Targeting uPAR with Antagonistic Recombinant Human Antibodies in Aggressive Breast Cancer

Pharmaceutical Chemistry, University of California, San Francisco.
Cancer Research (Impact Factor: 9.33). 02/2013; 73(7). DOI: 10.1158/0008-5472.CAN-12-3526
Source: PubMed


Components of the plasminogen activation system (PAS) which are overexpressed in aggressive breast cancer subtypes offer appealing targets for development of new diagnostics and therapeutics. By comparing gene expression data in patient populations and cultured cell lines, we identified elevated levels of the urokinase plasminogen activation receptor (uPAR, PLAUR) in highly aggressive breast cancer subtypes and cell lines. Recombinant human anti-uPAR antagonistic antibodies exhibited potent binding in vitro to the surface of cancer cells expressing uPAR. In vivo these antibodies detected uPAR expression in triple negative breast cancer (TNBC) tumor xenografts using near infrared (NIR) imaging and (111)In single-photon emission computed tomography (SPECT). Antibody-based uPAR imaging probes accurately detected small disseminated lesions in a tumor metastasis model, complementing the current clinical imaging standard (18)F-fluorodeoxyglucose (FDG) at detecting non-glucose-avid metastatic lesions. A monotherapy study using the antagonistic antibodies resulted in a significant decrease in tumor growth in a TNBC xenograft model. Additionally, a radioimmunotherapy (RIT) study, using the anti-uPAR antibodies conjugated to the therapeutic radioisotope (177)Lu, found that they were effective at reducing tumor burden in vivo. Taken together, our results offer a preclinical proof-of-concept for uPAR targeting as a strategy for breast cancer diagnosis and therapy using this novel human antibody technology.

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    • "To address the possible relevance of ligand-independent b 1 signalling in cells expressing (patho) physiological levels of uPAR, we also analysed the effect of b 1 inhibition in the triple negative MDA-MB-231 breast carcinoma cell line seeded on different substrates (Supplementary Fig S3A). These cells express high levels of uPAR, and their growth in xenograft models has been documented to be uPAR-and VN-dependent (Kunigal et al, 2007; Lebeau et al, 2013; Pirazzoli et al, 2013). These cells do not adhere to VN RAD independently of the addition of exogenous uPA (data not show), but do adhere and spread on an immobilised anti-uPAR antibody allowing us to isolate and analyse uPAR dependent cell adhesion "
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    ABSTRACT: The urokinase-type plasminogen activator receptor (uPAR) is a non-integrin vitronectin (VN) cell adhesion receptor linked to the plasma membrane by a glycolipid anchor. Through structure-function analyses of uPAR, VN and integrins, we document that uPAR-mediated cell adhesion to VN triggers a novel type of integrin signalling that is independent of integrin-matrix engagement. The signalling is fully active on VN mutants deficient in integrin binding site and is also efficiently transduced by integrins deficient in ligand binding. Although integrin ligation is dispensable, signalling is crucially dependent upon an active conformation of the integrin and its association with intracellular adaptors such as talin. This non-canonical integrin signalling is not restricted to uPAR as it poses no structural constraints to the receptor mediating cell attachment. In contrast to canonical integrin signalling, where integrins form direct mechanical links between the ECM and the cytoskeleton, the molecular mechanism enabling the crosstalk between non-integrin adhesion receptors and integrins is dependent upon membrane tension. This suggests that for this type of signalling, the membrane represents a critical component of the molecular clutch.
    The EMBO Journal 11/2014; · 10.43 Impact Factor
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    • "Once active, plasmin can activate a number of other proteases resulting in degradation of the extra-cellular matrix, cancer growth and metastasis. Over-expression of the plasminogen activation axis has been found in a number of cancers and is common to breast cancer with aggressive phenotypes 11, 13. In healthy mammary tissue, uPAR is virtually non-existent and its expression appears to be restricted only to diseased tissue 14, 15. "
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    ABSTRACT: Subtype-targeted therapies can have a dramatic impact on improving the quality and quantity of life for women suffering from breast cancer. Despite an initial therapeutic response, cancer recurrence and acquired drug-resistance are commonplace. Non-invasive imaging probes that identify drug-resistant lesions are urgently needed to aid in the development of novel drugs and the effective utilization of established therapies for breast cancer. The protease receptor urokinase plasminogen activator receptor (uPAR) is a target that can be exploited for non-invasive imaging. The expression of uPAR has been associated with phenotypically aggressive breast cancer and acquired drug-resistance. Acquired drug-resistance was modeled in cell lines from two different breast cancer subtypes, the uPAR negative luminal A subtype and the uPAR positive triple negative subtype cell line MDA-MB-231. MCF-7 cells, cultured to be resistant to tamoxifen (MCF-7 TamR), were found to significantly over-express uPAR compared to the parental cell line. uPAR expression was maintained when resistance was modeled in triple-negative breast cancer by generating doxorubicin and paclitaxel resistant MDA-MB-231 cells (MDA-MB-231 DoxR and MDA-MB-231 TaxR). Using the antagonistic uPAR antibody 2G10, uPAR was imaged in vivo by near-infrared (NIR) optical imaging and (111)In-single photon emission computed tomography (SPECT). Tumor uptake of the (111)In-SPECT probe was high in the three drug-resistant xenografts (> 46 %ID/g) and minimal in uPAR negative xenografts at 72 hours post-injection. This preclinical study demonstrates that uPAR can be targeted for imaging breast cancer models of acquired resistance leading to potential clinical applications.
    Theranostics 01/2014; 4(3):267-79. DOI:10.7150/thno.7323 · 8.02 Impact Factor
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    • "About 60 to 90% of invasive breast cancer tissues and 54% of the early stage of ductal carcinoma in situ (DCIS) expressed a high level of uPAR while normal breast tissues lack the expression 32, 39. Although uPAR may be a potential target for all breast cancer subtypes, a recent study showed that triple negative breast cancer tissues expressed a higher level of uPAR compared to luminal breast cancer subtype 40. Additionally, 86 to 90% of pancreatic cancer tissues had a high level of uPAR, but its expression was not found in the normal pancreas or pancreatic tissues with chronic pancreatitis 35, 41. "
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    ABSTRACT: Complete removal of tumors by surgery is the most important prognostic factor for cancer patients with the early stage cancers. The ability to identify invasive tumor edges of the primary tumor, locally invaded small tumor lesions, and drug resistant residual tumors following neoadjuvant therapy during surgery should significantly reduce the incidence of local tumor recurrence and improve survival of cancer patients. In this study, we report that urokinase plasminogen activator (uPA) and its receptor (uPAR) are the ligand/cell surface target pair for the development of targeted optical imaging probes for enhancing imaging contrasts in the tumor border. Recombinant peptides of the amino terminal fragment (ATF) of the receptor binding domain of uPA were labeled with near infrared fluorescence (NIR) dye molecules either as peptide-imaging or peptide-conjugated nanoparticle imaging probes. Systemic delivery of the uPAR-targeted imaging probes in mice bearing orthotopic human breast or pancreatic tumor xenografts or mouse mammary tumors led to the accumulation of the probes in the tumor and stromal cells, resulting in strong signals for optical imaging of tumors and identification of tumor margins. Histological analysis showed that a high level of uPAR-targeted nanoparticles was present in the tumor edge or active tumor stroma immediately adjacent to the tumor cells. Furthermore, following targeted therapy using uPAR-targeted theranostic nanoparticles, residual tumors were detectable by optical imaging through the imaging contrasts produced by NIR-dye-labeled theranostic nanoparticles in drug resistant tumor cells. Therefore, results of our study support the potential of the development of uPAR-targeted imaging and theranostic agents for image-guided surgery.
    Theranostics 12/2013; 4(1):106-18. DOI:10.7150/thno.7409 · 8.02 Impact Factor
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