An antibody molecule binding human upar

Abstract

The present invention relates to an antibody molecule or an antigen binding fragment targeting human urokinase plasminogen activator receptor (uPAR) and an antibody-conjugate comprising an antibody molecule or antigen binding fragment according to any aspect of the invention; and a coupling moiety. The invention further relates to the uses of these antibody molecules, antigen binding fragments or antibody-conjugates.

Description

[0001] AN ANTIBODY MOLECULE

[0002] The present invention relates to an antibody molecule or an antigen binding fragment targeting human urokinase plasminogen activator receptor (uPAR) and an antibodyconjugate comprising an antibody molecule or antigen binding fragment according to any aspect of the invention; and a coupling moiety. The invention further relates to the uses of these antibody molecules, antigen binding fragments or antibody-conjugates.

[0003] Thirty-five years after the first FDA approval, monoclonal antibodies (mAbs) are established modalities in oncology due to their unparalleled specificity and affinity for cancer related targets. There are currently over 870 antibodies in clinical development and in 2021 the 100th antibody has been approved by the FDA [1]. However, achieving effective intra-tumoral antibody concentrations remains a challenge due to their large molecular size (150 kilodalton (kDa)) which often results in heterogenous tissue penetration, poor diffusion and limited efficacy [2-4]. As such, there has been a growing call for antibody variants that overcome the limitations of full-sized antibodies.

[0004] Nature’s own alternative, single-domain antibodies (sdAb), are found in, amongst others, camelids and cartilaginous fish [5]. Where the variable domains of conventional antibodies rely on the synergy of heavy- and light-chain domains for antigen recognition, sdAbs possess only a single dedicated heavy chain domain, termed VHH, that, with a molecular weight of 15 kDa, is the smallest antibody-based fragment known to date. [6- 8]. VHH fragments profit from the desirable pharmacokinetic properties of small molecule drugs while retaining the binding specificity of conventional antibodies [9]. Therefore, as a next-generation moiety, VHHs are expected to play an important role in future targeted diagnostics and therapeutics [10, 11]. In 2019, the first VHH, caplacizumab, targeting van Willebrand factor, was approved by the FDA and EMA, and VHHs targeting, amongst others, HER2, PD-L1 and CEACAM6 can be expected to undergo approval as they are progressing the clinical trial pipeline [12, 13].

[0005] Ideally, VHHs are developed against targets whose expression is paramount for disease initiation, maintenance, and progression. Regarding cancer, these often consider pathways involved in the hallmarks of cancer

[0014] . Current insights place the urokinase plasminogen activator receptor (uPAR) as a central component in a potent proteolytic and cell-signaling activation cascade orchestrating multiple hallmarks of cancer: from resisting cell death to activating invasion and metastasis, and from avoiding immune destruction to sustaining proliferative signalling [15-17]. uPAR expression is upregulated at the invasive front of nearly every solid tumour, making it a well-established diagnostic, prognostic, and therapeutic target [18, 19]. A state-of-the-art imaging technique that could particularly exploit this ubiquitous expression at tumour margins is fluorescence guided surgery (FGS), which aims to assist surgeons in margin assessment using near-infrared (NIR) fluorescent labelled tumour targeted tracers

[0020] . The inventors have previously targeted uPAR for molecular imaging with a humanized mAb in preclinical in vivo models, whilst others have demonstrated the same in early clinical trials using a urokinase-based peptide [21 , 22]. However, both tracing vehicles show compound-related disadvantages, as the former is limited by its molecular size and the latter, as an urokinase competitor, depends on the presence of unoccupied uPAR [23, 24].

[0006] Brief summary of the disclosure

[0007] For the present invention, the inventors studied the selection and characterization of a novel uPAR targeting VHH, followed by subsequent humanization and in vivo validation. In addition, translational and clinical potential was demonstrated by NIR-imaging in an orthotopic colorectal cancer and a close-to-patient pancreatic ductal adenocarcinoma mouse model. The aim was to introduce a novel uPAR targeting tracer that circumvents the theoretical drawbacks of the previously presented humanized antibody and peptide for tumour imaging.

[0008] The inventors found that targeting uPAR using a novel epitope results in superior outcomes, with the most important result being that targeting this epitope results in more tumour uptake than current uPAR targeting agents. In combination with a payload delivery strategy, targeting this epitope can maximize tumour targeting and either result in better tumour visualization when using contrast payloads (such as but not limited to fluorophores, radio-active isotopes, gadolinium or micro-bubbles) or increased tumour destruction when using therapeutics / toxic payloads (such as, but not limited to, photodynamic fluorophore, radio-active isotope, nano-particle, any toxic drugs). In one aspect, the invention provides an antibody molecule, or an antigen binding fragment thereof, that binds to the amino acid sequence of SEQ ID NO. 1 of human uPAR.

[0009] Suitably, the fragment may be selected from the group consisting of a monovalent antibody, a Fab, a Fab’, a F(ab’)2, a single chain variable fragment (scFv), an antibody domain, a nanobody (VHH), a minibody and an scFv-FC; and variations thereof.

[0010] Suitably, the fragment may be a nanobody (VHH).

[0011] Suitably, the antibody molecule or antigen binding fragment may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO. 2, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 3 and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO. 4.

[0012] Suitably, the antibody molecule or antigen binding fragment may comprise the amino acid sequence of SEQ ID NO. 10.

[0013] Suitably, the antibody molecule or antigen binding fragment may be a nanobody (VHH) comprising the amino acid sequence of SEQ ID NO. 10, wherein the nanobody binds to the amino acid sequence of SEQ ID NO: 1 of human uPAR.

[0014] Suitably, the antibody molecule or antigen binding fragment may be humanised, optimised, and / or de-immunized.

[0015] In another aspect, the invention provides an antibody-conjugate which comprises: (a) an antibody molecule or antigen binding fragment according to any aspect of the invention; and (b) a coupling moiety coupled to the antibody molecule or antigen binding fragment.

[0016] Suitably, the coupling moiety may be a biologically active moiety and / or a diagnostic moiety.

[0017] Suitably, the coupling moiety may be selected from the group consisting of a detectable label, a toxin, a cytokine, a radionuclide, an enzyme, a photosensitizer and a combination thereof.

[0018] Suitably, the coupling moiety may comprise a photodynamic and / or a photoimmuno sensitizer. Suitably, the coupling moiety may comprise contrast payloads, optionally fluorophores, radio-active isotopes, gadolinium or micro-bubbles.

[0019] Suitably, the coupling moiety may comprise the dye IRDye800CW or variants of the dye ICG.

[0020] Suitably, the coupling moiety may comprise a drug.

[0021] Also provided herein is a nucleic acid encoding an antibody molecule or antigen binding fragment of any aspect of the invention.

[0022] Further provided herein is an expression vector comprising a nucleic acid according to any aspect of the invention.

[0023] Also provided herein is a host cell comprising an expression vector according to any aspect of the invention.

[0024] Another aspect of the invention provides a pharmaceutical composition comprising an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell according to any aspect of the invention; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0025] In a further aspect, the invention provides a method of producing an antibody molecule, antigen binding fragment or an antibody-conjugate according to any aspect of the invention, the method comprising culturing a host cell according to any aspect of the invention under conditions suitable for gene expression.

[0026] The invention also provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in therapy.

[0027] Further, the invention provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in treating diseases where extracellular matrix remodelling plays a pathophysiological role; optionally wherein the diseases are (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer. Suitably, the therapy or treatment may comprise administering therapeutics or toxic payloads, optionally a photo-dynamic fluorophore, radio-active isotope, nano-particle, or toxic drug, conjugated to an antibody molecule or antigen binding fragment according to any aspect of the invention.

[0028] Suitably, the therapeutic or drug may be tamoxifen, gefitinib or vemurafenib.

[0029] Suitably, the cancer may be selected from a group consisting of locally aggressive tumours; optionally wherein the cancer is selected from the group consisting of: pancreatic cancer, colorectal cancer, oesophageal cancer, head-and-neck cancer, breast cancer, lung cancer, kidney cancer (renal cell carcinoma), bladder cancer, prostate cancer, ovarium cancer, endometrium cancer, liver cancer, and melanoma.

[0030] Suitably, the pancreatic cancer may be pancreatic ductal adenocarcinoma.

[0031] Suitably, the treatment may also target tumour-associated stromal cells, as well as malignant cells in the cancer.

[0032] Suitably, the treatment may comprise treatment stratification in oncology wherein patients with high uPAR uptake on radioactive imaging are offered more aggressive treatment regimens and patients with low uptake are spared these modalities.

[0033] Suitably, the treatment may be administered to patients who have failed initial therapy or developed resistance to initial therapies, or in combination with initial therapies in order to prevent the development of therapy resistance.

[0034] Suitably, the treatment may comprise last-line therapy in aggressive arthritis using antibody-drug conjugates.

[0035] Suitably, the treatment may comprise photodynamic or photoimmuno therapy.

[0036] In another aspect, the invention provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in diagnosis.

[0037] Suitably, the diagnosis may comprise the identification of atherosclerotic plaques that are at risk of rupture using a PET tracer. Suitably, the diagnosis may comprise monitoring aggressive arthritis using radioactive tracers.

[0038] The invention further provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in imaging.

[0039] Additionally, the invention provides a method of imaging a subject, comprising administering an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention.

[0040] Suitably, the imaging may be image-guided surgery, optionally fluorescence guided surgery (FGS), and / or optionally uses near-infrared intraoperative imaging.

[0041] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0042] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0043] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0044] Various aspects of the invention are described in further detail below.

[0045] Brief description of the Figures

[0046] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which: Figure 1. Characterization of uPAR binding VHHs. (A) Affinity of Nb1 - Nb6 to uPAR as determined by ELISA. (B) Binding of Nb1 - Nb6 to HEK cells transfected with an empty vector (left; HEK EV) or wild type uPAR (right; HEK uPAR WT) measured by flow cytometry. (C) Binding of Nb1 - Nb6 to HEK cells transfected with uPAR domain 2-3 isotype (HEK uPAR D2-3) determined by flow cytometry. (D) Change in MFI of Nb1 - Nb6 after addition of the amino-terminal fragment (ATF) of uPA on urokinase-devoid HT- 29 human colon cancer cells. ATN-617 was used as positive control as it is a urokinase / ATF competitor.

[0047] Figure 2. Flow cytometry histograms showing that Nb2 does not bind mouse uPAR on mouse uPAR positive 4T1 breast (left) and AT84 head-and-neck (right) cancer cells.

[0048] Figure 3. Flow cytometry histograms showing no difference in uPAR binding between Nb2 and humanized Nb2 (huNb2) on HEK cells transfected with an empty vector (HEK EV; left) and with uPAR domain 2-3 isotype (HEK uPAR D2-D3; right).

[0049] Figure 4. Scatterplot showing that across multiple runs the KD between huNb2 and huNb2-IRDye800CW did not differ significantly as determined by surface plasmon resonance.

[0050] Figure 5. Flow cytometry histograms showing that the VHH J3Rsc does not bind HEK cells transfected with an empty vector (HEK EV; left), with human wild type uPAR (HEK uPAR WT; middle), and with human uPAR domain 2-3 isotype (HEK uPAR D2-3; right).

[0051] Figure 6. Characterization of fluorescent huNb2-IRDye800CW (A) Fluorescent signal on SDS-page gel of varying concentrations of huNB2-IRDye800CW at 15 kDa. (B) Sensogram of huNb2-IRDye800CW binding to recombinant uPAR. (C) Graph showing the correlation between uPAR copy number, ranging from below 1000 uPAR copies for MIA PaCa-2 until nearly 30,000 for NCI-H1299, and fluorescence from huNb2- IRDye800CW measured using an NIR flow cytometer.

[0052] Figure 7. (A) Representative NIR-images of subcutaneous HT-29 bearing mice one hour after 1.0 nmol huNb2-IRDye800CW or 1.0 nmol J3Rsc-IRDye800CW administration. NIR-images were taken with the Pearl Trilogy System. Tumours are indicated by the white arrows and kidneys by the asterisk. (B) in vivo tumour MFI’s and (C) TBR’s determined using the Pearl Trilogy after intravenous injection of 0.1 , 0.25, 0.5 and 1.0 nmol tracer.

[0053] Figure 8. Ex vivo biodistribution of 1.0 nmol huNb2-IRDye800CW one hour after intravenous injection in an orthotopic HT-29_luc2 colorectal cancer mouse model showing that the majority of the fluorescence accumulates in the kidneys, liver and tumours.

[0054] Figure 9. Representative images of bioluminescence and NIR-imaging using the Pearl Trilogy and clinical Artemis Hand Held Imaging System in an orthotopic HT-29-luc2 colorectal model (A) one hour after 1.0 nmol huNB2-IRDye800CW administration and (B) 72 hours after 1.0 nmol humAb-IRDye800CW administration. Fluorescence is matched in the images.

[0055] Figure 10. (A) Immunohistochemical staining for uPAR on FNA005 pancreatic cancer biopsy tissue using two mAb's against different uPAR domains. (B) Representative NIR images taken with the Pearl Trilogy one hour after administration of 2.0 nmol huNb2- IRDye800CW. Tumours are indicated by the white arrows. Line graphs showing the development of (C) TBRs and (D) MFIs after injection of different dosis of huNb2- IRDye800CW or J3Rsc-IRDye800CW. (D) post-mortem H&E and fluorescence scanning (Odyssey) 120 minutes after administration of huNb2-IRDye800CW or J3Rsc- IRDye800CW showing fluorescence localization towards tumours cells for the former but not for the latter. Black bar represents 500 micrometers and white bar represents 50 micrometers.

[0056] Figure 11. Hematoxylin & eosin staining and NIR fluorescence scanning of FNA005 close-to-patient pancreatic ductal adenocarcinoma tumours with a necrotic core showing that fluorescence specifically accumulates in the tumour cell rich areas and not in the necrotic core 2 hours after 1.0 nmol huNb2-IRDye800CW administration (top row). In comparison, fluorescence remains low in both the tumour cell rich areas and necrotic core 2 hours after 1.0 nmol J3Rsc-IRDye800CW administration (bottom row). Black bar represent 500 pm. Fluorescence is matched between the two NIR images.

[0057] Figure 12. Hematoxylin & eosin, conjugate control immunohistochemical staining and anti-human uPAR immunohistochemical staining showing tumour cell specific expression uPAR expression of the FNA005 close-to-patient pancreatic ductal adenocarcinoma tumours. The black bar represents 1000 pm and the white bar represents 200 pm.

[0058] Figure 13. Live-cell imaging of cells incubated with Nb2-800CW or the control VHH J3RSC-800CW. The uPAR-positive cell line is HEK-CL7 and AT-84 is a mouse cell line lacking human uPAR expression. Cells were incubated for 1 hour in the dark with 100 nM of the probe and imaged in PBS after washing. A fluorescence signal of 800CW was detected in the HEK-CL7 cell line, but not in the AT-84 cell line, when cells were incubated with Nb2-800CW. The signal was specifically detected at the cell membrane of cells of the HEK-CL7 cell line. No fluorescence was detected in either cell line when cells were incubated with the control VHH J3RSC-800CW or were unstained

[0059] Figure 14. In silico multimer prediction of Nb2, uPA, and uPAR. The molecular interaction between Nb2 and uPAR, both in the presence and absence of uPA, was computationally predicted using AlphaFold2 (AF2). The highest-quality predicted structural model was further refined and visualized with ICM Pro. (a) Model prediction of uPAR in complex with Nb2. The CDRs of Nb2 mainly interacted with Dll of uPAR, more specifically with Meth103 to His110 and Asn177 to Asn190. (b) Model prediction of uPAR in complex with uPA and Nb2. Nb2 was still interacting with DII-DIII of uPAR, particularly interacting with Arg25 to Glu68 in Dll and Thr85 to Pro151 . Nb2 did not appear to compete with uPA for binding sites on uPAR, nor did it interact with identical regions of uPAR as those engaged by uPA.

[0060] AlphaFold is an artificial intelligence-based tool that predicts the three-dimensional structure of a protein and its interactions with other proteins, using only the amino acid sequence and structural information from more than 200 million known protein conformations. Therefore, the molecular interaction between Nb2 and uPAR, both in the presence and absence of uPA, was computationally predicted using AlphaFold2 (AF2). The highest-quality predicted structural model was further refined and visualized with ICM Pro. In models where Nb2 and uPAR were simulated in the absence of uPA, the complementarity-determining regions (CDRs) of Nb2 mainly interacted with Dll of uPAR, mainly interacting with Meth103 to His110 and Asn177 to Asn190 (Figure 14a). In model predictions where uPA was added to the simulation, Nb2 was still interacting with DII-DIII of uPAR, particularly interacting with Arg25 to Glu68 in Dll and Thr85 to Pro151. All three CDRs of Nb2 were in contact with Dll-Dll I of uPAR (Figure 14b). In both prediction models, Nb2 did not appear to compete with uPA for binding sites on uPAR, nor did it interact with identical regions of uPAR as those engaged by uPA. These model predictions confirmed the described flow cytometry experiment results.

[0061] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0062] Various aspects of the invention are described in further detail below.

[0063] Detailed Description

[0064] In one aspect, the invention provides an antibody molecule, or an antigen binding fragment thereof, that binds to the amino acid sequence of SEQ ID NO. 1 of human uPAR.

[0065] The term “antibody molecule” or “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0066] In a natural antibody molecule, there are two heavy chains and two light chains. Each heavy chain and each light chain has at its N-terminal end a variable domain. Each variable domain is composed of four framework regions (FRs) alternating with three complementarity determining regions (CDRs). The residues in the variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et aL, 1987, in Sequences of proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter "Kabat et al. (supra)"). This numbering system is used in the present specification except where otherwise indicated. The term “binds to" refers to the process by which an antibody molecule or an antigen binding fragment interacts with and attaches to its target. The antibody molecule or antigen binding fragment thereof is capable of binding the amino acid sequence of SEQ ID NO. 1 of human uPAR with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting uPAR. An antibody that “binds to uPAR" is intended to refer to an antibody that binds to human uPAR with a dissociation constant (KD) of about 5 x 10-7 M or less, about 1 x 10-7 M or less, about 5 x 10-8 M or less, about 1 x 10-8 M or less, about 5 x 10-9 M or less, about 1 x 10-9 M or less, about 5 x 10-10 M or less, about 1 x 10-10 M or less, about 5 x 10-11 M or less, or about 1 x 10-11 M or less.

[0067] An “antibody that competes for binding" or “antibody that cross-competes for binding” with a reference antibody for binding to an antigen, e.g., uPAR, refers to an antibody that blocks binding of the reference antibody to the antigen (e.g., uPAR) in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to the antigen (e.g., uPAR) in a competition assay by 50% or more. An exemplary competition assay is described in “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0068] The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen (e.g., a uPAR polypeptide).”

[0069] The antibody molecules, or antigen binding fragments thereof according to any aspect of the invention bind a novel epitope in human uPAR (the amino acid sequence of SEQ ID NO. 1 of human uPAR). This epitope is not uPA dependent, isotype dependent or integrin dependent. The antibody molecules, or antigen binding fragments thereof according to any aspect of the invention binds uPAR independent of the presence of uPA. uPAR (urokinase-type plasminogen activator receptor), also known as CD87, is a glycosylphosphatidylinositol-anchored protein. uPAR is cysteine-rich and consists of three tandem LU domains, which bind urokinase-type plasminogen activator (uPA). (Kessler et aL, J. Neurochem. (2017); 142: 7-18; Llinas et aL, EMBOJ. (2005);24(9): 1655-63; Huai et al., Science (2006);311 (5761):656-9; Chelsea et al., Human Genomics (2016); 10:10). uPAR also interacts with several other proteins, including vitronectin, the uPAR associated protein (uPARAP) and the integrin family of membrane proteins. uPAR is associated with tumour growth or metastasis in various different types of cancers, including breast cancer (including triple negative breast cancer), endometrial cancer, ovarian cancer, colon cancer, rectal cancer, lung cancer, stomach cancer, prostate cancer, renal cancer, pancreatic cancer, rectal cancer, cervical cancer, head and neck cancer, liver cancer, gastric cancer, urothelial cancer, melanoma, brain cancer (including glioblastoma multiforme), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and acute myeloid leukemia (AML). It also plays a role in aging, such as its association with senescence-related diseases associated with aging. It can also regulate immune response and cell-matrix interaction and promote tumour cell proliferation and emergence from dormancy. uPAR is induced during the process of cellular senescence, which can be elicited by certain cancer agents and accumulates in a range of age-related and tissue damage pathologies (LIST). Elimination of senescent cells can improve the response of therapy, and ameliorate symptoms of the tissue damage pathologies including fibrosis. uPAR can be cleaved at its base resulting in soluble uPAR (suPAR) or between domain 1 and 2 resulting in a released domain 1 (suPAR D1) and cell-bound domain 2 and 3 (uPAR D2-3). The latter can be cleaved at its based resulting in soluble domain 2 and 3 (suPAR D2-3). Soluble urokinase plasminogen activator receptor (suPAR) is found upregulated in a number of pathologies noted above, also in chronic obstructive pulmonary disease, asthma, liver failure, heart failure, cardiovascular disease, and rheumatoid arthritis. (Desmedt et al., Crit. Rev. Clin. Lab. Sei. (2017);54(2): 117-133). uPAR is found to be highly expressed on senescent cells. (Wagner et aL, Nature (2020);583 (7814): 37-38, Amor et aL, Nature (2020 Jul);583(7814): 127- 132). Thus, uPAR (e.g., suPAR) can be used as a disease stage biomarker.

[0070] Mechanistically, uPAR plays a multifaceted and pivotal role in almost any process where migration of cells and tissue-remodelling is involved, such as inflammation, wound-healing and diseases such as arthritis, atherosclerosis and cancer. It achieves these effector functions by localizing urokinase’s proteolytic activity towards the cellmembrane, and influencing intracellular processes through its interaction with a broad range of cell membrane proteins, like vitronectin and various integrins. In healthy individuals, tissue-remodelling is kept at a minimal and as such uPAR expression is practically absent in healthy tissue. On the other hand, in disease processes where tissue-remodelling is extensive cells over-express uPAR. This difference in receptor expression is attractive for both disease imaging and therapy. uPAR is an attractive target due to its universal nature and expression pattern. Unlike targets such as Her2, CEA or EPCAM which are overexpressed in specific tumour types (i.e. breast, colorectal and gastro-intestinal respectively), uPAR is practically overexpressed in almost every solid tumour as it plays a fundamental role in disease progression. As such, a successful uPAR targeting agent has wide implications for a large group of patients. In addition, its expression pattern is not limited to the malignant cells but also found on tumour-associated stromal cells. In tumours, such as pancreas cancer, where the stromal component often inhibits treatment success, treatment success could be influenced by also targeting this secondary compartment. Due to uPAR’s expression pattern both the primary malignant cells and the stromal compartment can be treated with uPAR targeting agents.

[0071] Many oncogenic signalling pathways and tumour microenvironmental conditions such as hypoxia can activate transcription factors that in turn regulate uPAR. uPAR can regulate proteolysis by associating with the outer layer of the plasma membrane by a glycosyl phosphatidylinositol (GPI) anchor, but it can also be secreted or shed from the cell surface. (Harvey et aL, Nat. Rev. Mol. Cell Biol (2010); 11 , 23-36). uPAR expression directly correlates with the invasive potential of endometrial carcinomas. (Foca et aL, Gynecol. Oncol. (2000);79(2):244-50). uPAR is implicated in several hematological malignancies, particularly acute leukemia and multiple myeloma. (Hata et aL, Blood (1993); 81 : 3357-3364; MC Bene et aL, Leukemia (2004); 18, 394-400). uPAR is reported to be associated with poor prognosis in breast cancer patients. (Bo et aL, OncoL Rep. (2005); 14(1): 105-12; Foekens et aL, Cancer Res. (2000); 60(3): 636-43).

[0072] In certain embodiments, uPAR is human uPAR comprising or consisting of the amino acid sequence with a UniProt Reference No: Q03405-1 (SEQ ID NO. 9) or a fragment thereof.

[0073] In certain embodiments, the uPAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, at least about 100% identical to the amino acid sequence set forth in SEQ ID NO. 9 or a fragment thereof. The anti-uPAR antibodies or antigen-binding fragments thereof bind to a portion of human uPAR. In certain embodiments, the anti-uPAR antibodies or antigen binding fragments thereof bind to at least one of domain 1 , domain 2, and domain 3. In certain embodiments, the anti-uPAR antibodies or antigen-binding fragments thereof bind to domain 2. In certain embodiments, the anti-uPAR antibodies or antigen-binding fragments thereof bind to domain 3. In certain embodiments, the anti-uPAR antibodies or antigen-binding fragments thereof bind to both domain 2 and domain 3.

[0074] In certain embodiments, a presently disclosed antibody or antigen-binding fragment binds to uPAR (e.g., human uPAR) with a binding affinity, for example with a dissociation constant (KD) of 1 x 10-6 M or less, e.g., about 1 x 10-7 M or less, about 1 x 10-8 M or less, about 1 x 10-9 M or less, about 1 x 10-10 M or less, or about 1 x 10-11 M or less.

[0075] Suitably, the antigen binding fragment or “fragment” may be selected from the group consisting of a monovalent antibody, a Fab, a Fab’, a F(ab’)2, a single chain variable fragment (scFv), an antibody domain, a nanobody (VHH), a minibody and an scFv-FC; and variations thereof.

[0076] The term “antigen-binding fragment” or “antigen-binding region” of an antibody, as used herein, refers to that region or fragment of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a uPAR polypeptide). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of antigen-binding fragments encompassed within the term "antibody fragments" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL , VH , CL and CH1 domains; a F(ab)z fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et aL, Nature 1989;341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).

[0077] An “antigen binding fragment”, “antibody fragment” or “fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv), antibody domains, nanobodies (VHH), minibodies and scFv-FC; and multispecific antibodies formed from antibody fragments.

[0078] A monovalent antibody is an antibody with affinity for one epitope, antigen, or strain of microorganism i.e. is an antibody fragment with one antigen-binding site.

[0079] As used herein, “F(ab)” refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).

[0080] Fab’ fragments are similar to Fab fragments but include some of the Fc portion or hinge of the antibody. Fab’ fragments are formed by reducing F(ab’)2 fragments and therefore contain a small portion of Fc. As opposed to F(ab) fragments, Fab’ fragments contain a free sulfhydryl group that can be alkylated or used in a conjugation with another compound of interest (such as a protein, toxin or dye). As used herein, “F(ab’)2 ” refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab1) (bivalent) regions, wherein each (ab1) region comprises two separate amino acid chains, a part of a H chain and a light (L) chain linked by an S-S bond for binding an antigen and where the remaining H chain portions are linked together. A “F(ab’)2 ” fragment can be split into two individual Fab’ fragments. As used herein, the term “vector” refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences into cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors and plasmid vectors.

[0081] Single-chain variable fragments (scFv) are single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody. These two chains are connected by a flexible linker peptide that is usually 15-20 amino acids long.

[0082] Single-domain antibodies, also known as nanobodies (VHH), are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

[0083] Suitably, the fragment may be a nanobody (VHH).

[0084] A “minibody” is a class of bispecific fragments which are scFv-derived bispecific molecules. It is a bivalent fusion molecule with two scFvs fused to CH3. The scFv targeting antigen A is fused to the N-terminus of one of the CH3 domains and the scFv targeting antigen B to the other CH3.

[0085] An scFv-FC is two scFv fragments linked to Fc region. It is around 50 kDa lighter than a full-size IgG antibody.

[0086] As understood by persons skilled in the art, the Fc fragment of an IgG is all of the lower base of the antibody's Y-shape, which includes a sulfhydryl-bridged hinge region and CH2 and CH3 domains. The Fc fragment may be homodimeric. Cargo molecules may be attached to the hinge region via chemical conjugation to cysteine residues.

[0087] Fab fragments, scFv fragments or protein fragments or domains may be linked to immunoglobulin CH3 domains to form a homodimer. Protein fragments or domains may be linked to the N-termini of an Fc fragment to form homodimers. Protein fragments or domains may be linked to the C-termini of an Fc fragment to form homodimers or to both the N- and C-termini of an Fc fragment to form homodimers.

[0088] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. coli or phage).

[0089] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0090] The antibody constant region domains of an antibody of the present disclosure, if present, for example in a full length antibody or a multispecific molecule, may be selected having regard to the proposed function of the multispecific antibody molecule, and in particular the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the lgG1 and lgG4 isotypes.

[0091] In some embodiments, the antibody or antibody fragment may comprise a heavy chain constant region selected from lgG1 , lgG2, lgG3 and lgG4, preferably lgG1 and lgG4, most preferably lgG1.

[0092] In some embodiments, the antibody or antibody fragment may comprise a light chain constant region of kappa.

[0093] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs or CDRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0094] Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991). The term “Fc region” herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0095] Suitably, the antibody molecule or antigen binding fragment may comprise a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO. 2, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 3 and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO. 4.

[0096] Suitably, the antibody molecule or antigen binding fragment of any of claims 1-3, comprising a framework region 1 (FR1) amino acid sequence of SEQ ID NO. 5, a framework region 2 (FR2) amino acid sequence of SEQ ID NO. 6, a framework region 3 (FR3) amino acid sequence of SEQ ID NO. 7, and a framework region 4 (FR4) amino acid sequence of SEQ ID NO. 8.

[0097] Suitably, the antibody molecule or antigen binding fragment may comprise the amino acid sequence of SEQ ID NO. 10. Such an antibody molecule or antigen binding fragment may be referred to herein as ‘Nb2’.

[0098] Suitably, the antibody molecule or antigen binding fragment may be a nanobody (VHH) comprising the amino acid sequence of SEQ ID NO. 10, wherein the nanobody binds to the amino acid sequence of SEQ ID NO. 1 of human uPAR.

[0099] In certain embodiments, a presently disclosed antibody or antigen-binding fragment thereof comprises heavy and light chain variable regions comprising amino acid sequences that are homologous or identical to the amino acid sequences of the antibodies described herein, and wherein the antibodies or antigen-binding fragments thereof retain the desired functional properties of the anti-uPAR antibodies or antigenbinding fragments thereof of the presently disclosed subject matter.

[0100] In certain embodiments, the VHCDR1 , VHCDR2 and / or VHCDR3 amino acid sequences can be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous or identical to the sequences set forth above. In certain embodiments, the antibody molecule or antigen binding fragment may comprise an amino acid sequence at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% complementary to the amino acid sequence of SEQ ID NO. 10.

[0101] An antibody or antigen binding fragment having VHCDR1 , VHCDR2 and / or VHCDR3 amino acid sequences having high (i.e. , 80% or greater) homology or identity to the VHCDR1 , VHCDR2 and / or VHCDR3 amino acid sequences set forth above, can be obtained by mutagenesis (e.g., site- directed or PCR-mediated mutagenesis), followed by testing of the encoded altered antibody for retained function (i.e., the binding affinity) using the binding assays described herein.

[0102] As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity or homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = # of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0103] The percent homology or identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput Appl Biosci (1988); 14: 11-17) which has been incorporated into the ALIGN program (version 2.0), using a P AMI 20 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol (1970);48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.

[0104] Additionally or alternatively, the protein sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul et al., J Mol Biol (1990):215:403- 10. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et ah, Nucleic Acids Res (1997);25( 17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. 43.4. Antibodies with Conservative Modifications.

[0105] Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. In certain embodiments, a sequence disclosed herein, e.g., a CDR sequence, a VH sequence or a VL sequence, can have up to about one, up to about two, up to about three, up to about four, up to about five, up to about six, up to about seven, up to about eight, up to about nine or up to about ten amino acid residues that are modified and / or substituted.

[0106] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, y, and p, respectively.

[0107] In some embodiments, the antibody or antibody fragment may be a human antibody or antibody fragment.

[0108] A “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0109] In some embodiments, the antibody may be a full length antibody. The terms “full length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0110] In some embodiments, the antibody or antibody fragment may comprise wherein the antibody or antibody fragment is monospecific or bispecific.

[0111] “Monospecific” as employed herein refers to the ability to bind only one target antigen.

[0112] Multispecific antibodies are antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for the amino acid sequence of SEQ ID NO. 1 of human uPAR and the other is for any other antigen. In certain embodiments, bispecific antibodies may bind to two different epitopes of human uPAR, one being the amino acid sequence of SEQ ID NO. 1. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express uPAR. Bispecific antibodies can be prepared as full length antibodies or antibody fragments.

[0113] Suitably, the antibody molecule or antigen binding fragment may be humanised, optimised, and / or de-immunized.

[0114] A “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human hypervariable regions (HVRs) and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0115] “Optimised” as employed herein is intended to refer to where one, two, three, four, five or more amino acids in a naturally occurring sequence have been replaced or deleted, for example to optimize the properties of the domain such as by eliminating undesirable properties but wherein the characterizing feature(s) of the domain is / are retained.

[0116] “De-immunized” refers to a technology for location and removal of T-cell epitopes through the combined use of immunological and molecular biology techniques. In the case of deimmunization of antibodies, mutations to remove T-cell epitopes can generally be introduced without significantly reducing the binding affinity of the antibody. Typically, "deimmunized" antibodies are created with human constant regions and by expression of genes encoding these antibodies in mammalian cells.

[0117] A functionally equivalent residue of an amino acid used herein typically refers to other amino acid residues having physiochemical and stereochemical characteristics substantially similar to the original amino acid. The physiochemical properties include water solubility (hydrophobicity or hydrophilicity), dielectric and electrochemical properties, physiological pH, partial charge of side chains (positive, negative or neutral) and other properties identifiable to a person skilled in the art. The stereochemical characteristics include spatial and conformational arrangement of the amino acids and their chirality. For example, glutamic acid is considered to be a functionally equivalent residue to aspartic acid in the sense of the current disclosure. Tyrosine and tryptophan are considered as functionally equivalent residues to phenylalanine. Arginine is considered to be a functionally equivalent residue to lysine.

[0118] The term “dissociation constant” or “KD” refers to a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, such as when a complex falls apart into its component molecules. Equilibrium dissociation constants (KDS) can be determined using surface plasmon resonance. The lower the dissociation constant, the more tightly bound the component molecules, or the higher the affinity between the component molecules.

[0119] According to some embodiments, Kd is measured using surface plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5 chips at -10 response units (RU).

[0120] In another aspect, the invention provides an antibody-conjugate which comprises: (a) an antibody molecule or antigen binding fragment according to any aspect of the invention; and (b) a coupling moiety coupled to the antibody molecule or antigen binding fragment.

[0121] The term "antibody-conjugate" as used herein refers to antibody-based conjugates that are configured to deliver a “conjugate”, “coupling moiety” or “cargo” to a cell.

[0122] The term as used herein refers to an antibody that includes a targeting component linked to a cargo component. Accordingly, the targeting component may include an antibody Fab fragment, an antibody variable domain, nanobody, or a protein that binds to a cell surface receptor or cell surface molecule. The cargo component may include a protein, one or more imaging labels, such as radiolabels, fluorescent molecules or other labelled molecules.

[0123] “Conjugated” or “coupled to” generally means joined or bound to another molecule. The terms “conjugating,” “joining,” “bonding” or “linking” refer to making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a radionuclide or other molecule to a polypeptide, such as an scFv. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule. The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

[0124] As described herein, the terms “cargo component", “cargo molecule", “coupling moiety” and “conjugate” all refer to functional molecules bound to or conjugated to the antibody or antibody fragment of the invention, which have a functional use and may cause an effect in a target cell.

[0125] Cargo molecules as described herein can include any molecule having a function useful to cause an effect in a target cell. For example, in addition to cytotoxic drugs or imaging labels, additional cargo molecules could be radiolabels that kill cells through radiation damage i.e. can be used therapeutically rather than for imaging. Examples of such radiolabels are Yttrium (Y)-90 and Iodine (I)- 131 . Additional types of cargo molecules are identifiable by skilled persons upon reading the present disclosure.

[0126] The antibody molecule or antigen binding fragment and the coupling moiety or conjugate may be linked by a covalent bond or may be non-covalently associated with each other.

[0127] A cargo component, cargo molecule, coupling moiety or conjugate may comprise a linker. “Linker” refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody to a coupling moiety. Nonlimiting exemplary linkers are described herein. In one aspect, a linker has a functionality that is capable of reacting with a free cysteine present on an antibody to form a covalent bond. Nonlimiting exemplary such reactive functionalities include maleimide, haloacetamides, a-haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates.

[0128] In some embodiments, a linker has a functionality that is capable of reacting with an electrophilic group present on an antibody. Exemplary such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, a heteroatom of the reactive functionality of the linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Nonlimiting examples of such reactive functionalities include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.

[0129] A linker may comprise one or more linker components. Exemplary linker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl (“MP"), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (a “PAB"), N — Succinimidyl 4-(2-pyridylthio) pentanoate (“SPP"), and 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“MCC”). Various linker components are known in the art.

[0130] A linker may be a “cleavable linker," facilitating release of the coupling moiety, such as a drug. Nonlimiting exemplary cleavable linkers include acid-labile linkers (e.g., comprising hydrazone), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No. 5,208,020).

[0131] Suitably, the coupling moiety may be a biologically active moiety and / or a diagnostic moiety.

[0132] The term “biologically active moiety" means the core molecule or ion of a drug (i.e., the drug molecule without certain appendages) that is responsible for the physiological or pharmacological action of a drug substance. Examples include actinomycin D, bleomycin, anthracyclines, epirubicin and doxorubicin as biologically active anti-cancer chemicals.

[0133] The term “diagnostic moiety” means any molecule that is used to aid in diagnosing, characterizing or identifying disease. Examples include a fluorophore for during fluorescence guided surgery, a radioactive isotype for nuclear imaging, or horse-radish peroxidase for immunohistochemical stainings. Suitably, the coupling moiety may be selected from the group consisting of a detectable label, a toxin, a cytokine, a radionuclide, an enzyme, a photosensitizer and a combination thereof.

[0134] A conjugate may be generated by conjugation of an imaging label identifiable by persons skilled in the art to the antibody or fragment of the invention. Non-limiting examples of imaging labels include near infrared dyes such as IRDye800CW, or radiolabels such as 1-124, Cu-64 or Zr-89. Conjugation to Cu-64 or Zr-89 can be achieved through chelation to 1 ,4,7, 10-tetraazacyclododecane- 1 ,4,7, 10-tetraacetic acid (DOTA) which chelates these radiolabels, among other methods identifiable by persons skilled in the art.

[0135] In further examples, the conjugate can be a cytotoxic radiolabel (e.g. Yttrium-90, Y-90, or iodine-131 , 1 -131 ) or drug or other agent that modifies the behaviour of the targeted cell. For example, the drug could be an antagonistic ligand for the androgen receptor (AR) and could be used to downregulate AR activity.

[0136] A “toxin” is a naturally occurring organic poison produced by metabolic activities of living cells or organisms. They occur especially as proteins, often conjugated.

[0137] A “cytokine” is a type of protein that is made by certain immune and non-immune cells and has an effect on the immune system. Some cytokines stimulate the immune system and others slow it down. They can also be made in the laboratory and used to help the body fight cancer, infections, and other diseases.

[0138] A “radionuclide" is a an unstable form of a chemical element that releases radiation as it breaks down and becomes more stable. Radionuclides may occur in nature or be made in a laboratory. In medicine, they are used in imaging tests and in treatment. Also called radioisotope or radioactive isotope.

[0139] An “enzyme” is a biological catalyst that is usually a protein but could be RNA. Enzymes are proteins that act upon substrate molecules and decrease the activation energy necessary for a chemical reaction to occur by stabilizing the transition state. This stabilization speeds up reaction rates and makes them happen at physiologically significant rates. Enzymes bind substrates at key locations in their structure called active sites. They are typically highly specific and only bind certain substrates for certain reactions. “Photosensitizers” are agents that absorb light of a specific wavelength and transform it into useful energy. Photosensitizers can alter the course of a photochemical reaction. They are usually catalysts. They can function by many mechanisms, sometimes they donate an electron to the substrate, sometimes they abstract a hydrogen atom from the substrate. At the end of this process, the photosensitizer returns to its ground state, where it remains chemically intact, poised to absorb more light

[0140] Suitably, the coupling moiety may comprise a photodynamic and / or a photoimmuno sensitizer.

[0141] “Photodynamic sensitizers” have the property of intensifying or inducing a toxic reaction to light (as in the destruction of cancer cells stained with a light-sensitive dye) in a living system. Photodynamic therapy (PDT) is a form of phototherapy involving light and a photosensitizing chemical substance used in conjunction with molecular oxygen to elicit cell death (phototoxicity).

[0142] A “photoimmuno sensitizer”, "photosensitizer" or “photodynamic fluorophore” is an agent that absorbs light at a specific wavelength and is activated to a higher energy state from which free radicals and reactive oxygen are formed which subsequently cause cell death. An example is Foscan® which is administered intravenously to patients with head and neck cancer and have failed prior therapies. Over a period of four days it localizes towards the tumour after which the cancer is illuminated with light which causes a chemical reaction that destroys tumour cells.

[0143] Suitably, the coupling moiety may comprise contrast payloads, optionally fluorophores, radio-active isotopes, gadolinium or micro-bubbles.

[0144] Antibody-drug conjugates (ADCs) are designed to deliver highly toxic cargoes (payloads) to clinical targets. In ADCs, payloads are the pharmaceutically active component. For this reason, they dictate the overall effectiveness of these therapies. Up to recently, the development of payloads for ADCs has focused almost exclusively on cytotoxic drugs and cancer targets. But as the safety and efficacy of linker chemistry continue to increase, so does the diversity of payloads that can be used for the generation of new ADCs.

[0145] “Contrast payloads” or “detectable labels ’’are molecules that when visualized / imaged using an imaging modality create increased contrast between structures or fluids within the body. For example, in x-ray imaging contrast payloads (i.e. iodine or barium) enhance radiodensity, in magnetic resonance imaging contrast payloads (i.e. gadolinium) shorten or increase the relaxation times of nuclei within the body tissue and in ultrasound imaging contrast agents (micro-bubbles) alter the scattering and reflectance of ultrasound.

[0146] A “fluorophore” (or fluorochrome) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores are molecules that, upon absorbing light energy, can reach an excited state, then emit light energy. The three-stage process of excitation, excited lifetime, and emission is called fluorescence. Fluorophores absorb a range of wavelengths of light energy, and also emit a range of wavelengths.

[0147] Gadolinium (Gd) is a silvery-white, malleable, and ductile rare-earth metal. Gd has paramagnetic properties and, therefore, solutions of organic Gd complexes and Gd compounds are commonly used intravenously for magnetic resonance imaging (MRI) as contrast agents. Gadolinium-based contrast agents (GBCAs) are a form of chelated Gd3+ ion that are used in combination with magnetic resonance (MR) imaging.

[0148] Microbubbles are small, gas-filled bubbles, typically between 0.5pm and 10pm in diameter, that are widely used as contrast agents in medical imaging and as carriers for targeted drug delivery.

[0149] Suitably, the coupling moiety may comprise the dye IRDye800CW or variants of the dye ICG.

[0150] The dye IRDye800CW is an 800nm near-infrared dye characterized by high water solubility and salt tolerance, low-nonspecific binding to cellular components, high signal- to-noise ratio and favourable clinical safety profile. It can be conjugated to other proteins via an NHS-ester or maleimide conjugation.

[0151] The dye Indocyanine green (ICG) is a water-soluble tricarbocyanine dye that is used in medical diagnostics. ICG itself is chemically unreactive and cannot form conjugates but variations of ICG have been produced that have introduced carboxylic acids that allow for covalent binding to other molecules.

[0152] Suitably, the coupling moiety may comprise a drug. A “drug” or “therapeutic” is any chemical substance that affects the functioning of living things and the organisms (such as bacteria, fungi, and viruses) that infect them. A “toxic drug” is a drug that is toxic to target cells, such as cancer cells, and kills them.

[0153] The presently disclosed invention provides an anti-uPAR antibody or an antigen-binding fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates may be referred to herein as “immunoconjugates”. Immunoconjugates that include one or more cytotoxins are referred to as “immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Non-limiting examples of cytotoxins include taxol (such as ricin, diphtheria, gelonin), cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 - dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents also include, for example, calecheamicin, aureastatin, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC)), hypomethylating agents (azacytidine and decitabine), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0154] A cargo component, cargo molecule, coupling moiety or conjugate may comprise a drug. The drug may be a cytotoxic drug.

[0155] The term “cytotoxic agent” or “cytotoxic drug” as used herein refers to a substance that inhibits or prevents a cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211 , 1131 , 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various antitumor or anticancer agents.

[0156] A cytotoxic drug may be conjugated to the antibody or antibody fragment of the invention through chemical conjugation. Examples of chemical coupling that may be used are: amine-to-amine (NHS esters), sulfhydryl-to-sulfhydryl (maleimide), amine-to-sulfhydryl (NHS ester / maleimide), sulfhydryl-to-carbohydrate (maleimide / hydrazide), or attachment via an unnatural amino acid with the desired chemical reactivity, among other approaches identifiable by skilled persons. The unnatural amino acid may be inserted during recombinant production of the targeting component. Polyethyleneglycol (PEG) spacers may be inserted between the chemically conjugated proteins, protein fragments or other molecules. Linkers may be cleavable, such as valine-citrulline to enable release of the cytotoxic drug in the late endosomes or lysosomes by resident proteases such as cathepsins. In cases where the linkage is not cleavable, such as for trastuzumab-DMI, the antibody may be proteolysed to release the drug. Linkage chemistry, sites of linkage and choice of peptide can be guided by molecular modeling, and can be designed to minimize loss of binding activity of the antibody-drug conjugate (ADC) for cell surface receptor or other cell surface molecule, as would be understood by skilled persons.

[0157] A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal l and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. EngL, 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzi nostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2- pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); thiotepa; taxoids, e.g., paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™ Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, HL), and docetaxel (TAXOTERE®; Rhone- Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine (NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; CVP, an abbreviation for a combined therapy of cyclophosphamide, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0158] In addition, anti-uPAR antibodies or antigen-binding fragments thereof of the presently disclosed subject matter can be conjugated to an agent that induces senescence. In certain embodiments, the agent that induces senescence is a senogenic agent. Nonlimiting examples of senescence-inducing agents include Cdk4 / 6 inhibitors, Cdk2 inhibitors, MEK inhibitors, inhibitors of CDC7 and chemotherapy drugs. Non-limiting examples of MEK inhibitors include trametinib, cobimetinib, binimetinib, selumetinib, PD- 325901 , TAK-733, CI-1040 (PD 184352), PD0325901 , MEK162, AZD833O, GDC-0623, refametinib, pimasertib, R04987655, R05126766, WX-554, HL-085, ClnQ-03, G-573, PD184161 , PD318088, PD98059, R05068760, U0126, and SL327. Non-limiting examples of CDK4 / 6 inhibitors include palbociclib, ribociclib, and abemaciclib. Nonlimiting examples of chemotherapy drugs include cisplatin, doxorubicin, cyclophosphamide, and etoposide. Anti-uPAR antibodies or antigen-binding fragments thereof of the presently disclosed subject matter also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Non-limiting examples of radioactive isotopes that can be conjugated to antibodies for use diagnostically or therapeutically include90 Y,131 1 ,225 Ac, 213 Bi, 223 Ra and227 Th. Methods for preparing radioimmunconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. The antibody conjugates of the presently disclosed subject matter can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor (TNF) or interferon-Y; or, biological response modifiers such as, for example, lymphokines, interleukin- 1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), or other growth factors.

[0159] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et aL, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243- 56 (Alan R. Liss, Inc. 1985); Hellstrom et aL, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies ’84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et aL, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119-58 (1982).

[0160] Also provided herein is a nucleic acid encoding an antibody molecule or antigen binding fragment of any aspect of the invention.

[0161] A “nucleic acid” as described herein (or “isolated nucleic acid”) refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0162] Further provided herein is an expression vector comprising a nucleic acid according to any aspect of the invention. The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0163] Also provided in herein is a host cell comprising an expression vector according to any aspect of the invention.

[0164] The terms “host cell," “host cell line,” and “host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0165] Another aspect of the invention provides a pharmaceutical composition comprising an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell according to any aspect of the invention; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

[0166] “Pharmaceutically acceptable carriers”, “pharmaceutically acceptable vehicles”, “diluents” or “excipients” are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0167] An “adjuvant” is a drug or other substance, or a combination of substances, that is used to increase the efficacy or potency of certain drugs. It is an ingredient in a medicine that increases or modifies the activity of the other ingredients.

[0168] In a further aspect, the invention provides a method of producing an antibody molecule, antigen binding fragment or an antibody-conjugate according to any aspect of the invention, the method comprising culturing a host cell according to any aspect of the invention under conditions suitable for gene expression.

[0169] Recombinant proteins, such as antibodies, are conventionally generated by transfecting the recombinant DNA into a host cell, following which the host cells are cultured and the transfected DNA transcribed and translated. Different host cells can be chosen for recombinant protein production, the choice of which depends on the type of protein that needs to be generated, its functional activity and requisite yield. Culturing a host cell under conditions suitable for gene expression allows for the expression of the recombinant DNA in the host cell and production of the required recombinant proteins.

[0170] The invention also provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in therapy.

[0171] An antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of treating a subject.

[0172] Any of the antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, and / or host cell, and / or pharmaceutical composition according to any aspect of the invention described herein may be used in methods, e.g., therapeutic methods.

[0173] Any of the antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, and / or host cell, and / or pharmaceutical composition according to any aspect of the invention described herein may be administered using any suitable method to deliver them to a subject, such as a cell, a plurality of cells, or a multicellular organism, in particular an animal or a human, and in particular an animal or a human that may have one or more tumours, such as via injection, particularly intravenous, subcutaneous or intramuscular injection, among other methods identifiable by skilled persons.

[0174] The antibody, antibody fragment or antibody-conjugate of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein.

[0175] The antibody, antibody fragment or antibody-conjugate of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody, antibody fragment or antibody-conjugate need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody, antibody fragment or antibody-conjugate present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically / clinically determined to be appropriate. For the prevention or treatment of disease, the appropriate dosage of an antibody, antibody fragment or antibody-conjugate of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, antibody fragment or antibody-conjugate, the severity and course of the disease, whether the antibody, antibody fragment or antibody-conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, antibody fragment or antibody-conjugate, and the discretion of the attending physician. The antibody, antibody fragment or antibody-conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg / kg to 15 mg / kg (e.g. 0.1 mg / kg-10 mg / kg) of antibody, antibody fragment or antibodyconjugate can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody, antibody fragment or antibody-conjugate would be in the range from about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0176] The antibody, antibody fragment, antibody-conjugate, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered at suitable time intervals, for example weekly, monthly or, for example, whenever 50% of subjects are expected to have shown tumour regression.

[0177] The antibody, antibody fragment or antibody-conjugate of the invention can be used either alone or in combination with other agents in a therapy. For instance, an antibody, antibody fragment or antibody-conjugate of the invention may be co-administered with at least one additional therapeutic agent. Further, the invention provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in treating diseases where extracellular matrix remodelling plays a pathophysiological role; optionally wherein the diseases are (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer.

[0178] Further, the invention provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in treating a disease selected from the list consisting of: (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer.

[0179] An antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of treating diseases where extracellular matrix remodelling plays a pathophysiological role; optionally wherein the diseases are (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer.

[0180] An antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of treating a disease selected from the list consisting of: (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer.

[0181] As used herein the terms “treat”, “treating" or “treatment” refer to a clinical improvement of a disease, for example cancer, in a subject with this disease or, for example, preventing the development of a benign tumour to a malignant tumour. A clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the disease. In some embodiments, effective treatment may be demonstrated by slowing or halting the progression of the disease in the subject, or reversing the disease. Suitably, the disease may be reversed partially, or completely. In some embodiments, complete reversal of the diseases may be sufficient to result in curing of the disease.

[0182] In certain embodiments, the disease or disorder is associated with uPAR. In certain embodiments, the disease or disorder is associated with overexpression of uPAR. In certain embodiments, the disease or disorder is selected from the group consisting of tumours, senescence-associated pathologies, and tissue decline associated with aging. Non-limiting examples of senescence- associated pathologies include lung fibrosis, atherosclerosis, Alzheimer's disease, diabetes, osteoarthritis, liver fibrosis, chronic kidney disease, cardiac fibrosis, and Parkinson’s disease.

[0183] Where the disease is cancer or pre-cancer, a clinical improvement may be demonstrated by an improvement of the pathology and / or symptoms associated with the cancer or pre-cancer. Suitably, therapeutic effect (or “a therapeutically effective amount”) may be demonstrated by preventing the development of the cancer or pre- cancer in a subject, slowing or halting the progression of the cancer or pre-cancer in the subject, or reversing the cancer or pre-cancer. Suitably, the cancer or pre-cancer may be reversed partially, or completely. Clinical improvement of the pathology may be demonstrated by one or more of the following: reduced cancer or pre-cancer biomarker levels in the subject, reduced cancer or pre-cancer cell number in the subject, increased time to regrowth of cancer upon stopping of treatment, prevention or delay of pre-cancer development into cancer, prevention of regrowth of cancer upon stopping treatment, decreased tumour invasiveness, reduction or complete elimination of metastasis, increased cancer cell differentiation, or increased survival rate. In some embodiments, anti-tumour effects may be demonstrated by inhibition of tumour growth, reduced speed of tumour growth, or a partial or complete reduction in tumour mass / lump. Other suitable indications of clinical improvement in the pathology will be known to the skilled person. It will be appreciated that indications of clinical improvement of the pathology will vary depending on the type of cancer. Clinical improvement of symptoms associated with cancer may be, but are not limited to, partial or complete alleviation of pain and / or swelling, increased appetite, reduced weight loss, and / or reduced fatigue.

[0184] Suitably, in the methods of the invention, the antibody, antibody fragment, antibodyconjugate, the nucleic acid, the expression vector, the host cell and / or the pharmaceutical composition herein described may be administered in a therapeutically effective amount. The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in a clinical improvement of diseases, such as cancer.

[0185] In the context of the present disclosure, the term “subject” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). In suitable embodiments, subjects are mammals, particularly primates, especially humans. In suitable embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats. In certain embodiments (e.g., particularly in research contexts) subject mammals will be, for example, rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like.

[0186] Herein, the terms, “patients” and “subjects” may be used interchangeably.

[0187] The “extracellular matrix” is a large network of proteins and other molecules that surround, support, and give structure to cells and tissues in the body. The extracellular matrix helps cells attach to, and communicate with, nearby cells, and plays an important role in cell growth, cell movement, and other cell functions. The extracellular matrix is also involved in repairing damaged tissue. Abnormal changes in the extracellular matrix may lead to the development of certain diseases, such as cancer. The extracellular matrix of cancer cells can affect how they grow and spread. Also called ECM.

[0188] The extracellular matrix (ECM) is a highly dynamic structure that is present in all tissues and continuously undergoes controlled remodelling. This process involves quantitative and qualitative changes in the ECM, mediated by specific enzymes that are responsible for ECM degradation, such as metalloproteinases.

[0189] Pathophysiology refers to disordered physiological processes associated with disease or injury.

[0190] Diseases where extracellular matrix remodelling plays a pathophysiological role include (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and cancer.

[0191] Arthritis is an acute or chronic joint inflammation that often co-exists with pain and structural damage. Atherosclerosis, the formation of fibrofatty lesions in the artery wall, causes much morbidity and mortality worldwide, including most myocardial infarctions and many strokes, as well as disabling peripheral artery disease. Atherosclerosis is initiated by endothelium activation and, followed by a cascade of events (accumulation of lipids, fibrous elements, and calcification), triggers the vessel narrowing and activation of inflammatory pathways. The resultant atheroma plaque, along with these processes, results in cardiovascular complications. Atherosclerosis is the main risk factor for cardiovascular disease (CVD), which is the leading cause of mortality worldwide.

[0192] Inflammatory bowel disease (IBD) is a term that describes disorders involving longstanding (chronic) inflammation of tissues in the digestive tract. Types of IBD include Ulcerative colitis and Crohn's disease. Ulcerative colitis involves inflammation and sores (ulcers) along the lining of the large intestine (colon) and rectum. Crohn's disease is characterized by inflammation of the lining of the digestive tract, which often can involve the deeper layers of the digestive tract. Crohn's disease most commonly affects the small intestine. However, it can also affect the large intestine and uncommonly, the upper gastrointestinal tract.

[0193] Suitably, the therapy or treatment may comprise administering therapeutics or toxic payloads, optionally a photo-dynamic fluorophore, radio-active isotope, nano-particle, or toxic drug, conjugated to an antibody molecule or antigen binding fragment according to any aspect of the invention.

[0194] Toxic payloads are highly toxic cargoes that are designed to be delivered to clinical targets.

[0195] A nano-particle is a therapeutic nanoscale particle.

[0196] Suitably, the therapeutic or drug may be tamoxifen, gefitinib or vemurafenib.

[0197] Tamoxifen is a synthetic drug. It is a selective oestrogen receptor modulator used to prevent breast cancer in women and men. Tamoxifen (Nolvadex® or Soltamox®) is a drug that treats hormone receptor-positive (hormone-positive) breast cancer.

[0198] Gefitinib, sold under the brand name Iressa, is a medication used for certain breast, lung and other cancers. Gefitinib is an EGFR inhibitor, like erlotinib, which interrupts signalling through the epidermal growth factor receptor in target cells. Gefitinib can be used to treat non-small cell lung cancer.

[0199] Vemurafenib, sold under the brand name Zelboraf, is a medication used for the treatment of late-stage melanoma. It is an inhibitor of the B-Raf enzyme.

[0200] Suitably, the cancer may be selected from a group consisting of locally aggressive tumours; optionally wherein the cancer is selected from the group consisting of: pancreatic cancer, colorectal cancer, oesophageal cancer, head-and-neck cancer, breast cancer, lung cancer, kidney cancer (renal cell carcinoma), bladder cancer, prostate cancer, ovarium cancer, endometrium cancer, liver cancer, and melanoma.

[0201] A “cancer” or a “tumour” is an abnormal growth of cells that may form a mass or lump, but may be distributed diffusely. A “tumour” can be a benign tumour or a malignant tumour. Benign tumours are those that stay in their primary location without invading other sites of the body. They do not spread to local structures or to distant parts of the body. Benign tumours are caused by the presence of noncancerous cells. Specific types of benign tumours can turn into malignant tumours. Malignant tumours are caused by the presence of cancerous cells or precancerous cells, i.e. cells that grow uncontrollably and can spread locally and / or to distant sites within the body. Malignant tumours have the ability to invade other sites of the body. They may spread to distant sites via the bloodstream or the lymphatic system. This spread is called metastasis.

[0202] A malignant tumour or neoplasm may comprise a mixture of cancerous cells (and / or pre-cancerous cells) and healthy (i.e. non-cancerous) cells. When a subject has a malignant tumour it can be said that the subject has cancer.

[0203] The term “tumour” as used herein, encompasses the tumour in its entirety, i.e. the cells present within the tumour including cancerous, pre-cancerous cells, and / or healthy cells (for example stromal cells), as well as the tumour microenvironment which typically comprises immune cells and interstitial fluid.

[0204] “Cancer cells” may be defined by one or more of the following characteristics: reduced differentiation, self-sufficiency in growth signalling, insensitivity to anti-growth signals, evasion of apoptosis, enabling of a limitless replicative potential, induction and sustainment of angiogenesis, and / or activation of metastasis and invasion of tissue. As used herein, “pre-cancer” or a “pre-cancerous condition" is an abnormality that has the potential to become cancer (such a cancer mentioned hereinabove), wherein the potential to become cancer is greater than the potential if the abnormality was not present, i.e., was normal. Examples of pre-cancer include but are not limited to adenomas, hyperplasias, metaplasias, dysplasias, benign neoplasias (benign tumours), premalignant carcinoma in situ, and polyps. In one example, the pre-cancer is a pre- cancer tumour. Such a tumour may comprise pre-cancerous and healthy cells.

[0205] A cancer may be a solid cancer (solid cancer cells that grow in an organ system) or a liquid cancer (cancers that develop in the blood, bone marrow or lymph nodes). Suitably, a cancer or the tumour may be selected from the group consisting of pancreatic cancer, colorectal cancer, oesophageal cancer, head-and-neck cancer, breast cancer, lung cancer, kidney cancer (renal cell carcinoma), bladder cancer, prostate cancer, ovarium cancer, endometrium cancer, liver cancer, and melanoma.

[0206] Locally aggressive tumours or locally advanced tumours are tumours that have spread outside the body part the tumour first started in or spread to lymph nodes but have not (yet) metastasized to other parts of the body. For example, for prostate cancer it means the cancer has broken through the capsule of the prostate gland and may have spread to the tissue around the prostate, the seminal vesicles, body organs nearby such as the rectum or the bladder, or lymph nodes near the prostate. For breast cancer it entails a tumour that is larger than 50 mm and may have spread to the adjacent skin, muscles of the chest wall or more than 3 lymph nodes. In the TNM staging system, these are defined as T3 or T4 and / or N positive.

[0207] Suitably, the pancreatic cancer may be pancreatic ductal adenocarcinoma. PDAC is a type of exocrine pancreatic cancer. It develops from cells lining small tubes in the pancreas called ducts.

[0208] Suitably, the treatment may also target tumour-associated stromal cells, as well as malignant cells in the cancer.

[0209] The tumour microenvironment (TME) is made up of cells and extracellular matrix (non- cellular component), and cellular components include cancer cells and non-malignant cells such as immune cells and stromal cells. These three types of cells establish complex signals in the body and further influence tumour genesis, development, metastasis and participate in resistance to anti-tumour therapy. Tumour-associated stromal cells include tumour-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), tumour-associated adipocytes (CAAs), tumour endothelial cells (TECs), and pericytes (PCs).

[0210] Malignant cells are cells that grow uncontrollably and can spread locally and / or to distant sites within the body. Malignant cells have the ability to invade other sites of the body. They may spread to distant sites via the bloodstream or the lymphatic system. This spread is called metastasis.

[0211] Suitably, the treatment may comprise treatment stratification in oncology wherein patients with high uPAR uptake on radioactive imaging are offered more aggressive treatment regimens and patients with low uptake are spared these modalities.

[0212] “Treatment stratification” refers to the process of systematically arranging or categorizing patients based on their health status and other factors. In oncology, this is the ability to stratify, or group, cancer patients based on the specific characteristics of their cancer type.

[0213] Patients with high uPAR uptake have tumours that show high uPAR uptake upon radioactive imaging compared to tumours with no or low uPAR uptake upon radioactive imaging.

[0214] Patients with low uPAR uptake have tumours that show no or low uPAR uptake upon radioactive imaging compared to tumours with high uPAR uptake upon radioactive imaging.

[0215] Radioactive imaging is a method that uses radioactive substances to make pictures of areas inside the body. The radioactive substance is injected into the body, and locates and binds to specific cells or tissues, including cancer cells. Images are made using a special machine that detects the radioactive substance. Also called radioimaging.

[0216] “Aggressive treatment regimens” or “aggressive treatment modalities” describes treatments that are more severe, radical or intense than usual with a high level of intervention. Examples are initiating treatment before symptoms develop (as opposed to a watch-and-wait strategy or active surveillance strategy) or combining different treatment regimens or modalities. Often aggressive treatment regimens come at a higher rate of complications or morbidity with the intent of having a higher chance at curing. Suitably, the treatment may be administered to patients who have failed initial therapy or developed resistance to initial therapies, or in combination with initial therapies in order to prevent the development of therapy resistance.

[0217] Initial therapies may include surgery, chemotherapy, radiation therapy, immunotherapy, targeted therapy, and / or hormone therapy. “Failing” initial therapy means the patient has not sufficiently responded to the initial therapy and therefore not attained the desired suppression of disease symptoms. They have not demonstrated a significant clinical improvement, which may be demonstrated by an improvement of the pathology and / or symptoms associated with the disease.

[0218] Therapy resistance can occur when cancer cells contain molecular changes that make them insensitive to a particular drug before treatment even begins. Because cancer cells within the same tumour often have a variety of molecular changes, this so-called intrinsic resistance is common. In other cases of resistance, cancer cells may adapt to the drug while it is being administered, acquiring molecular changes that allow them to escape its effects. Molecular alterations that contribute to intrinsic or acquired treatment resistance include mutation of the drug's molecular target, changes in the way the drug interacts with the tumour, broad cellular changes, and changes in the tumour microenvironment, among others. To complicate matters, many of these factors can be at play simultaneously in a single tumour. When cancer cells resist the effects of drugs used for treatment, they can grow and reform tumours, a process known as recurrence or relapse. Sometimes resistance develops quickly, within a matter of weeks of starting treatment. In other cases, it develops months, or even years, later.

[0219] The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition according to any aspect of the present invention may be administered in combination with other cancer therapies. For example, the treatment may be administered in combination with other therapies to prevent the development of therapy resistance.

[0220] Suitably, the treatment may comprise last-line therapy in aggressive arthritis using antibody-drug conjugates.

[0221] Last-line therapy is the final treatment option available to a patient. Aggressive arthritis is a particularly severe presentation of the disease. Rheumatoid arthritis (RA) and progressive rheumatoid arthritis (PRA) are more aggressive forms of the disease.

[0222] Antibody-drug conjugates refer to antibody-based conjugates that are configured to deliver a drug to a cell.

[0223] Suitably, the treatment may comprise photodynamic or photoimmuno therapy.

[0224] In another aspect, the invention provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in diagnosis.

[0225] An antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of diagnosis.

[0226] Diagnosis is the process of identifying a disease, condition, or injury from its signs and symptoms. Diagnosis also includes prognosis i.e. the likely outcome or course of a disease and the chance of recovery or recurrence.

[0227] In one embodiment, an antibody molecule, antigen binding fragment or antibodyconjugate for use in a method of diagnosis or detection is provided. In certain embodiments, the method comprises contacting the biological sample with an antibody molecule, antigen binding fragment or antibody-conjugate as described herein under conditions permissive for binding of the antibody molecule, antigen binding fragment or antibody-conjugate to human uPAR, and detecting whether a complex is formed between the antibody molecule, antigen binding fragment or antibody-conjugate and human uPAR in the biological sample. Such method may be an in vitro or in vivo method. In one embodiment, the antibody molecule, antigen binding fragment or antibody-conjugate is used to select subjects eligible for therapy with said antibody molecule, antigen binding fragment or antibody-conjugate. In a further embodiment, the biological sample is a cell or tissue (e.g., cancerous or potentially cancerous tissue). In diagnostic / theranostic imaging, it is expected that the administration to a subject of an antibody molecule, antigen binding fragment or antibody-conjugate that includes an imaging label such as a radiolabel, near infrared label, or fluorescent label may be followed by a period of 1-7 days to allow localization of the target cell in the subject, for example, tumour localization in the subject. Following this period, the subject may be imaged using positron emission tomography or other suitable imaging modality, such as localized or whole-body imaging, to allow detection of the location of the target cell such as the tumour.

[0228] Suitably, the diagnosis may comprise the identification of atherosclerotic plaques that are at risk of rupture using a PET tracer.

[0229] An atherosclerotic plaque is composed of fat, cholesterol, calcium and inflammatory cells that build up on the inside of the body's arteries. Plaque rupture occurs when intraplaque stress exceeds the material strength of the overlying fibrous cap; increased plaque structural stress (PSS) is therefore a potential mechanism that determines rupture of a higher risk lesion. Atherosclerotic plaques that are at risk of rupture can be identified. Plaque rupture is the most common cause of myocardial infarction, occurring particularly in higher risk lesions such as fibroatheromas.

[0230] A PET radiotracer (also known as PET tracer) is a positron-emitting radiopharmaceutical used in positron emission tomography (PET). Positron emission tomography (PET) is a type of nuclear medicine procedure that measures metabolic activity of the cells of body tissues.

[0231] Suitably, the diagnosis may comprise monitoring aggressive arthritis using radioactive tracers.

[0232] Aggressive arthritis may be monitored by regular assessment of the disease state.

[0233] A radioactive tracer is a chemical compound in which one or more atoms have been replaced by a radioisotope. Monitoring its radioactive decay, a radiotracer can be used to explore the mechanism of chemical reactions. They are also used for flow visualisation through different technologies, such as Single Photon Emission Computed Tomography (SPECT), Positron Emission Tomography (PET) and Computed Radioactive Particle Tracking (CARPT).

[0234] The invention further provides an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention for use in imaging. An antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention may be used in a method of imaging.

[0235] Medical imaging is the technique and process of imaging the interior of a body for clinical analysis and medical intervention, as well as visual representation of the function of some organs or tissues (physiology).

[0236] A method of imaging may include the steps of: administering a subject with an effective dose of the antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell, and / or pharmaceutical composition, wherein the cargo molecule or conjugate is an imaging label; and performing an imaging method suitable for detecting the imaging label in the subject. In the method, the administering is performed at an effective dose to provide a sufficient concentration of the imaging label that is detectable by the imaging method, as would be identifiable by skilled persons.

[0237] In a further embodiment, an antibody molecule, antigen binding fragment, or antibodyconjugate is used in vivo to detect, e.g., by in vivo imaging, a cancer expressing uPAR in a subject, e.g., for the purposes of diagnosing, prognosing, or staging cancer, determining the appropriate course of therapy, or monitoring response of a cancer to therapy. One method known in the art for in vivo detection is immuno-positron emission tomography (immuno-PET), as described, e.g., in van Dongen et al., The Oncologist 12:1379-1389 (2007) and Verel et aL, J. Nucl. Med. 44:1271-1281 (2003). In such embodiments, a method is provided for detecting a cancer in a subject, the method comprising administering a labelled antibody molecule, antigen binding fragment, or antibodyconjugate to a subject having or suspected of having a cancer, and detecting the labelled antibody molecule, antigen binding fragment, or antibody-conjugate in the subject, wherein detection of the labeled an antibody molecule, antigen binding fragment, or antibody-conjugate indicates a cancer in the subject. In certain of such embodiments, the labeled antibody molecule, antigen binding fragment, or antibody-conjugate comprises an antibody molecule, antigen binding fragment, or antibody-conjugate conjugated to a positron emitter, such as 68Ga, 18F, 64Cu, 86Y, 76Br, 89Zr, and 1241. In a particular embodiment, the positron emitter is 89Zr.

[0238] In certain embodiments, any of the antibody molecule, antigen binding fragment, or antibody-conjugate provided herein may be useful for detecting the presence of uPAR in a biological sample. The term “detecting" as used herein encompasses quantitative or qualitative detection. A “biological sample” comprises, e.g., a cell or tissue (e.g., biopsy material, including cancerous or potentially cancerous).

[0239] Additionally, the invention provides a method of imaging a subject, comprising administering an antibody molecule or antigen binding fragment, an antibody-conjugate, a nucleic acid, an expression vector, and / or a host cell, and / or a pharmaceutical composition according to any aspect of the invention.

[0240] Suitably, the imaging may be image-guided surgery, optionally fluorescence guided surgery (FGS), and / or optionally uses near-infrared intraoperative imaging.

[0241] Image-guided surgery is any surgical procedure where the surgeon uses tracked surgical instruments in conjunction with preoperative or intraoperative images in order to directly or indirectly guide the procedure.

[0242] Fluorescence guided surgery (FGS), also called fluorescence image-guided surgery, or in the specific case of tumour resection, fluorescence guided resection, is a medical imaging technique used to detect fluorescently labelled structures during surgery. Similarly to standard image-guided surgery, FGS has the purpose of guiding the surgical procedure and providing the surgeon of real time visualization of the operating field.

[0243] “Near-infrared intraoperative imaging” or “intraoperative near-infrared fluorescence (NIR) imaging” allows surgeons to identify and localize specific structures and boundaries, which can facilitate the optimization of surgical procedures. For example, intraoperative fluorescence imaging can identify invasive margins of tumours during surgical resection in order to aid surgeons in attaining adequate tumour margins. In order to create fluorescence, fluorescent contrast agents, such as Indocyanine Green, are administered pre / peri-operatively that accumulate in the tissue of interest resulting in high fluorescent signal in the tissue of interest. Given its capacity for intraoperative, real-time anatomical navigation and identification, near-infrared intraoperative imaging is considered to possess great potential for clinical practical in the future of minimally invasive surgery.

[0244] The presently disclosed subject matter provides kits for treatment or ameliorating a disease or disorder, and / or detecting uPAR. In certain embodiments, the kit comprises the anti-uPAR antibodies or antigen-binding fragments thereof, the immunoconjugate, the multi-specific molecule, or the composition disclosed herein. In certain embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0245] In certain embodiments, the kit further comprises instructions for administering the anti- uPAR antibodies or antigen-binding fragments thereof, the immunoconjugate, the multispecific molecule, or the composition disclosed herein to a subject in need the treatment. The instructions can generally include information about the use of the anti- uPAR antibodies or antigen-binding fragments thereof, the immunoconjugate, the multispecific molecule, and the composition disclosed herein for the treatment or ameliorating a disease or disorder. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment and / or prevention of a tumor or neoplasm or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0246] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 31orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0247] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and / or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and / or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and / or the use of the term in the present document shall prevail.

[0248] Aspects of the invention are demonstrated by the following non-limiting examples.

[0249] Examples

[0250] Materials and Methods

[0251] Anti-uPAR VHH production

[0252] In order to generate VHHs, two llamas were immunized with full-sized human recombinant uPAR derived from HEK293 cells (cat # 10925-H08H, Sino Biological, China), on day 0, 14, 28 and 35. Peripheral blood lymphocytes were purified for RNA isolation and library construction on day 43. 400-basepair fragments were cloned in a phage display vector and transformed into Escherichia coli TG1 followed by two rounds of phage display selection to identify binders to recombinant uPAR, confirmed by ELISA. VHHs were purified via his-tag using Talon beads. Purity was confirmed by SDS-PAGE and binding by ELISA and flow cytometry.

[0253] Llama immunization and library construction

[0254] Immunizations and RNA preparation were performed by Eurogentec (Belgium). In order to generate uPAR specific VHHs, two llamas were immunized with full-sized recombinant uPAR (cat# 10925-H08H, Sino Biological, China) on day 0, 14, 28 and 35. Blood samples were collected on day 0, day 28, and day 43 for analysis of the immune response. Large bleeds were performed on day 43 for RNA isolation and library preparation.

[0255] Before use, 5pl of the obtained RNA was precipitated and loaded onto gel to confirm the intactness of 28S and 18S rRNA. The remaining RNA was stored in 70% ethanol, containing 200mM sodium acetate at -80°C. About 40pg RNA was transcribed into cDNA using SuperScript III Reverse Transcriptase Kit (Invitrogen). The cDNA was cleaned on Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). Immunoglobulin H (both conventional and heavy chain) fragments were amplified using primers annealing at the leader sequence region and at the CH2 region. 5pl was loaded onto a 1% TBE agarose gel to confirm amplification. The rest of the samples was loaded onto a 1 % TAE agarose gel. The 700-bp fragment was excised from the gel and purified. About 80ng was used as a template for the nested PCR. The amplified fragment was cleaned on Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit and eluted in 120pl. The eluted DNA was digested first with Sfil and next with BstEIL Restriction digestion was confirmed by agarose gel electrophoresis using 1.5% TBE agarose gel. After the restriction digestion, the samples were loaded onto a 1 .5% TAE agarose gel. The 400-bp fragment was excised from the gel and purified on Macherey-Nagel NucleoSpin Gel and PCR Clean-up kit. The 400-bp fragments were ligated into the phagemid pUR8100 vector and transformed into Escherichia coli TG1. The transformed E. coli TG1 were titrated using 10-fold dilutions. 5pl of the dilutions were spotted on LB-agar plates supplemented with 100pg / ml of ampicillin and 2% glucose.

[0256] The number of transformants was calculated from the spotted dilutions of the rescued E. coli TG1 culture. The transformants were stored in 2xYT (Sigma-Aldrich) medium supplemented with 20% glycerol, 2% glucose and 100pg / ml ampicillin at-80°C. The insert frequency was determined by picking 24 different clones from transformations from each library and performing a colony PCR. Bands of ~700bp indicate a cloned VHH fragment.

[0257] Phage production and selection

[0258] Phages were produced from the libraries as outlined below. E. coli TG1 containing libraries from each library were diluted from the glycerol stock up to an OD600 of 0.05 in 2xYT medium containing 2% glucose and 100pg / ml ampicillin and grown at 37°C for 2 hours to reach an OD600 of ~0.5. Subsequently, about 7ml of the cultures were infected with helper phage VCS M13 using a MOI (multiplicity of infection) of 100 for 30 minutes at 37°C. E. coli TG1 were spun down and resuspended into 50ml fresh 2xYT medium supplemented with both ampicillin (100pg / ml) and kanamycin (25pg / ml) and grown overnight at 37°C with shaking. Produced phages were precipitated from the supernatant of the cultures using PEG-NaCI precipitation. Titers of the produced phages were calculated by serial dilution of the phage sample and infection of E. coli TG1.

[0259] Twenty microliters of the precipitated phages (~1011 phages, which is >1000-fold the diversity of the libraries) were applied to wells coated with ruPar. In short, for each library, 10OpI antigen was coated on the MaxiSorp plate overnight at 2 concentrations 5pg / ml and 0.5pg / ml. As a negative control, one well was incubated with PBS only. Next day, after removal of non-bound antigen, the plate was washed three times with PBS and blocked with 4% MPBS. At the same time, freshly precipitated phages were pre-blocked with 2% MPBS for 30 minutes. Pre-blocked phages were incubated directly with coated antigen for 2 hours. Upon extensive washing with PBS-Tween and PBS, bound phages were eluted with 0.1 M TEA solution and subsequently neutralized with 1 M Tris / HCI, pH 7.5. Eluted phages were serially diluted and then used to infect E. coli TG1 bacteria and spotting on LB agar plates supplemented with 2% glucose and 100pg / ml ampicillin and incubated overnight at 37°C.

[0260] Screening and sequence analysis of VHHs

[0261] After the 2nd round of phage display selection, glycerol stocks were prepared from all outputs rescued by infection of E. coli TG1 and stored at -80°C in the same way as for the outputs obtained after the 1st round of phage display selection. Subsequently, all rescued outputs of the 2nd round of selection were plated out in order to pick single colonies, which were grown in a 96-wells plate. These master plates were used to produce periplasmic fractions containing monoclonal VHHs for screening of binders. To test the binding specificity, monoclonal VHHs were tested using 25pl of the periplasmic fractions exactly as described below. Based on the ELISA results, clones from the master plate were selected for sequence determination. uPAR ELISA

[0262] 96-wells plates (cat # 44-2404-21 , ThermoFisher Scientific, USA) were coated with 2 pg / ml uPAR (cat# 10925-H08H, Sino Biological, China) in PBS. Plates were washed with PBS and blocked with 4% BSA in PBS. Plates were incubated with VHHs diluted in 2% PBSA for 1 hour, anti-VHH mouse anti-FLAG (clone M2, Sigma-Aldrich, USA) or rabbit anti-VHH (clone QE19, QVQ, The Netherlands) for another hour and peroxidase conjugated donkey anti mouse or anti rabbit, respectively, for another hour. Between each incubation step plates were washed. Visualization was initiated by o-phenylenediamine, supplemented with 0.03% H2O2 and stopped using 1 M H2SO4. Signals were measured at 490 nm using a plate reader (Cytation 5, BioTek, USA).

[0263] Cloning, production, purification, and analysis of selected VHHs

[0264] From all the clones that were sequenced, the VHHs were produced as described below. Pre-cultures were prepared by growing the bacteria containing the plasmids containing the selected VHH in 8ml 2xYT medium supplemented with 2% glucose and 100pg / ml ampicillin overnight at 37°C. The pre-cultures were diluted into 800ml fresh 2xYT that was pre-warmed at 37°C and supplemented with 100pg / ml ampicillin and 0.1 % glucose. The bacteria were grown for 2 hours at 37°C before induction of the VHH expression with 1 mM IPTG. The VHHs were expressed for 4 hours at 37°C and bacteria were harvested by centrifugation. Bacteria pellets were resuspended into 30ml PBS and frozen at -20°C.

[0265] Frozen bacteria were thawed at room temperature and centrifugated to separate cell debris and soluble fraction, which contains the VHH. VHH were purified from the soluble fraction using immobilized metal affinity chromatography resin charged with cobalt (TALON beads). Bound VHHs were eluted with 150mM imidazole and dialyzed against PBS. The protein concentration was measured using absorption at 280nm and corrected according to the molar extinction coefficient and the molecular weight of different VHHs. About 1 pg of the purified VHH was loaded onto an SDS-PAGE.

[0266] VHH humanization

[0267] The lead VHH was humanized by comparing the sequence and the predicted structure of the VHH to human variants of VH domain resembling the sequence and structure of the lead compound. Clones with several mutations were synthetically made (Twist Biosciences), cloned, expressed, and purified and the mutants were tested for loss of affinity in binding to recombinant uPAR in an ELISA. From these mutants the lead humanized clone was cloned in yeast expression vectors and produced in Saccharomyces cerevisiae. The filtered supernatant of a 1 L batch was purified on an Akta start FPLC system with affinity columns and subsequently the purified VHH was used for further experiments.

[0268] Cells Vendors and culture conditions of the cell lines AT84 (mouse origin), A549, BxPC-3, COLO 205, HCC1954, HCT-15, HEK EV (empty vector), HEK uPAR D2-3 (isotype domain 2-3), HEK uPAR WT (wildtype), HT-1197, HT-29, HT-29-luc2, J82, MCF-7, MDA- MB-231 , MIA PaCa-2, NCI-H1299, Panc-1 , SK-OV-3, UM-UC-3 and 4T1 (mouse origin) are described in Table 1. Primary pancreatic tumour cells (FNA005) were derived from endoscopic-ultrasound-guided fine-needle biopsies after written informed consent was obtained (CCMO B20.015). Biopsies were dissociated using a mix (3:1 ratio) of collagenase (Gibco / Thermo Fisher Scientific, The Netherlands) and dispase II (Roche, Switzerland) for two hours at 37 °C. After expansion, FNA005 was shown to harbor mutations commonly found in pancreatic cancer (KRASG12D, TP53A306*, homozygous loss of CDKN2A) using the AmpliSeq Cancer Hotspot Panel V2 (Thermo Fisher Scientific). Low-passage cells (p6) were used in the experiments. All cells were routinely screened for mycoplasma infection.

[0269] Table 1 : Cell lines and culture conditions

[0270] Flow cytometry

[0271] Flow cytometry for VHHs was performed using standard flow cytometry methodology in a three-step protocol (for details see

[0021] ). Briefly, cells were consecutively incubated with 100 nM VHH, 2 pg / ml mouse anti-FLAG (clone M2, Sigma-Aldrich, USA) or 1 pg / ml rabbit anti-VHH (clone QE19, QVQ, The Netherlands) and 2.5 pg / ml rabbit anti-mouse AF488 (cat# A21121 , Invitrogen, USA) or 2.5 pg / ml goat anti-rabbit AF488 (cat# A-11070, Invitrogen, USA). Flow cytometry for cells with human- or mouse-uPAR was performed using a two-step protocol with 5 pg / ml mouse anti-human uPAR ATN617 or 2.5 pg / ml goat anti-mouse uPAR (cat# AF534, R&D systems, USA) as primary antibodies and rabbit anti-mouse AF488 (cat# A21121 , Invitrogen, USA) or 2.5 pg / ml rabbit anti-goat AF488 (cat# A11078, Invitrogen, USA) as secondary antibodies. Quantitative flow cytometry for human-uPAR was performed using ATN617 and Qifi-kit (Agilent Technologies, USA) per suppliers instructions. All samples were measured using a LSRII flow cytometer (BD Biosciences, USA) except for those requiring a NIR laser, for which a dedicated 800 nm LSRFortessa flow cytometer (BD Biosciences, USA) was used.

[0272] Surface plasmon resonance

[0273] Binding kinetics were determined using a Biacore T200 (Cytiva, Uppsala, Sweden) using a NiHC1500M sensor chip (Xantec Bioanalytics, Dusseldorf, Germany) to which recombinant human uPAR was immobilized exploiting the polyhistidine-tag. Each analysis was done at least in triplicate at 25°C in a running buffer of 10 mM HEPES, 150 mM NaCI, pH 7.4 with 0.05% Tween-20 and 2% BSA at a flow speed of 30 ml / min. A single-cycle kinetic method was used to test five increasing concentrations of labelled or non-labelled VHH with a 20 minutes dissociation phase at the end. The kinetic analysis was performed using the Biacore T200 Evaluation software version 3.2.1 (Cytiva, Uppsala, Sweden) using a single-cycle kinetic 1 :1 binding model generating association, dissociation, and affinity constants. The overall correctness of the binding model was expressed with the Chi-square goodness of fit.

[0274] Animal models

[0275] All animal experiments were approved by the Dutch Central Commission for Animal Experimentation (AVD1160020172925) and performed in accordance with the code of practice ‘Dierproeven In Het Kankeronderzoek’. Experimental groups consisted of three- to-five six-to-ten-weeks old female BALB / c-Nude mice (Charles River Laboratories, France). Two to four subcutaneous tumours were obtained by injection of 0.5 x 106 HT- 29 cells or 1.0 x 106 FNA005 in 50% Matrigel (cat# 354230, Corning, USA). Orthotopic colorectal tumour models were induced by the implantation of subcutaneously grown HT- 29-luc2 tumours onto the cecum of a healthy mouse as described in detail by Verbeek et al.

[0025] . Orthotopic tumour growth was monitored using bioluminescence imaging (BLI) using the PerkinElmer MS ® Spectrum Preclinical In Vivo Imaging System (Spectrum, PerkinElmer, USA) as described earlier by Baart et al

[0021] .

[0276] Imaging

[0277] NIR-imaging was performed on anaesthetized mice (1.5-4% isoflurane; Teva Pharmachemie BV, The Netherlands) using the preclinical Pearl Trilogy imager (LI-COR Biosciences, USA) and the clinical Artemis Hand Held Imaging System (Quest Medical Imaging, The Netherlands). Serial imaging of subcutaneous HT-29 colon tumours was performed at one, two, four, eight, and twelve hours after injection. FNA005 tumours were imaged at 30, 60, 90 and 120 minutes after injection. Orthotopic tumours were imaged after sacrification and surgical tumour exposure at one hour after tracer administration. Time-lapse images were gathered using the serial imaging function of the Pearl Trilogy set at two minutes. VHH J3Rsc-IRDye800CW, targeting the human immunodeficiency virus, was used in in vivo experiments as a negative control, whereas the uPAR targeting humanized mAb huATN658-IRDye800CW, referred to as humAb-IRDye800CW, was used as a positive control

[0021] .

[0278] Histology and immunohistochemistry

[0279] After imaging, tumours were resected, fixed, and embedded in paraffin, sectioned, scanned for 800 nm fluorescence (Odyssey; LI-COR Biosciences, USA), and stained with hematoxylin and eosin or uPAR. Immunohistochemistry (IHC) was performed using standard IHC methods using 1.2 pg / ml ATN617 as primary antibody, anti-mouse monoclonal antibody conjugated with HRP (cat# K4001 , Agilent Technologies Inc., USA) as secondary antibody, and visualized with 3,3’-Diaminobenzidine (brown staining). Stained sections were digitalized using the Panoramic Digital Slide Scanner and viewed with Caseviewer 2.3 (both 3D Histech, Hungary).

[0280] Image analysis and statistics

[0281] Pearl Trilogy images were analysed using Image Studio 5.2 (LI-COR Biosciences, USA) and Artemis Hand Held Imaging System images using Spectrum Capture Suite 1.4.3 (Quest Medical Imaging, The Netherlands). Tumour-to-background ratios (TBRs) were calculated by dividing the mean fluorescence intensity (MFI) of the tumour with that of appropriate adjacent normal tissue, i.e. surrounding tissue in subcutaneous models and the cecum in orthotopic models. Means were reported with standard deviations and statistically compared by two-way repeated measurement ANOVA (GraphPad Prism 8; GraphPad Software, USA). Significance levels were set at p < 0.5.

[0282] Results

[0283] Example 1 - In vitro VHH selection

[0284] To obtain VHHs recognizing uPAR, two llamas were immunized with recombinant wild type human uPAR. Subsequently, phage display libraries were created and VHHs selected for their ability to bind immobilized recombinant uPAR. Two VHHs were identified by ELISA with sub-nanomolar affinities (Nb2, SEQ ID NO. 10 and Nb5, SEQ ID NO. 13) and three with nanomolar affinities (Nb1 , SEQ ID NO. 11 ; Nb3, SEQ ID NO. 15 and Nb4, SEQ ID NO. 12) (Figure 1A). Nb6 (SEQ ID NO. 14) was included as a control. After recloning in a standard E. coli expression vector, Nb1-Nb6 were produced and purified, and binding to human uPAR on HEK uPAR WT cells was verified by flow cytometry. Nb2, Nb4 and Nb5 resulted in the highest signal after binding to HEK uPAR WT, Nb1 resulted in lower signal, and Nb3 and Nb6 did not bind at all (Figure 1 B). Due to cleavage of domain 1 (D1) from uPAR by proteases and the resulting release into the extra-cellular matrix, D1 is an unfavourable target when aiming to trace overall uPAR presence on cells. To this end, VHH binding to domain D2-D3 was determined by flow cytometry with HEK uPAR D2-D3 cells, lacking domain D1 . Nb2, Nb4, and Nb5 bound similarly to HEK uPAR D2-3 cells whereas Nb1 , Nb3, and Nb6 did not (Figure 1C). The effect of the occupancy of uPAR on the binding of the anti-uPAR Nb's was studied using HT-29 colonic cancer cells without / with addition of the amino-terminal fragment of urokinase and subsequent flow cytometry. Nb2 showed the highest signal out of all VHHs and competition with the amino-terminal fragment of uPA at ten-fold higher concentrations did not result in a decrease of signal for Nb2 (p = 0.67; Figure 1 D). As the interaction between uPAR and its ligands uPA is known to be species-specific, Nb2 binding to mouse uPAR was determined per flow cytometry with uPAR-positive mouse 4T1 breast cancer and AT84 head-and-neck cancer cells. Nb2 did not bind to mouse uPAR (Figure 2).

[0285] Example 2 - In vitro characterization of huNb2-IRDye800CW

[0286] For future translational purposes Nb2 was humanized and subsequently conjugated to IRDye800CW, resulting in huNb2-IRDye800CW (Figure 6A). uPAR recognition of the conjugate remained unaffected compared to parent Nb2 (Figure 3). SPR analysis revealed the KD of huNb2-IRDye800CW to be 2.21 x 10-9 ± 5.8 x 10-10 with a Ka and Kd of 3.3 x 105 ± 2.3 x 104 and 7.2 x 10-4 ± 1 .3 x 10-4, respectively (Figure 6B). KDs for huNb2 and huNb2-IRDye800CW did not differ significantly (Figure 4). On a panel of 15 human cancer cell lines huNb2-IRDye800CW fluorescence (800 nm) correlated significantly with uPAR receptor expression on the cell wall, as determined by Qifikit (r2 = 0.93, p < 0.0001 ; Figure 6C).

[0287] In vivo targeting of uPAR with 0.1 -1.0 nmol huNb2-IRDye800CW was assessed using the robust subcutaneous HT-29 colorectal mouse model with J3Rsc-IRDye800CW as a non-binding control (Figure 5). Fluorescence accumulated in tumours as early as one hour after huNb2-IRDye800CW intravenous administration, while tumour fluorescence did not, or only minimally, surpass background fluorescence after injection of the not tumour specific VHH J3Rsc-IRDye800CW (Figure 7A). As the metabolizing organ, kidneys were brightly fluorescent throughout the whole imaging period. No transcutaneous liver signal was seen. Absolute signal in the tumour corresponded with injected dose (Figure 7B). Tumour-to-background ratios (TBRs) were consistently higher with huNb2-IRDye800CW compared to J3Rsc-IRDye800CW across all dose groups. The largest difference, with an average TBR increase of 1 .4, was present in the 1 .0 nmol group. All differences were statistically significant in multiple comparison analyses except at the 8 and 12 hour timepoint in the 0.1 nmol dose group (Figure 7C).

[0288] Example 3 - In vivo imaging of huNb2-IRDye800CW in an orthotopic colorectal model and comparison to hu-mAb-IRDye800CW

[0289] A more translational model was achieved by orthotopic implanting of subcutaneously grown HT-29 colon tumours on the cecum of tumour naive mice. Mice were either injected with 1.0 nmol of huNb2-IRDye800CW and imaged 1 hour post-injection or 1.0 nmol hu- mAb-IRDye800CW at 72 hours post injection. Tumours could be easily visualized using the preclinical Pearl Trilogy imager and clinical Artemis Hand Held Imaging System. Both huNB2-IRDye800CW and hu-mAb-IRDye800CW fluorescence corresponded with the BLI-signal (Figure 9A-B). In vivo TBRs were 3.6 ± 0.6 and 4.6 for the VHH and antibody, respectively. Ex vivo TBRs were confirmed to be similar (3.6 ± 0.6. and 4.7, respectively). Further ex vivo biodistribution analysis of the VHH huNb2-IRDye800CW at 1 hour after administration revealed, besides the high tumour signal, low background signals in all organs except for the metabolizing organs (Figure 8).

[0290] Example 4 - In vivo imaging of huNb2-IRDye800CW and J3Rsc-IRDye800CW in a close-to-patient pancreatic ductal adenocarcinoma tumour model

[0291] Patient-derived primary tumour cells more closely represent original tumours with presumably heterogenous receptor expression compared to cloned cancer cell lines. Therefore, huNb2-IRDye800CW targeting was subsequently assessed in a more realistic tumour model in mice, consisting of subcutaneously inoculated primary tumour cells, isolated from endoscopic-ultrasound guided fine needle aspirates of a patient with pancreatic ductal adenocarcinoma (FNA005). Immunohistochemistry on the original FNA005 biopsy showed heterogeneously localized uPAR towards both malignant epithelium and tumour-associated stromal cells using two mAb's detecting different domains of uPAR (Figure 10A). After intravenous administration, 1.0 nmol huNb2- IRDye800CW rapidly accumulated in the tumours with background signal rapidly diminishing, while tumour and background signals could not be differentiated after 1.0 nmol of J3Rsc-IRDye800CW administration (Figure 10B). TBRs after 30 minutes were 1.8 ± 0.3 for huNb2-IRDye800CW and 1.1 ± 0.1 for 1.0 nmol huNb2-IRDye800CW (p < 0.01 ). After 60 minutes the TBRs surpassed the ratio value of two and remained higher than two for the remainder of the imaging period, whereas the TBRs did not surpass 1.1 ± 0.1 for J3Rsc-IRDye800CW (Figure 10C). An additional set of mice received 2.0 nmol huNb2-IRDye800CW to determine whether MFI’s could be improved while retaining TBRs. The higher dose improved visualization of tumours, while kidney background levels remained visually consistent (Figure 10B). TBRs from as early as 30 minutes postinjection were 1.9 ± 0.2 (Figure 10C). The MFI, however, was significantly higher in the 2.0 nmol group compared to the 1.0 nmol group at all time points (p < 0.01 ; Figure 10D). On post-mortem analyses of tumours two hours after huNb2_IRDye800CW injection the majority of the fluorescent signal located towards areas rich in tumour cells, some fluorescence was found in the skin and almost no fluorescence was seen in subcutaneous tissue or necrotic areas of the tumour. In the case of J3Rsc-IRDye800CW, highest signals of fluorescence were seen in the skin, followed by that of tumour necrotic areas. Areas rich in tumour cells barely showed fluorescence (Figure 10E, Figure 11 ). Lastly, human uPAR expression on tumour cells within subcutaneous FNA005 was confirmed per IHC (Figure 12).

[0292] Discussion

[0293] VHHs are excellent candidate moieties for molecular diagnostics and therapeutics

[0010] . They possess a unique set of characteristics combining the precision of an antibody and pharmacokinetics of a peptide that makes previously hard-to-reach targets accessible, both at tissue level (e.g. tumours with elevated interstitial fluid pressure or behind the blood-brain barrier) and protein level (e.g. hidden or intracellular epitopes) [8, 11 , 13]. The lead VHH identified in this study, huNb2, has a low nanomolar affinity for the D2-D3 epitope of uPAR, which is independent of uPAR occupancy, resulting in fast tumour accumulation on uPAR-positive cancer cells, together with rapid background clearance, when administered in vivo in cell-line based and close-to-patient murine models. huNb2 is the first VHH targeting uPAR, a welcome addition both in the VHH and uPAR-targeting landscape [16, 26]. uPAR has previously been targeted in prognostic and therapeutic settings in (pre-)clinical studies using antibodies, nanoparticles, and peptides

[0016] . Amongst the most advanced are a humanized antibody, huATN658, targeting domain 3 of uPAR (uPAR D3, amino acids 268 - 275) and a 9-mer peptide, AE-105, targeting the urokinase-binding cavity with a prominent role for uPAR D1 [27, 28]. Both tracers have specific advantages. In a therapeutic setting, monoclonal antibodies benefit from their extended circulation times, increasing absolute tracer deposition in tumours and subsequent greater reduction in tumour burden [29, 30]. Indeed, multiple molecular imaging studies show that, although smaller antibody-fragments without an Fc portion have unaltered TBRs compared to the parent mAb, they have significantly reduced MFIs [23, 31-33]. However, the long “washout times” needed before suitable TBRs are reached, make full size antibodies suboptimal for imaging, where a rapid imaging window is desirable

[0034] . For this purpose peptides, with their molecular weights far below the renal threshold (approximately 20kDa), are a suitable alternative, as they are practically cleared first-pass and have improved extravasation and intra-tumoral diffusion compared to full-sized mAbs [35, 36].

[0294] Next to structural differences between uPAR targeting tracers, also the targeting site within uPAR determines the efficiency of a tracer for a specific purpose. As the central player in an extensive network, interacting with 42 proteins, uPAR shows various 3- dimensional conformations [15, 16, 37]. A pronounced example are the almost opposite immunohisto-chemical staining patterns (epithelial vs. stromal) shown by Ahn et aL, using two uPAR targeting antibodies against different domains

[0038] . Similarly, using two different mAb's, ATN617 which is a urokinase competitor and ATN615 which binds uPAR independent of the presence of urokinase, shows discrete uPAR staining patterns on the FNA005 biopsy. The (pre-)clinical consequences of these epitopal differences, both for therapy and imaging, has been and remains an area of investigation. For instance, mAb ATN658 exerts a robust anti-tumour effect in vivo by interfering with integrin signalling, whereas ATN-615 fails to elicit an antitumor effect in vivo as it binds a different epitope on the same domain

[0027] . With respect to imaging, co-injection of urokinase, often upregulated in tumour tissue, with the urokinase-based peptide ICG-Glu-Glu-AE-105 led to a 47 ± 4% reduction in vivo in tumour accumulation of the tracer in an in vivo model

[0024] . In our present study, huNb2 was identified to bind uPAR D2-D3 independent of urokinase, implying that more signal will be obtained, including detection of uPAR occupied by uPA. Immunohistochemistry studies are not only indicative of the importance of epitopal differences, they also demonstrate the diverse cell populations within a tumour, including but not limited to neo-angiogenic endothelium, cancer-associated fibroblasts and tumour- associated macrophages, which are all shown to express uPAR [16, 18]. The relevance and potential of the tumour micro environment (TME) for imaging has already been demonstrated with stromal specific tracers such as those targeting neo-angiogenesis like anti-VEGF(R)-directed antibodies or RGD-based peptides

[0039] .

[0295] The current study introduces novel uPAR targeting VHHs of which the humanized lead compound Nb2 has been shown to specifically target domain 2-3 of uPAR independent of urokinase. In addition, huNb2’s potential as a molecular imaging compound has been demonstrated by fluorescence imaging in a close-to-patient pancreatic-ductal adenocarcinoma model. As such, huNb2 is invaluable as a new molecular imaging and therapeutic compound.

[0296] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0297] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0298] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0299] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0300] Sequences

[0301] References

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Claims

Claims1 . An antibody molecule, or an antigen binding fragment thereof, that binds to the amino acid sequence of SEQ ID NO. 1 of human uPAR.

2. The antigen binding fragment of claim 1 , wherein the fragment is selected from the group consisting of a monovalent antibody, a Fab, a Fab’, a F(ab’)2, a single chain variable fragment (scFv), an antibody domain, a nanobody (VHH), a minibody and an scFv-FC; and variations thereof.

3. The antigen binding fragment of claim 2, wherein the fragment is a nanobody (VHH).

4. The antibody molecule or antigen binding fragment of any of claims 1-3, comprising a heavy chain complementarity determining region 1 (VHCDR1) amino acid sequence of SEQ ID NO. 2, a heavy chain complementarity determining region 2 (VHCDR2) amino acid sequence of SEQ ID NO. 3 and a heavy chain complementarity determining region 3 (VHCDR3) amino acid sequence of SEQ ID NO. 4.

5. The antibody molecule or antigen binding fragment of any of claims 1-4, comprising the amino acid sequence of SEQ ID NO. 10.

6. The antibody molecule or antigen binding fragment of any of claims 1-5, wherein the antibody molecule or antigen binding fragment is a nanobody (VHH) comprising the amino acid sequence of SEQ ID NO. 10, wherein the nanobody binds to the amino acid sequence of SEQ ID NO. 1 of human uPAR.

7. The antibody molecule or antigen binding fragment of any of claims 1-6, wherein the antibody molecule or antigen binding fragment is humanised, optimised, and / or deimmunized.

8. An antibody-conjugate which comprises:(a) an antibody molecule or antigen binding fragment according to any of claims 1-7; and (b) a coupling moiety coupled to the antibody molecule or antigen binding fragment.

9. The antibody-conjugate of claim 8, wherein the coupling moiety is a biologically active moiety and / or a diagnostic moiety.

10. The antibody-conjugate of claim 8 or claim 9, wherein the coupling moiety is selected from the group consisting of a detectable label, a toxin, a cytokine, a radionuclide, an enzyme, a photosensitizer and a combination thereof.11 . The antibody-conjugate of any of claims 8-10, wherein the coupling moiety comprises a photodynamic and / or a photoimmuno sensitizer.

12. The antibody-conjugate of any of claims 8-11 , wherein the coupling moiety comprises contrast payloads, optionally fluorophores, radio-active isotopes, gadolinium or micro-bubbles.

13. The antibody-conjugate of any of claims 8-12, wherein the coupling moiety comprises the dye IRDye800CW or variants of the dye ICG.

14. The antibody-conjugate of any of claims 8-10, wherein the coupling moiety comprises a drug.

15. A nucleic acid encoding an antibody molecule or antigen binding fragment according to any of claims 1-7.

16. An expression vector comprising a nucleic acid according to claim 15.

17. A host cell comprising an expression vector according to claim 16.

18. A pharmaceutical composition comprising an antibody molecule or antigen binding fragment according to any of claims 1 -7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17; and further comprising a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

19. A method of producing an antibody molecule or antigen binding fragment according to any of claims 1-7 or an antibody-conjugate according to any of claims 8-14, the method comprising culturing a host cell according to claim 17 under conditions suitable for gene expression.

20. An antibody molecule or antigen binding fragment according to any of claims 1-7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17, and / or a pharmaceutical composition according to claim 18 for use in therapy.21 . An antibody molecule or antigen binding fragment according to any of claims 1-7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17, and / or a pharmaceutical composition according to claim 18 for use in treating diseases where extracellular matrix remodelling plays a pathophysiological role; optionally wherein the diseases are (osteo)arthritis, fibrosis, cardiomyopathy, atherosclerosis, chronic obstructive pulmonary disorder, inflammatory bowel disease, neurodegenerative diseases such as Alzheimer’s disease, and / or cancer.

22. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 20 or 21 , wherein the therapy or treatment comprises administering therapeutics or toxic payloads, optionally a photo-dynamic fluorophore, radio-active isotope, nano-particle, or toxic drug, conjugated to an antibody molecule or antigen binding fragment according to any of claims 1-7.

23. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to any of claims 20-22, wherein the therapeutic or drug is tamoxifen, gefitinib or vemurafenib.

24. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 21 , wherein the cancer is selected from a group consisting of locally aggressive tumours; optionally wherein the cancer is selected from the group consisting of: pancreatic cancer, colorectal cancer, oesophageal cancer, head-and-neck cancer, breast cancer, lung cancer, kidney cancer (renal cell carcinoma), bladder cancer, prostate cancer, ovarium cancer, endometrium cancer, liver cancer, and melanoma.

25. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 24, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma.

26. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to any of claims 20-25, wherein the treatment also targets tumour-associated stromal cells, as well as malignant cells in the cancer.

27. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 21 , wherein the treatment comprises treatment stratification in oncology wherein patients with high uPAR uptake on radioactive imaging are offered more aggressive treatment regimens and patients with low uptake are spared these modalities.

28. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 21 , wherein the treatment is administered to patients who have failed initial therapy or developed resistance to initial therapies, or in combination with initial therapies in order to prevent the development of therapy resistance.

29. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 21 , wherein the treatment comprises last-line therapy in aggressive arthritis using antibody-drug conjugates.

30. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 21 , wherein the treatment comprises photodynamic or photoimmuno therapy.31 . An antibody molecule or antigen binding fragment according to any of claims 1-7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17, and / or a pharmaceutical composition according to claim 18 for use in diagnosis.

32. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 31 , wherein the diagnosis comprises the identification of atherosclerotic plaques that are at risk of rupture using a PET tracer.

33. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 31 , wherein the diagnosis comprises monitoring aggressive arthritis using radioactive tracers.

34. An antibody molecule or antigen binding fragment according to any of claims 1-7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17, and / or a pharmaceutical composition according to claim 18 for use in imaging.

35. A method of imaging a subject, comprising administering an antibody molecule or antigen binding fragment according to any of claims 1 -7, an antibody-conjugate according to any of claims 8-14, a nucleic acid according to claim 15, an expression vector according to claim 16, and / or a host cell according to claim 17, and / or a pharmaceutical composition according to claim 18.

36. The antibody molecule or antigen binding fragment, antibody-conjugate, nucleic acid, expression vector, host cell and / or pharmaceutical composition for use according to claim 34, wherein the imaging is image-guided surgery, optionally fluorescence guided surgery (FGS), and / or optionally uses near-infrared intraoperative imaging.

37. The method of claim 35, wherein the imaging is image-guided surgery, optionally fluorescence guided surgery (FGS), and / or optionally uses near-infrared intraoperative imaging.

Images