the amphiphilic polymer coating of ATF-IONPs via hydrophobic interactions (top) [236,237]. Similarly, the chemotherapeutic cisplatin (middle) was conjugated on the surface of ATF-PEG-IONPs via a coordinate bond between the positive charge of platinum (Pt+) with the negative charge of the coating polymer’s carboxylate groups (O=C-O-, Lewis base) [239]. In both cases, drug release is triggered at acidic pH values, typical of the hypoxic tumor milieu or intracellular lysosomes. Alternatively, more controlled drug release may be achieved via linker-mediated adsorption [242]. Using a pH-sensitive lysosomally cleavable peptide linker, gemcitabine, a hydrophilic drug, was covalently conjugated on the polymer surface of ATF-IONPs, achieving cathepsin B-mediated intracellular drug release (bottom) [236]. Created with BioRender.com.CC BY · PubMed Central
Figure 10 Theranostic application of uPAR-targeted IONPs. (A) Schematic representation of optical-MRI imaging trackable Dox-loaded ATF-IONPs [238,239]. The near infrared (NIR)-830 dye was conjugated to the targeting moiety, ATF (PDB accession number: 1URK), for optical imaging. Incorporating multiple imaging modalities on the same nanocarrier system provides a valuable way to validate and track the IONPs and, therefore, better investigate tumor targeting and biodistribution of nanoconstructs in animal models. (B) In vivo, these theranostic-targeted nanoparticles allowed for real-time and non-invasive MRI and NIR-optical imaging assessment of targeted drug delivery and distribution, tumor response to treatment, and detection of small drug-resistant residual tumors in orthotopic human pancreatic (shown in 2, reproduced from [238] under the Creative Commons Attribution (CC BY-NC) License) and breast cancer xenograft models, thus supporting the translation potential of these agents for image-guided therapy of cancer. Created with BioRender.com.
Figure 10 Theranostic application of uPAR-targeted IONPs. (A) Schematic representation of optical-MRI imaging trackable Dox-loaded ATF-IONPs [238,239]. The near infrared (NIR)-830 dye was conjugated to the targeting moiety, ATF (PDB accession number: 1URK), for optical imaging. Incorporating multiple imaging modalities on the same nanocarrier system provides a valuable way to validate and track the IONPs and, therefore, better investigate tumor targeting and biodistribution of nanoconstructs in animal models. (B) In vivo, these theranostic-targeted nanoparticles allowed for real-time and non-invasive MRI and NIR-optical imaging assessment of targeted drug delivery and distribution, tumor response to treatment, and detection of small drug-resistant residual tumors in orthotopic human pancreatic (shown in 2, reproduced from [238] under the Creative Commons Attribution (CC BY-NC) License) and breast cancer xenograft models, thus supporting the translation potential of these agents for image-guided therapy of cancer. Created with BioRender.com.CC BY · PubMed Central
Figure 12 Schematic structure and in vivo antitumor effects of dual stromal and tumor-targeted oncolytic MV-CD46-muPA. (a) MV-CD46-muPA was generated by displaying the ATF of murine uPA as a C-terminal extension of unmodified MV-H glycoprotein (highlighted in the MV genome and structure) to allow simultaneous binding and infection of human cancer cells via the MV receptor CD46 and murine cells via murine uPAR. (b) The contribution of stromal and tumor targeting on MV’s overall anticancer efficacy in vivo was evaluated in human colon cancer xenograft mouse models, comprising CD46+ human cancer cells and uPAR+ murine stromal cells [264]. Systemically administered oncolytic MV-CD46-muPA led to improved antitumor effects and outcomes compared to vehicle or CD46 only targeted MVs. These effects resulted from the mouse uPAR-mediated stromal targeting ability of MV-CD46-muPA, lacking in the monospecific counterpart, and were associated with increased tumor viral penetration (via stromal-tumor viral transfer), apoptosis, decrease in murine stromal CAFs and endothelial cells (ECs), and viral-induced modulation of tumor-stroma interactions [264]. Created with BioRender.com.
Figure 12 Schematic structure and in vivo antitumor effects of dual stromal and tumor-targeted oncolytic MV-CD46-muPA. (a) MV-CD46-muPA was generated by displaying the ATF of murine uPA as a C-terminal extension of unmodified MV-H glycoprotein (highlighted in the MV genome and structure) to allow simultaneous binding and infection of human cancer cells via the MV receptor CD46 and murine cells via murine uPAR. (b) The contribution of stromal and tumor targeting on MV’s overall anticancer efficacy in vivo was evaluated in human colon cancer xenograft mouse models, comprising CD46+ human cancer cells and uPAR+ murine stromal cells [264]. Systemically administered oncolytic MV-CD46-muPA led to improved antitumor effects and outcomes compared to vehicle or CD46 only targeted MVs. These effects resulted from the mouse uPAR-mediated stromal targeting ability of MV-CD46-muPA, lacking in the monospecific counterpart, and were associated with increased tumor viral penetration (via stromal-tumor viral transfer), apoptosis, decrease in murine stromal CAFs and endothelial cells (ECs), and viral-induced modulation of tumor-stroma interactions [264]. Created with BioRender.com.CC BY · PubMed Central

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  1. Sampson, J.H. (2017). Chapter 10-Immunotoxin Therapy for Brain Tumors. Translational Immunotherapy of Brain Tumors, Academic Press.

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Metadata

Authors
Metrangolo, V., Ploug, M., & Engelholm, L. H.
Affiliations
University of Copenhagen (DK)
Corresponding author
Engelholm, Lars H.
ORCID
Venue
Cancers
Publisher
MDPI AG
Published
2021-10-27
Volume
13
Issue
21
Pages
5376
ISSN
2072-6694 (ISSN-L)
DOI
10.3390/cancers13215376
PMC
PMC8582577
OpenAlex
W3209572831
Cited by
44
References
267
Citations by year
2026: 4 · 2025: 11 · 2024: 8 · 2023: 11 · 2022: 10
FWCI
0.87
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancers (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Research at the University of Copenhagen (University of Copenhagen) (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska Curie (801481)
  • Danish Cancer Society (R231-A13832)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Protease and Inhibitor Mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biochemistry › Peptidase Inhibition and Analysis
  • Health Sciences › Medicine › Hematology › Blood Coagulation and Thrombosis Mechanisms
Keywords
urokinase-type plasminogen activator receptor, uPAR, targeted cancer therapy, precision oncology, plasminogen activation, extracellular matrix degradation, metastasis, cell migration, off-target toxicity, prognostic factors

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