WO2007134274A2 - Antibodies to urokinase- type plasminogen activator receptor(upar)bind cancer stem cells: use in diagnosis and therapy - Google Patents

Antibodies to urokinase- type plasminogen activator receptor(upar)bind cancer stem cells: use in diagnosis and therapy Download PDF

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Publication number
WO2007134274A2
WO2007134274A2 PCT/US2007/068845 US2007068845W WO2007134274A2 WO 2007134274 A2 WO2007134274 A2 WO 2007134274A2 US 2007068845 W US2007068845 W US 2007068845W WO 2007134274 A2 WO2007134274 A2 WO 2007134274A2
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upar
cells
cascs
antibody
tumor
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PCT/US2007/068845
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French (fr)
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WO2007134274A3 (en
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Andrew P. Mazar
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Attenuon, Llc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention in the field of biochemistry, immunology and medicine relates to the use of antibodies ("Abs") or other ligands specific for the urokinase-type plasminogen activator receptor (uPAR) to bind, detect, inactivate or purge uPAR-expressing cancer stem cells.
  • Abs or other non-Ab ligands are used in method for diagnosing and treating cancer.
  • uPA urokinase plasminogen activator
  • uPAR is selectively expressed on metastatic tumor cells and angiogenic endothelial cells ("ECs"), but not on other cells;
  • uPAR is an important participant in several extracellular and intracellular pathways required for metastasis that are currently the object of intense drug development efforts;
  • uPA and uPAR are promising targets for the development of diagnostics and therapeutics useful against many different types of tumors/cancers.
  • ECM extracellular matrix
  • BasM basement membrane
  • the uPA “system” which comprises the serine protease uPA, its receptor uPAR, and its specific serpin inhibitor, plasminogen activator inhibitor-type 1 (PAI-I) plays a central role in many of these activities which are responsible for:
  • proMMPs pro- metalloproteases
  • FGF-2 fibroblast growth factor-2
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • TGF ⁇ transforming growth factor- ⁇
  • the uPA system can also initiate localized fibrin turnover which may play a role in angiogenesis.
  • uPA and uPAR have been demonstrated in numerous tumor types including glioblastoma, prostate, breast, colon, hepatocellular, and renal cell carcinoma.
  • uPA is chemotactic for monocytes and mediates both adhesion and migration of these cells. Adhesion and migration require only uPAR occupancy but not uPA catalytic activity. Thus, the uPA/uPAR system is believed to contribute to tumor progression by acting on multiple tumor-associated cell types.
  • Several identified peptide inhibitors of the uPA-uPAR interaction would suffer from the typically poor pharmacological properties of peptides and have not demonstrated the requisite levels of activity even in cell-based assays (Ploug M et al. (2001) Biochemistry 40:12157-68).
  • tumors include a rare cell population with stem cell-like properties, defined by self-renewal and proliferative capacity, which are responsible for growth and progression of the tumor.
  • AML acute myelogenous leukemia
  • HSC normal hematopoietic stem cells
  • LSC leukemia stem cells
  • neural stem cell biology had its origins in the discovery that culturing mammalian brain cells in serum- free conditions (in the presence of epidermal growth factor (EGF) and bovine fibroblast growth factor (bFGF), yielded clonally derived colonies of undifferentiated neural cells and that cells in these colonies showed basic properties of stem cells: self-renewal and multi-lineage differentiation (Reynolds BA et al. , Science, 1992, 255:1707-10. Investigators began to question whether this approach could be used to isolate stem cells from brain tumors.
  • EGF epidermal growth factor
  • bFGF bovine fibroblast growth factor
  • brain tumors contain a small population of cells with stem cell properties (as measured in vitro).
  • the self-renewal and differentiation properties of the candidate brain tumor stem cells varied among tumors of different histologic types.
  • Clonogenic brain tumor cells were prospectively enriched by cell-sorting techniques employing the same cell-surface marker CD 133 (of unknown function) that was useful for enriching stem cells from normal human brain (Uchida, N et al., Proc Natl Acad Sci, 2000, 97: 14720-5). Human brain tumors of distinct types from adults and children were found to contain a subpopulation of CD133 + brain tumor cells with stem cell properties in vitro and that further defined a stem cell in vivo (Singh, SK et al, Nature, 2004, 452:396-401).
  • CD133 + cells Malignant brain tumors tended to have a relatively high frequency of CD133 + cells (up to 30%), whereas less aggressive tumors had fewer such cells (0.5-5.0%). As few as 100 malignant CD133 + cells could in some cases initiate growth of a serially transplantable brain tumor following intracerebral engraftment into immunodeficient (NOD/SCID) mice. Tumors that resulted from these CD133 + cell injections were a phenocopy of the original patient tumor. Moreover, injection of 10 5 CD133 " cells survived when engrafted into the mouse brain, but these cells did not form brain tumor masses.
  • cancer stem cells These various cancer stem cells are referred to (sometimes interchangeably) as cancer stem cells (“CaSC”), “cancer progenitor” cells, “tumor stem” cells (TuSC) or “tumor progenitor” cells.
  • CaSC cancer stem cells
  • TuSC tumor stem cells
  • TuSC tumor progenitor progenitor progenitor
  • Stem cells in adult organisms are responsible for tissue renewal and repair of aged or damaged tissue.
  • An important characteristic of stem cells is their ability for self-renewal without loss of proliferative capacity with each cell division. They can differentiate to form specific types of tissue under the influence of microenvironmental and other factors.
  • Stem cells are believed to divide asymmetrically producing two daughter cells - one of which is a new stem cell and the second is more committed progenitor cell with the ability to differentiate and proliferate, but without the capacity for self-renewal.
  • Cancer stem cells are in many aspects similar. Cells making up a tumor are heterogeneous and comprise (a) rare tumor initiating cells and (b) abundant non- tumor initiating cells. Tumor initiating cells - the CaSCs - can self-renew and proliferate, express typical stem cell markers, and are relatively resistant to drugs. It is not clear whether CaSCs
  • DC:50410124.1 « ATT-17 originate from normal stem cells as a consequence of genetic and epigenetic changes and/or whether they arise by de-differentiation of somatic tumor cells to a stem-like cell stage. It is likely that both mechanisms contribute to the origin of CaSCs. Dysregulation of stem cell self-renewal is a likely precursor to the development of cancer. CaSCs have been identified in, and isolated from, human tumors and in tumor cell lines (see references cited above). To date, the existence of CaSCs has been demonstrated in acute and chronic myeloid leukemia, breast cancer, brain tumors, lung cancer and gastrointestinal tumors.
  • the CaSC model is also consistent with certain clinical observations. Although standard chemotherapy kills most cells in a tumor, CaSCs survive. Despite their small number, they may be the cause of tumor recurrence, which sometimes takes place years after "successful" treatment of a primary tumor. Furthermore, growth of metastases at distinct sites of body and their cellular heterogeneity may be consequence of CaSC differentiation and/or dedifferentiation and asymmetric division. There is a recognized need in the art to increase our understanding of CaSCs in order to find approaches to destroying them, a step which may contribute significantly to the therapeutic management of malignant tumors. The present invention is directed to this objective. uPAR on Cancer Stem Cells
  • uPAR is expressed or upregulated on the surface of CaSCs (Gutova, M et al, Proc Amer Assoc Cancer Res 2006; 47: Abstr #3986, 2006, April). These investigators identified a small population of such cells in small cell lung cancer (SCLC). These CaSCs expressed high levels of the uPAR/CD87 gene, which is known to contribute to development of the most invasive phenotypes in several cancers, e.g.
  • BM transplantation has been employed to induce or maintain a state of remission in patients with various malignancies, primarily leukemias.
  • BM cells of a patient with a malignancy are removed during remission and stored for re-administration after the patient relapses into active disease (Gale, RP et al, 1987, Sem Hematol 24:40-54).
  • This obviates the need for an allogeneic HLA-matched donor from whom BM transplants are burdened with other genetic incompatibilities.
  • a significant drawback for autologous BM is that malignant cells, or CaSCs, may remain in the BM obtained during remission.
  • US Pat. 5,087,636 describes methods to purge malignant cells from the bone marrow or other hemopoietic cell populations of leukemia patients with active disease (not in remission) or from patients with other (nonleukemic) malignancies who would benefit from such a purging procedure employing photoactivation of green porphyrins added to the hemopoietic cells.
  • U.S. Pat. 7,001,770 (February 21, 2006 ; to Atencio et al. describes a method for purging or ablating neoplastic cells that contaminate a population of normal cells ex vivo by administering a recombinant adenoviral vectors encoding p53.
  • Stem cell preparations are conventionally obtained by apheresis of mobilized or non-mobilized peripheral blood. This document discusses "3-log purges” ⁇ i.e. removal of approximately 99.9% of the tumor cells from the stem cell preparation) and more efficient “5-log purges” (removal of approximately 99.999% of tumor cells from the preparation.
  • U.S. Pat. 6,331,175 discloses methods and compositions for detecting or treating normal, hypoplastic, ectopic or remnant tissue, an organ or cells in a mammal.
  • the method involves parenteral administration to the subject, at a locus and by a route providing access to above-mentioned tissue or organ, of a composition comprising Abs or Ab fragments which specifically bind to targeted organ, tissue or cell.
  • the antibody/fragment may be administered alone, or labeled or conjugated with an imaging agent, a therapeutic moiety, a cytoprotective agent or an activating agent.
  • the art recognizes the utility of removing selected populations of cells from a mixture, particularly malignant cells, before administering the remaining non-malignant and presumably therapeutic cells to the donor or a third party subject, as a therapeutic measure.
  • the present invention is also directed to such an approach by using antibodies or other ligands to target uPAR on cancer cells and, more particularly, CaSCs, and remove them from a mixed population.
  • anti-uPAR Abs preferably monoclonal antibodies (mAbs) to identify CaSCs, positively select (isolate) such cells from mixed populations for study, diagnose the presence of CaSCs with these Abs, to treat primary or metastatic tumors by inhibiting the proliferation of or by killing such CaSCs.
  • the antibodies can be used to treat cell populations that are being administered to a subject, and that are suspected of comprising uPAR+ CaSCs ex vivo to purge such cells from the populations before the cells are infused or implanted in the subject.
  • An example is to treat autologous bone marrow cells or enriched hematopoietic stem cells (HSCs) from cancer patients, primary leukemia patients, prior to re- infusion of these sources of HSCs.
  • Treatment of such a cell population with uPAR Abs according to this invention may be performed in conjunction with other treatments that are intended to positively selected HSCs or negatively select remaining leukemic (or leukemic progenitor) cells.
  • the invention is therefore directed in part to a method for functionally inactivating or killing cancer stem cells (CaSCs), comprising providing to said CaSCs an effective amount of a ligand that binds to uPAR.
  • CaSCs cancer stem cells
  • DC:50410124.1 O ATT-17 Also provided is method for treating a subject having or susceptible to a malignant disease characterized by the presence of CaSCs, comprising administering to the subject functionally- inactivating amount of a therapeutic composition that comprises a ligand that binds to uPAR.
  • the ligand is preferably an antibody or an antigen binding fragment thereof.
  • anti-uPAR Abs preferably mAbs that are known in the art, and some of which are commercially available
  • the present inventors have produced a set of anti-uPAR mAbs with the property that they bind to uPA-uPAR complexes and that serve to inhibit the interactions of these proteins and downstream effects (such as further interactions with integrins).
  • the action of such mAbs can inhibit tumor growth and metastasis by blocking angiogenesis, tumor invasion and proliferation.
  • such mAbs also act by interfering with self- renewal or differentiation of CaSCs.
  • these mAbs may be used to targeting therapeutic agents or diagnostic (imaging) agents to tumors, and via their binding to uPAR on CaSCs, to permit the identification, localization and/or elimination of these cells.
  • antibodies that target uPAR are effective in animal models of cancer growth (such as the A2780 ovarian cancer model and the A549 lung cancer model).
  • the antibodies that are useful in the present invention preferably recognized peptide epitopes of uPAR as expressed on CaSCs.
  • uPA-uPAR Malignant tumor cells and CaSCs (as well as angiogenic ECs) gain a selective advantage in cell migration and invasiveness, resulting at least in part from the cells' expression of uPAR on their surface. These uPAR molecules are saturated by binding the endogenously produced ligand, uPA.
  • mAbs or other binding partners
  • Preferred downstream ligands of uPA-uPAR, or of uPAR alone include integrins, low-density lipoprotein receptor-related protein (LRP) and others. Such downstream ligands mediate or play a role in cell signaling, migration and/or invasion.
  • LRP low-density lipoprotein receptor-related protein
  • two mAbs specifically bind uPA-occupied uPAR and thus serve as exemplary molecules that can bind uPAR regardless of the presence of uPA ligand.
  • the mAbs can detect both occupied and unoccupied uPAR in a tumor or other diseased tissue where the uPA
  • DC:50410124.1 Q ATT-17 system plays a role in the pathobiology.
  • Preferred Abs or other non-Ab ligands for uPAR are those that do not bind to the uPA-binding site of uPAR.
  • the present inventors have identified the epitopes to which these Abs bind.
  • Such peptides or natural or synthetic peptides or peptide derivatives that retain the 3D structure of these epitopes are useful as therapeutic and/or diagnostic agents.
  • Abs antigen binding fragments
  • scFv single chain Abs
  • non-Ab polypeptides and peptides aptamers, etc.
  • small organic molecules to target CaSCs.
  • the above Ab may be a mAb, or an antigen binding fragment thereof.
  • Preferred mAbs are humanized chimeric or human mAbs.
  • the above Ab or other uPAR ligand is (a) diagnostically labeled (with a detectable label); or (b) labeled with, conjugated to, or fused to (in the case of a polypeptide), a therapeutically active moiety, rendering the ligand therapeutically active.
  • a diagnostic composition comprising (a) the diagnostically labeled ligand, primarily an Ab, as above; and (b) a diagnostically acceptable carrier.
  • the ligand is preferably labeled with a radionuclide, a PET- imageable agent, an MRI-imageable agent, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer.
  • a radionuclide is 3 H, 14 C, 35 S,
  • fluorescers or fluorogens are fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green and Texas Red.
  • the present invention provides a therapeutic anti-tumor pharmaceutical composition that inhibits tumor growth and/or tumor metastasis comprising (a) an effective amount of the therapeutically active ligand above, and (b) a pharmaceutically acceptable carrier.
  • This composition is preferably in a form suitable for injection.
  • the therapeutically active moiety may be conjugated directly to, or bound indirectly to, the ligand.
  • a preferred therapeutic moiety is a chemotherapeutic drug, a toxin or a therapeutic radionuclide - described below.
  • the therapeutically active moiety may be a peptide or polypeptide, e.g., a toxin, which is fused to the uPAR-binding Ab or other ligand.
  • This invention is directed to a method for inhibiting CaSC migration, invasion, proliferation or the process of tumor angiogenesis in which CaSCs play a role. Also included is a method for inducing apoptosis in uPAR+ CaSCs, comprising contacting these cells with an effective amount of a therapeutically active Ab or other uPAR-ligand, as above. Also included is a method for treating a subject having a disease, disorder or condition characterized by undesired tumor growth and/or tumor metastasis comprising administering to the subject an effective amount of the above therapeutic pharmaceutical composition that bind to tumor CaSCs.
  • One embodiment is an assay method for detecting uPAR-expressing cells in a cell mixture suspected of including CaSCs in a sample of tumor, tissue, organ, or cell population.
  • Abs preferably mAbs, peptides or other chemical entities that target the uPAR can bind CaSCs and thereby be used to in the treatment and/or diagnosis of cancer or precancerous states.
  • the present inventors generated a panel of mAbs targeting uPAR which is an ideal target for antibodies because it is expressed on the cell surface.
  • uPAR is also expressed at the tumor- vasculature interface (on invasive tumor cells, angiogenic endothelial cells, or tumor-associated macrophages).
  • anti-uPAR Abs would be able to enter tumors, where CaSCs may be found, and serve as diagnostic agents or exert therapeutic effects.
  • uPAR is not normally expressed on quiescent tissues, which should minimize the potential for toxicity when employing a therapeutic Ab and minimize non-specific signals (or false positives) when employing a diagnostic Ab.
  • suPAR fragment of the soluble form of uPAR
  • DC:50410124.1 ⁇ ⁇ ATT-17 an immunogen is expected to overcome heterogeneous binding to uPAR, a property associated with most other anti-uPAR mAbs.
  • studies performed as part of a Leukocyte Antigen Workshop compared anti-uPAR antibodies available in 1995-1996 and found all of them to be specific for carbohydrate, not protein, epitopes (Manupello, J. et al, (1996) Tiss. Antigens 48: 368.).
  • uPAR expressed in tumors is highly and heterogeneously glycosylated, and the glycosylation pattern and representation of different iso forms change in response to various signals (Stoppelli MP et al. (1985) Proc. Natl. Acad. Sci. USA 82 4939-4943).
  • anti-uPAR antibodies raised against carbohydrate epitopes are unlikely to recognize all iso forms of uPAR and may cross- react undesirably with other proteins that comprise glycosylation structures similar to those present on uPAR.
  • Use of S2 has led to the identification of mAbs that recognize the protein epitopes within suPAR.
  • a mutant form of suPAR has been expressed in which all glycosylation sites have been mutated.
  • the existing murine mAb clones may be humanized or primatized.
  • the present inventors' ability to generate conformationally intact domain fragments of suPAR has allowed them to produce mAbs against isolated domains of suPAR (isolated Dl and isolated D2D3).
  • This invention is thus directed in part to methods using a mAb that binds to uPAR and is produced by a process comprising the initial step of immunizing a mammal, preferably a mouse, with
  • DC:50410124.1 y ⁇ ATT-17 Abs may be tested preferably in xenogeneic tumor models, two preferred examples of which are the A2780 and A549 models (described in more detail below).
  • an Ab or mAb has "essentially the same antigen- binding characteristics" as a reference mAb if it demonstrates a similar specificity profile (e.g., by rank order comparison), and has affinity for the relevant antigen (e.g., a uPAR epitope) within 1.5 orders of magnitude, more preferably within one order of magnitude, of the reference Ab.
  • the antibodies are evaluated for action on CaSCs by any of a number of assays. These may include functional assays, for example, inhibition of self-renewal or proliferation of CaSCs (including CaSC lines). This can be measured in clonogenic assays in vitro (see, for example Jacobs, P et al, Hematology, 2005, 70:321-6). Antibody internalization may also be measured.
  • the present invention comprises a method of ablating or killing CaSC by contacting them with a polypeptide molecule, preferably an anti-uPAR mAb, which, recognizes the uPAR on the surface of the CaSCs, and results in the binding, and, preferably the functional inactivation of the CaSC.
  • a polypeptide molecule preferably an anti-uPAR mAb, which, recognizes the uPAR on the surface of the CaSCs, and results in the binding, and, preferably the functional inactivation of the CaSC.
  • “Functional inactivation” refers to a statistically significant reduction in the ability of the CaSC to (a) proliferate and undergo self-renewal, (b) differentiate and form colonies of tumor/cancer cells in vitro or in vivo, (c) migrate, extravasate, metastasize, and the like.
  • the Ab or uPAR-binding fragment thereof can be used alone, or can be combined with other natural or artificial agents that kill cells to which the Abs have bound, such as complement, or can be chemically linked or otherwise associated with another substance that is effective in killing or ablate the CaSCs after the Ab has bound.
  • the Ab may, but is not required to, be internalized after binding cell surface uPAR.
  • the methods of the present invention are particularly useful in killing or ablating any type of CaSC that expresses uPAR on its surface.
  • every type of tumor has associated with it a CaSC, although these have been demonstrated definitively thus far in a limited number of cancer types.
  • the present methods are particularly useful for binding to and functionally inactivating CaSCs associated with acute myeloid leukemia, chronic myeloid leukemia, breast cancer, various brain tumors, lung cancer and gastrointestinal cancer.
  • Another aspect of the present invention relates to a method of detecting the presence, or quantitating, CaSCs in a mixed cell population including a tumor/cancer cell population, whether in the form of a cell suspension or cell culture in vitro, or a solid tumor or a leukemia in a subject.
  • the present Ab When contacted with CaSCs, the present Ab will bind to them, and if bound to a detectable label, will permit detection of these cells. Abs immobilized to a solid support, for example a resin or magnetic beads, can also be used to remove CaSCs from BM or whole blood (or any cell population). If bound to a therapeutic moiety, the Ab will bind to and inactivate or kill the target cells, for example, within a cancerous tissue.
  • the method is directed to detecting CaSCs or inactivating/killing such cells in a tumor or cancerous tissue
  • the Abs of the present invention bind to living CaSCs therapeutic methods for eliminating or inactivating these cells are more effective than those which require that target cells be lysed or their proteins be denatured for Ab recognition and binding. For the same reasons, diagnostic and imaging methods which determine the location of living CaSCs are improved by employing the present Abs.
  • the present method permits the monitoring of the efficacy of a treatment regimen directed to eliminating or inactivating CaSCs in vivo by permitting identification and quantification of these cells in a subject after the therapy.
  • Anti-idiotypic antibodies are described, for example, in Idiotypy in Biology and Medicine, Academic Press, New York, 1984; Immunological Reviews Vol. 79, 1984 and Vol. 90, 1986; Curr. Top. Microbiol, Immunol. Vol. 119, 1985; Bona, C. et al, CRC Crit. Rev. Immunol, pp. 33-81 (1981); Jerne, NK, Ann. Immunol. 725C:373-389 (1974); Urbain, J et al, Ann. Immunol. I33D ⁇ 79- (1982); Rajewsky, K. et al, Ann. Rev. Immunol. 7:569-607 (1983).
  • the present invention provides Abs, both polyclonal and monoclonal, reactive with uPAR including those that bind uPA-uPAR complexes that inhibit interactions of uPAR with integrins or other downstream targets.
  • the Abs may be xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized or chimeric Abs.
  • Antiidiotypic Abs specific for the idiotype of, for example, an anti- uPAR Ab are also included.
  • antibody is meant to include both intact immunoglobulin (Ig) molecules as well as fragments and derivative thereof, that may be produced by proteolytic cleavage of Ig molecules or engineered genetically or chemically. Fragments include, for example, Fab, Fab', F(ab') 2 and Fv, each of which is capable of binding antigen. These fragments lack the Fc fragment of intact Ab and have an additional advantage, if used therapeutically, of clearing more rapidly from the circulation and undergoing less non-specific tissue binding than intact Abs. Papain treatment of Ig's produces Fab fragments; pepsin treatment produces F(ab') 2 fragments. These fragments may also produced by genetic or protein engineering using methods well known in the art. A Fab fragment is a multimeric protein consisting of the portion of an Ig molecule containing the immunologically active portions
  • Fab fragments are typically prepared by proteolytic digestion of substantially intact Ig molecules with papain using methods that are well known in the art. However, a Fab fragment may also be prepared by expressing in a suitable host cell the desired portions of Ig H chain and L chain using methods well known in the art.
  • a (Fab') 2 fragment is a tetramer that includes a fragment of two H and two L chains.
  • the Fv fragment is a multimeric protein consisting of the immunologically active portions of an Ig H chain variable (V) region (V H ) and an Ig L chain V region (V L ) covalently coupled together and capable of specifically combining with antigen.
  • Fv fragments are typically prepared by expressing in suitable host cell the desired portions of Ig V H region and V L region using methods well known in the art.
  • Single-chain antigen-binding protein or single chain Ab is a polypeptide composed of an Ig V L amino acid sequence tethered to an Ig V H amino acid sequence by a peptide that links the C-terminus of the V L sequence to the N-terminus of the V H sequence.
  • Polyclonal Abs are obtained as sera from immunized animals such as rabbits, goats, rodents, etc. and may be used directly without further treatment or may be subjected to conventional enrichment or purification methods such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography.
  • An immunogen for generation of the anti-uPAR Abs of this invention may comprise uPAR, suPAR, uPA/uPAR or uPAR-integrin complexes/ or an epitope-bearing fragments or derivative thereof.
  • Useful immunogens are produced in a variety of ways known in the art, e.g., expression of cloned genes using conventional recombinant methods, isolation from cells of origin, cell populations expressing high levels of e.g., uPA or uPAR, etc. In the case of shorter fragments, they may be chemically synthesized.
  • a preferred immunogen is the D2D3 fragment of suPAR.
  • the mAbs may be produced using conventional hybridoma technology, such as the procedures introduced by Kohler and Milstein ⁇ Nature, 256:495-97 (1975)),-and modifications thereof (see above references).
  • An animal preferably a mouse is primed by immunization with an immunogen as above to elicit the desired Ab response in the primed animal.
  • B lymphocytes from the lymph nodes, spleens or peripheral blood of a primed, animal are fused with myeloma cells, generally in the presence of a fusion promoting agent such as polyethylene glycol (PEG).
  • a fusion promoting agent such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • Any of a number of murine myeloma cell lines are available for such use: the P3-NSl/l-Ag4-l, P3-x63-k0Ag8.653, Sp2/0-Agl4, or HL1-653 myeloma lines (available
  • Subsequent steps include growth in selective medium so that unfused parental myeloma cells and donor lymphocyte cells eventually die while only the hybridoma cells survive. These are cloned and grown and their supernatants screened for the presence of Ab of the desired specificity, e.g., by immunoassay techniques. Positive clones are subcloned, e.g., by limiting dilution, and the mAbs are isolated.
  • Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (see generally Fink et ah, Prog. Clin. Pathol., 9:121-33 (1984)).
  • the individual cell line is propagated in culture and the culture medium containing high concentrations of a single mAb can be harvested by decantation, filtration, or centrifugation.
  • uPAR immobilized onto plastic is preferred for the primary screening.
  • Cells that overexpress uPAR may be used to demonstrate cell binding of an anti- uPAR mAb.
  • Many tumor cell lines overexpressing uPAR are well-known and publicly available; these may be used for screening.
  • Cells are generally plated in 96-well microplates. The cells may be fixed, e.g.,, with methanol/acetone (50/50), and the binding detected by immunofluorescence staining.
  • the mAbs may be radiolabeled and binding detected by measurement of radioactivity.
  • Hybridoma supernatants are preferably studied using methods employing immunofluorescence or ELISA.
  • the Ab is a mAb designated ATN-615 or ATN-658, both of which are IgGl Abs.
  • the Ab is a chimeric Ab that recognizes an epitope recognized by ATN-615 or ATN-658.
  • the chimeric Abs of the invention comprise individual chimeric H and L Ig chains.
  • the chimeric H chain comprises an antigen binding region derived from the H chain of a non-human Ab specific for e.g., uPA/uPAR or uPAR-integrin complex, for example, mAb ATN-615 or ATN-658, which is linked to at least a portion of a human C H region.
  • a chimeric L chain comprises an antigen binding region derived from the L chain of a non-human Ab specific for the target antigen, such as the hybridoma ATN-615 or ATN-658, linked to at least a portion of a human C L region.
  • a non-human Ab specific for the target antigen such as the hybridoma ATN-615 or ATN-658
  • the term "antigen binding region” refers to that portion of an Ab molecule which contains the amino acid residues that interact with an antigen and confer on the Ab its specificity and affinity for the antigen.
  • the Ab region includes the "framework” amino acid residues necessary to maintain the proper conformation of the antigen-binding (or "contact”) residues.
  • chimeric antibody includes monovalent, divalent or polyvalent Igs.
  • a monovalent chimeric Ab is an HL dimer formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain.
  • a divalent chimeric Ab is tetramer H 2 L 2 formed by two HL dimers associated through at least one disulfide bridge.
  • a polyvalent chimeric Ab can also be produced, for example, by employing a C R region that aggregates (e.g., from an IgM H chain, termed the ⁇ chain).
  • the invention also provides for "derivatives" of the mouse mAbs or the chimeric Abs, which term includes those proteins encoded by truncated or modified genes to yield molecular species functionally resembling the Ig fragments.
  • the modifications include, but are not limited to, addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins.
  • the fragments and derivatives can be produced from any of the hosts of this invention.
  • Abs, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears et al, Proc. Natl. Acad. ScL USA 72:353-357 (1975). With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the Ig chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled Ig, fragment or derivative.
  • the antigen binding region of the chimeric Ab (or a human mAb) of the present invention is derived preferably from a non-human Ab specific for e.g. , uPAR.
  • a non-human Ab specific for e.g. , uPAR examples of preferred chimeric Abs (or human Abs) are those described in WO 2005/116077.
  • the chimeric Abs of the present invention are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of an Ab of the invention, preferably non-human, and joining these DNA segments to DNA segments encoding human C H and C L regions, respectively, to produce chimeric Ig-encoding genes.
  • the humanized Ab may be the product of an animal having transgenic human Ig Constant region genes (see for example WO90/10077 and WO90/04036).
  • the Ab of interest may be genetically engineered to substitute the CHi, CH 2 , CH 3 , hinge domains, and/or the framework domain with the corresponding human sequence (see WO92/02190).
  • the Ab of the present invention may be produced as a single chain Ab or scFv instead of the normal multimeric structure.
  • Single chain Abs include the hypervariable regions from an Ig of interest and recreate the antigen binding site of the native Ig while being a fraction of the size of the intact Ig (Skerra, A. et al (1988) Science, 240: 1038-1041; Pluckthun, A. et al (1989) Methods Enzymol 178: 497-515; Winter, G. et al (1991) Nature, 349: 293-299); Bird et al, (1988) Science 242:423; Huston et al (1988) Proc. Natl Acad. Sci.
  • DNA sequences encoding the V regions of the H chain and the L chain are ligated to a linker encoding at least about 4 amino acids (typically small neutral amino acids).
  • the protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original Ab.
  • One method of producing the single chain Abs of the present invention is to link two or more peptides or polypeptides together by protein chemistry techniques known in the art and described in more detail in WO 2005/116077.
  • Abs can be selected for particular desired properties.
  • Ab screening procedures can include any of the in vitro or in vivo bioassays that measure binding to uPAR (or too uPA/uPAR or uPAR-integrin complexes), , to cells expressing the relevant polypeptide or peptide epitope.
  • the Abs may be screened in various of tumor models such as a xenogeneic mouse model in which a human tumor cell line expressing the antigen is grown in immunocompromised, e.g., nude, mice.
  • diagnostically labeled means that the present Ab has attached to it a diagnostically detectable label.
  • labels and methods of labeling known to those of ordinary skill in the art, described below.
  • General classes of labels which can be used in
  • the present invention include radioactive isotopes, paramagnetic isotopes, and compounds which can be imaged by positron emission tomography (PET), fluorescent or colored compounds, etc.
  • Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels.
  • Useful radiolabels (radionuclides), which are detected simply by gamma counter, scintillation counter or autoradiography include 3 H, 125 I, 131 1, 35 S and 14 C. 131 I is also a useful therapeutic isotope (see below).
  • U.S. patents disclose methods and compositions for complexing metals to larger molecules, including description of useful chelating agents.
  • the metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules.
  • These documents include: U.S. Patents 5,627,286; 5,618,513; 5,567,408; 5,443,816; and 5,561,220.
  • Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • the fluorophore such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR., 1996).
  • the long wavelength rhodamines which are basically Rhodamine GreenTM derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known.
  • spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluoresceins for many biological applications.
  • This group includes the tetramethylrhodamines, X-rhodamines and Texas RedTM derivatives.
  • Other preferred fluorophores for derivatizing the peptide according to this invention are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.
  • semiconductor nanocrystals comprising, for example, cadmium sulfate (Bruchez, M et al, Science 257:2013-2016 (1998), and quantum dots, e.g., zinc-sulf ⁇ de-capped Cd selenide (Chan, WC et al, Science 281 :2016-2018 (1998)).
  • the amino group of the Ab is allowed to react with reagents that yield fluorescent products, for example, fluorescamine, dialdehydes such as o-phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.
  • reagents that yield fluorescent products for example, fluorescamine, dialdehydes such as o-phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.
  • 7-nitrobenz-2-oxa-l,3-diazole (NBD) derivatives, both chloride and fluoride, are useful to modify amines to yield fluorescent products.
  • the Ab of the invention can also be labeled for detection using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the peptide using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA, see Example X, infra) or ethylenediaminetetraacetic acid (EDTA). DTPA, for example, is available as the anhydride, which can readily modify the NH2-containing peptides of this invention.
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • Radiolabeled antibodies labeled with ⁇ -emitters, positron-emitters, x-ray emitters and fluorescence-emitters are suitable for localization and/or therapy, while ⁇ -emitters and ⁇ -emitters may also be used for therapy.
  • Suitable radioisotopes for the methods of the present invention include: 211 At, 123 I, 125 I, 126 I, 131 I, 133 I, 212 Bi, 77 Br, 111 In, 113 In, 67 Ga, 68 , 95 Ru, 97 Ru, 103 Ru, 105 Ru, 107 Hg, 203 Hg, 186 Re, 188 Re, 121m Te, 122m Te, 125m Te, 165 Tm, 167 Tm, 168 Tm, 99m Tc, 18 Fl, 111 Ag, 197 Pt, 109 Pd, 67 Cu, 32 P, 33 P, 90 Y, 47 Sc, 153 Sm, 177 Lu, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 166 No, 199 Au, 57 Co, 58 Co, 51 Cr, 59 Fe, 75 Se, 201 Tl, and 169 Yb.
  • the radioisotope will emit in the 10-5,000 kev range, more preferably 50
  • those preferred for diagnostic use include 123 I, 131 I, 111 In, 67 Ga, 97 Ru,
  • the amount of labeled Ab needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.001 mg/kg to 100 mg/kg.
  • the Ab can also be made detectable by coupling to a phosphorescent or a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged peptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescers are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the peptides. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence
  • DC:50410124.1 21 ATT-17 of a bio luminescent protein is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • colorimetric detection is used, based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients.
  • In situ detection of the labeled peptide may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label.
  • histological methods such as staining procedures
  • the type of detection instrument available is a major factor in selecting a radionuclide.
  • the radionuclide chosen must have a type of decay which is detectable by a particular instrument.
  • any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention.
  • Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized.
  • a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
  • Imaging may be used to detect occult metastases which are not observable by other methods. Imaging could be used, for example, to stage tumors non-invasively.
  • One preferred assay for detecting the antigens in a tissue is by immunohistochemistry, using any conventional assay methods, with which the art is replete.
  • a preferred assay is the one described in the Examples below. For a description of such methods, see, for example, Dabbs, DJ, Diagnostic Immunohistochemistry, Churchill Livingstone, 2001, which is incorporated by reference in its entirety.
  • Preferred immunoassays are enzyme immunoassays (EIA' s) such as ELISA, which employ antigens or Abs immobilized to solid supports.
  • EIA' s enzyme immunoassays
  • the solid support is preferably any one of polystyrene, polypropylene, polyethylene, dextran, nylon,
  • the surface of polystyrene or other plastic multiwell plates serves as the solid support.
  • Multiwell plates in which the bottoms of the wells comprise nitrocellulose or a similar membrane material and through which liquid can be moved under pressure or vacuum may also be used.
  • Typical, and preferred, immunoassays include "forward" assays in which the Ab immobilized to a solid support is first contacted with the sample being tested to bind or "extract" the antigen from the sample by formation of a binary immobilized Ab-antigen complex. After suitable incubation, the solid support is washed to remove the residue of the fluid sample including unbound antigen, if any, and then contacted with the solution containing an unknown quantity of labeled Ab (which functions as a "reporter molecule").
  • This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the amount of immobilized labeled Ab with the amount immobilized when a standard sample containing a known quantity of antigen is used.
  • a simultaneous assay involves a single incubation step as the immobilized Ab and labeled Ab are added simultaneously to the sample. After appropriate incubation, the solid support is washed to remove residue of the sample and uncomplexed labeled Ab. The presence or amount of labeled Ab associated with the solid support is then determined as in the above conventional "forward" sandwich assay.
  • the uPAR-targeting Ab and/or conjugate thereof is readily tested for binding to uPAR, preferably by measuring inhibition of the binding of [ 125 I]DFP-uPA to uPAR in a competitive ligand-binding assay or by directly labeling the Ab with [ 125 I] .
  • the assay may employ whole cells that express uPAR, for example cells lines such as A2780 or HeLa.
  • a preferred assay is conducted as follows. Cells (about 5 x 10 4 /well) are plated in medium (e.g., MEM with Earle's salts/10% FBS + antibiotics) in 24-well plates, then incubated in a humid 5% CO 2 atmosphere until the cells reach 70% confluence.
  • Catalytically inactivated high molecular weight uPA (DFP-uPA) is radioiodinated using Iodo-gen ® (Pierce) to a specific activity of about 250,000 cpm/ ⁇ g.
  • the cell-containing plates are then chilled on ice and the cells are washed twice (5 minutes each) with cold PBS/ 0.05% Tween-80.
  • Test Abs and/or conjugates thereof are serially diluted in cold PBS/ 0.1 % BSA/ 0.01% Tween-80 and added to each well to a final volume of 0.3mL 10 minutes prior to the addition of the [ 125 I]DFP-uPA.
  • Each well then receives 9500 cpm of [ 125 I]DFP-uPA at a final concentration of 0.2 nM).
  • the plates are then incubated at 4°C for 2 hrs, after which time the cells are washed 3x (5 minutes each) with cold PBS/ 0.05% Tween-80.
  • NaOH (IN) is added to each well in 0.5 mL to lyse the cells, and the plate is incubated for 5 minutes at room temperature or until all the cells in each well are lysed as determined by microscopic examination.
  • the contents of each well are then aspirated and the total counts in each well determined using a gamma counter.
  • Each compound is tested in triplicate and the results are expressed as a percentage of the total radioactivity measured in wells containing [ 125 I]DFP-uPA alone, which is taken to represent maximum (100 %) binding.
  • the inhibition of binding of [ 125 I]DFP-uPA to uPAR is usually dose-related, such that the concentration of the test compound necessary to produce a 50% inhibition of binding (the IC50 value), which is expected to fall in the linear part of the curve, is easily determined.
  • Abs and/or conjugates thereof have IC50 values of less than about 10 "5 M.
  • Abs and/or conjugates thereof have IC50 values of less than about 10 "6 M, more preferably, less than about 10 "7 M.
  • transwells are coated with type I collagen (50 ⁇ g/mL) by adding 200 ⁇ L of the collagen solution per transwell, then incubating overnight at 37°C.
  • the transwells are assembled in a 24-well plate and a chemoattractant (e.g. , FGF -2) is added to the bottom chamber in a total volume of 0.8 mL media.
  • ECs such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using trypsin, are diluted to a final concentration of about 10 6 cells/mL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell.
  • a chemoattractant e.g. , FGF -2
  • Inhibitors to be tested may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37°C.
  • the transwells are removed from the plate stained using DiffQuik ® .
  • Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (40Ox) to determine the number of cells migrated.
  • ECs or tumor cells e.g., PC-3 human prostatic carcinoma cells
  • Matrigel® invasion assay system The ability of cells such as ECs or tumor cells (e.g., PC-3 human prostatic carcinoma cells) to invade through a reconstituted basement membrane (Matrigel®) in an assay known as a Matrigel® invasion assay system is well known (Kleinman et al, Biochemistry 1986, 25: 312-318; Parish et al, 1992, Int. J. Cancer 52:378-383).
  • Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan (which bind to and localize bFGF), vitronectin as well as transforming growth factor- ⁇ (TGF ⁇ ), urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA) and the serpin known as plasminogen activator inhibitor type 1 (PAI-I) (Chambers et al., Cane. Res. 1995, 55:1578-1585).
  • perlecan which bind to and localize bFGF
  • TGF ⁇ transforming growth factor- ⁇
  • uPA urokinase-type plasminogen activator
  • tPA tissue plasminogen activator
  • PAI-I plasminogen activator inhibitor type 1
  • Such assays employ transwell tissue culture inserts.
  • Invasive cells are defined as cells which traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane.
  • Transwells e.g., from Costar
  • Polycarbonate membranes 8.0 ⁇ m pore size
  • Matrigel® e.g., from Collaborative Research
  • the cells which are to be tested are trypsinized and resuspended in DMEM+antibiotics and added to the top chamber of the transwell at a final concentration of about 8x10 5 cells/mL.
  • the final volume of the upper chamber is adjusted to 200 ⁇ L.
  • the assembled plate is then incubated in a humid 5% CO 2 atmosphere for about 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane.
  • the membranes are detached from the transwell using an X-acto ® blade, mounted on slides using Permount ® and coverslips, then counted under a microscope using high power (e.g., 40Ox). A mean number of invading cells from 5-10 counted fields is calculated and plotted as a function of inhibitor concentration.
  • ECs for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 x 10 5 cells/mL with fibrinogen (5mg/mL in phosphate buffered saline (PBS) in a 1 :1 (v/v) ratio.
  • fibrinogen 5mg/mL in phosphate buffered saline (PBS) in a 1 :1 (v/v) ratio.
  • Thrombin is added (5 units/ mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well).
  • the fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound.
  • the cells are incubated at 37°C in 5% CO 2 for 4 days at which time the cells in each well are counted and classified as either rounded, elongated with no branches, elongated with one branch, or elongated with 2 or more branches. Results are expressed as the average of 5 different wells for each concentration of compound. Typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et al, Cancer Res. 1996, 56: 2428-2433).
  • EC tube formation is observed when ECs are cultured on Matrigel® (Schnaper HW et ⁇ /., J. Cell Physiol. 1995, 755:107-118). 10 4 EC /well are transferred onto Matrigel®-coated 24-well plates, and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the time of adding the ECs or at various time points thereafter. Tube formation can also be stimulated by adding (a) an angiogenic growth factor such as bFGF or VEGF, (b) a differentiation stimulating agent ⁇ e.g., PMA) or (c) a combination of these.
  • an angiogenic growth factor such as bFGF or VEGF
  • a differentiation stimulating agent ⁇ e.g., PMA
  • this assay models angiogenesis by presenting to the ECs a particular type of basement membrane, namely the layer of matrix which migrating and differentiating ECs would be expected to encounter first.
  • the matrix components found in Matrigel® (and in basement membranes in situ), or proteolytic products thereof may also be stimulatory for EC tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood, CH et ah, Biochim. Biophys. Acta 1990, 7052:89-118; Odedra, R et al., Pharmac. Ther. 1991, 4P:111-124).
  • the ability of the Abs and/or conjugates of this invention to inhibit the proliferation of ECs may be determined in a 96-well format.
  • Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x using PBS), 3-6 x 10 3 cells are plated per well and allowed to attach for 4 hrs (37°C/5% CO 2 ) in Endothelial Growth Medium (EGM; Clonetics ) or M199 medium supplemented with 0.1-2% FBS.
  • the medium and any unattached cells are removed at the end of 4 hrs and fresh medium supplemented with bFGF (1-10 ng/mL) or VEGF (1-10 ng/niL) is added to each well.
  • Antibodies and/or conjugates to be tested are added last, and the plate is allowed to incubate (37°C/5% CO 2 ) for 24-48 hrs.
  • the chromogenic compound MTS (Promega) is added to each well and allowed to incubate from 1-4 hrs.
  • the color developing in each well is directly proportional to the cell number, thereby serving as a surrogate for counting cells.
  • Absorbance read at 490nm is used to determine the differences in cell numbers, i.e., proliferation, between control wells and those containing test Abs and/or conjugates.
  • Tumor cells ⁇ e.g., 3-10 x 10 /well
  • Serum-free medium is then added, and the cells forced to synchronize for 24 hrs.
  • Medium + 10% FBS is then added to each well to stimulate proliferation.
  • DC:50410124.1 27 ATT-17 conjugates to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as above.
  • the anti-pro liferative and cytotoxic effects of Abs and/or conjugates thereof may be determined for various cell types including tumor cells, ECs, fibroblasts and macrophages. This is especially useful when testing a Ab which has been conjugated to a therapeutic moiety such as a radiotherapeutic or a toxin.
  • a conjugate of one of the Abs of the invention with Bolton-Hunter reagent which has been iodinated with 131 I would be expected to inhibit the proliferation of cells expressing uPAR (most likely by inducing apoptosis).
  • Anti-pro liferative effects would be expected against tumor cells and stimulated endothelial cells but, under some circumstances not quiescent endothelial cells or normal human dermal fibroblasts. Any anti-proliferative or cytotoxic effects observed in the normal cells may represent non-specific toxicity of the conjugate.
  • a typical assay would involve plating cells at a density of 5-10,000 cells per well in a 96-well plate.
  • the compound to be tested is added at a concentration 10x the IC50 measured in a binding assay (this will vary depending on the conjugate) and allowed to incubate with the cells for 30 minutes.
  • the cells are washed 3X with media, then fresh media containing [ 3 H]thymidine (1 ⁇ Ci/mL) is added to the cells and they are allowed to incubate at 37°C in 5% CO 2 for 24 and 48 hours.
  • Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a ⁇ -counter.
  • Proliferation may be measured non-radioactively using MTS reagent or CyQuant ® to measure total cell number.
  • MTS reagent or CyQuant ® for cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS (Genzyme).
  • the ability of the Abs and/or conjugates to promote apoptosis of EC's may be determined by measuring activation of caspase-3.
  • Type I collagen gelatin
  • 5xlO 5 ECs are seeded in EGM + 10% FBS.
  • EGM + 2% FBS 10 ng/ml bFGF and the desired test compound.
  • the cells are harvested after 6 hrs, cell lysates prepared in 1% Triton X-IOO detergent, and the lysates assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures' instructions.
  • Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
  • This assay is performed essentially as described by Nguyen et al. , Microvascular Res. 1994, 47:31-40.
  • Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain visible blood vessels.
  • This assay is performed essentially as described by Passaniti, A et al., 1992, Lab Invest. (57:519-528. Ice-cold Matrigel® (e.g., 500 ⁇ L) (Collaborative Biomedical Products, Inc., Bedford, MA) is mixed with heparin (e.g., 50 ⁇ g/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus.
  • the Matrigel® mixture is injected subcutaneously (s.c.) into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse.
  • the injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF2 + heparin), a negative control plug (e.g., buffer +heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2 + heparin + compound). All treatments groups are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned microscopically immediately at high resolution. Plugs are then dispersed in water and incubated at 37°C overnight. Hemoglobin (Hb) levels in the plugs are determined using Drabkin's
  • the amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample.
  • animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore.
  • a 0.1 ml buffer preferably PBS
  • the amount of fluorescence in the dispersed plug determined fluorimetrically, also serves as a measure of angiogenesis in the plug.
  • Staining with mAb anti-CD31 (CD31 is "platelet-endothelial cell adhesion molecule", "PECAM" may also be used to confirm neovessel formation and microvessel density in the plugs.
  • tumor cells for example 1-5 x 10 6 cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described above. A mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days.
  • the anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug. Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran- fluorophore conjugate injected prior to sacrifice). To measure Hb or fluorescence, the plugs are first homogenized with a tissue homogenizer.
  • A2780 human ovarian cancer line was established from tumor tissue from an untreated patient.
  • the A2780 cells are maintained as a monolayer in RPMI 1640 medium supplemented with 2 mM glutamine, 0.01 mg/mL bovine insulin, and 10% FBS.
  • Two million A2780 are inoculated in the right flank of nude Balb/c female mice.
  • the A2780 tumor is staged to 50 to 200 mm range before treatment is.
  • the IgG control Ab as well as the anti-D2D3 uPAR mAbs are administered by the intraperitoneal route at 10 mg/kg twice weekly on Monday and Friday.
  • the cisplatin treatment group was staged to 1000 mm 3 ; animals received 6 mg/kg once a week. Tumor volumes were measured twice a week. At the time of sacrifice, plasma is obtained and the tumor excised from each animal. Half of the tumor is snap frozen for biochemical assessment and the rest is placed in Zinc fixative for histological assessment.
  • A549 human lung carcinoma (ATCC Catalog No. CCL-185) cell line, was established through explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male (Giard, DJ et al., J. Natl. Cancer Inst. 57:1417-23 (1973)). A549 cells are maintained in Ham's F12K medium supplemented with 2 mM L-glutamine, 0.15% NaHCO 3 , and 10 % FBS.
  • A549 carcinoma cells are inoculated in the right flank of C.B-17/Sys (scid/scid) Severe Combined Immunodeficient (SCID) female mice. Treatment is preferably initiated the day after tumor inoculation.
  • the IgG control Ab (and the PBS control) as well as the anti-D2D3 uPAR mAb ATN-658 are administered intraperitoneally 10 mg/kg twice weekly on Monday and Friday. Initially tumor volumes are measured once a week. When the volume in any treatment group exceeds 300 mm 3 , measurements are obtained twice a week.
  • Late metastasis involves the steps wherein tumor cells attach and extravasate, invade locally, seed, proliferate and induce angiogenesis.
  • Human prostatic carcinoma cells PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice.
  • a reporter gene preferably the green fluorescent protein (GFP) gene
  • This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical colorimetric detection of the various enzymes) to follow the fate of these cells.
  • Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer.
  • GFP-expressing PC-3 cells (10 6 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at lOO ⁇ g/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
  • the compounds that may be employed in the pharmaceutical compositions of the invention include all uPAR ligands, typically polypeptide molecules, preferably Abs, described above, as well as pharmaceutically acceptable salts of various of these compounds.
  • Pharmaceutically acceptable acid addition salts of the compounds of the invention containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art.
  • Exemplary of the acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.
  • Pharmaceutically acceptable base addition salts of compounds of the invention containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hy droxymethy l)methy lamine .
  • nontoxic alkali metal and alkaline earth bases such as calcium, sodium, potassium and ammonium hydroxide
  • nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hy droxymethy l)methy lamine .
  • the compounds of the invention possess the ability to inhibit EC proliferation, motility, or invasiveness and angiogenesis, properties that are exploited in the treatment of cancer, in particular metastatic cancer.
  • a composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to the active form.
  • the mAbs describe herein are "therapeutically conjugated” or “therapeutically labeled” (terms which are intended to be interchangeable) and used to deliver a therapeutic agent to the site to which the compounds home and bind, such as sites of tumor metastasis or foci of infection/inflammation, restenosis or fibrosis.
  • therapeutically conjugated means that the modified mAb is conjugated to another therapeutic agent that is directed either to the underlying cause or to a "component" of tumor invasion, angiogenesis, inflammation or other pathology.
  • a therapeutically labeled polypeptide carries a suitable therapeutic "label” also referred to herein as a "therapeutic moiety.”
  • a therapeutic moiety is an atom, a molecule, a compound or any chemical component added to the peptide that renders it active in treating a target disease or condition, primarily one a associated with undesired angiogenesis.
  • the therapeutic moiety may be bound directly or indirectly to the mAb.
  • the therapeutically labeled mAb is
  • DC:50410124.1 32 ATT-17 administered as pharmaceutical composition which comprises a pharmaceutically acceptable carrier or excipient, and is preferably in a form suitable for injection.
  • isotopes were discussed more fully above. Those isotopes preferred for therapeutic use include: 125 I, 131 I, 186 Re, 188 Re, 111 Ag, 197 Pt, 109 Pd, 67 Cu, 32 P, 33 P, 90 Y, 47 Sc, 53 Sm, 177 Lu, 105 Rh, 142 Pr, 143 Pr, 161 Tb, 166 Ho, and 199 Au. These atoms can be conjugated to the peptide directly, indirectly as part of a chelate, or, in the case of iodine, indirectly as part of an iodinated Bolton- Hunter group. The radioiodine can be introduced either before or after this group is coupled to the peptide compound.
  • Preferred doses of the radionuclide conjugates are a function of the specific radioactivity to be delivered to the target site which varies with tumor type, tumor location and vascularization, kinetics and biodistribution of the peptide carrier, energy of radioactive emission by the nuclide, etc.
  • Those skilled in the art of radiotherapy can readily adjust the dose of the peptide in conjunction with the dose of the particular nuclide to effect the desired therapeutic benefit without undue experimentation.
  • boron neutron capture therapy where a boronated peptide is delivered to a desired target site, such as a tumor, most preferably an intracranial tumor (Barth, RF, Cancer Invest. 74:534-550 (1996); Mishima, Y (ed.), Cancer Neutron Capture Therapy, New York: Plenum Publishing Corp., 1996; Soloway, AH et al, (eds), J. Neuro-Oncol. 33:1-188 (1997).
  • the stable isotope 10 B is irradiated with low energy ( ⁇ 0.025eV) thermal neutrons, and the resulting nuclear capture yields ⁇ -particles and 7 Li nuclei which have high linear energy transfer and respective path lengths of about 9 and 5 ⁇ m.
  • This method is predicated on 10 B accumulation in the tumor with lower levels in blood, endothelial cells and normal tissue ⁇ e.g., brain).
  • Such delivery has been accomplished using epidermal growth factor (Yang. W et al, Cancer Res 57:4333-4339 (1997).
  • Other therapeutic agents which can be coupled to the mAbs according to the method of the invention are drugs, prodrugs, enzymes for activating pro-drugs, photosensitizing agents, nucleic acid therapeutics, antisense vectors, viral vectors, lectins and other toxins.
  • the present invention contemplates dyes used, for example, in photodynamic therapy, conjugated to anti-uPAR Abs and fragments, and used in conjunction with appropriate nonionizing radiation (U.S. Pat. 6,331,175).
  • Many drugs and toxins with known cytotoxic and apoptotic effects on cells can be found in compendia or textbooks of drugs and toxins, such as the Merck Index (most recent edition),
  • Such agents include taxol, nitrogen mustards, such as, mechlorethamine, cyclophosphamide, melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as, thiotepa; alkyl sulfonates, such as, busulfan; nitrosoureas, such as, carmustine, lomustine, semustine and streptozotocin; triazenes, such as, dacarbazine; folic acid analogs, such as, methotrexate; pyrimidine analogs, such as, fluorouracil, cytarabine and azaribine; purine analogs, such as, mercaptopurine and thioguanine; vinca alkaloids, such as, vinblastine and vincristine; antibiotics, such as, dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin and mito
  • Lectins are proteins, commonly derived from plants, that bind to carbohydrates. Among other activities, some lectins are toxic. Some of the most cytotoxic substances known are protein toxins of bacterial and plant origin (Frankel, AE et ah, Ann. Rev. Med. 57:125-142 (1986)). These molecules binding the cell surface and inhibit cellular protein synthesis. The most commonly used plant toxins are ricin and abrin; the most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A. In ricin and abrin, the binding and toxic functions are contained in two separate protein subunits, the A and B chains.
  • the ricin B chain binds to the cell surface carbohydrates and promotes the uptake of the A chain into the cell. Once inside the cell, the ricin A chain inhibits protein synthesis by inactivating the 60S subunit of the eukaryotic ribosome Endo, Y. et al, J. Biol. Chem. 262: 5908-5912 (1987)).
  • Other plant derived toxins which are single chain ribosomal inhibitory proteins, include pokeweed antiviral protein, wheat germ protein, gelonin, dianthins, momorcharins, trichosanthin, and many others (Strip, F. et al, FEBS Lett. 7P5: 1-8 (1986)).
  • Diphtheria toxin and Pseudomonas exotoxin A are also single chain proteins, and their binding and toxicity functions reside in separate domains of the same protein Pseudomonas exotoxin A has the same catalytic activity as diphtheria toxin. Ricin has been used therapeutically by binding its toxic ⁇ -chain, to
  • Bacterial toxins have also been used as anti-tumor conjugates. As intended herein, a toxic peptide chain or domain is conjugated to a compound of this invention and delivered in a site-specific manner to a target site where the toxic activity is desired, such as a metastatic focus. Conjugation of toxins to protein such as Abs or other ligands are known in the art (Olsnes, S. et al., Immunol. Today 70:291-295 (1989); Vitetta, ES et al, Ann. Rev. Immunol. 5:197-212 (1985)).
  • the compounds of the invention may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or are more preferably used as injectable preparations.
  • Solid or liquid pharmaceutically acceptable carriers may be employed.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid ⁇ e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • a solution such as an ampoule
  • aqueous or nonaqueous liquid suspension sterile injectable liquid
  • the pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration.
  • the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
  • the present invention may be used in the diagnosis or treatment of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine.
  • the pharmaceutical compositions can be used to treat domestic and commercial animals, including birds and more preferably mammals, as well as humans.
  • systemic administration refers to administration of a composition or agent such as the polypeptide, described herein, in a manner that results in the introduction of the composition into the subject's circulatory system or otherwise permits its spread throughout the body, such as intravenous (i.v.) injection or infusion.
  • Regular administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. Examples include intravaginal, intrapenile, intranasal, intrabronchial(or lung instillation), intracranial, intra-aural or intraocular.
  • local administration refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous (s.c.) injections, intramuscular (i.m.) injections.
  • s.c. subcutaneous
  • i.m. intramuscular
  • injectables or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions.
  • the pharmaceutical composition may be administered topically or transdermally, e.g. , as an ointment, cream or gel; orally; rectally; e.g. , as a suppository.
  • the compound may be incorporated into topically applied vehicles such as a salve or ointment.
  • the carrier for the active ingredient may be either in sprayable or nonsprayable form.
  • Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water.
  • Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • Preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
  • an effective amount of the compound for the preferred topical applications, especially for humans, it is preferred to administer an effective amount of the compound to an affected area, e.g., skin surface, mucous membrane, eyes, etc.
  • an affected area e.g., skin surface, mucous membrane, eyes, etc.
  • This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, the severity of the symptoms, and the nature of the topical vehicle employed.
  • compositions of the present invention are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active polypeptide is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • phospholipids such as lecithin and sphingomyelin
  • steroids such as cholesterol
  • more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid
  • compositions for treating tumors and cancer may comprise, in addition to the peptide, one or more additional anti-tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase, topoisomerase inhibitors such as etoposide; or biological response modifiers, e.g., interferons or interleukins.
  • mitotic inhibitors e.g., vinblastine
  • alkylating agents e.g., cyclophosphamide
  • folate inhibitors e.g., methot
  • compositions comprising any known cancer therapeutic in combination with the peptides disclosed herein are within the scope of this invention.
  • the pharmaceutical composition may also comprise one or more other medicaments to treat additional symptoms for which the target patients are at risk, for example, anti-infectives including antibacterial, anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • the therapeutic dosage administered is an amount which is therapeutically effective, as is known to or readily ascertainable by those skilled in the art.
  • the dose is also dependent upon the age, health, and weight of the recipient, kind of concurrent treatment(s), if any, the frequency of treatment, and the nature of the effect desired, such as, for example, anti-inflammatory effects or anti-bacterial effect.
  • the methods of this invention may be used to inhibit tumor growth and invasion in a subject or to suppress angiogenesis induced by tumors by inhibiting endothelial cell growth and migration.
  • the methods result in inhibition of tumor metastasis.
  • a vertebrate subject preferably a mammal, more preferably a human, is administered an amount of the compound effective to inhibit tumor growth, invasion or angiogenesis.
  • the compound or pharmaceutically acceptable salt thereof is preferably administered in the form of a pharmaceutical composition as described above.
  • Doses of the proteins preferably include pharmaceutical dosage units comprising an effective amount of the peptide.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of, and sensitivity of, individual subjects
  • an effective amount is meant an amount sufficient to achieve a steady state concentration in vivo which results in a measurable reduction in any relevant parameter of disease and may include growth of primary or metastatic tumor, any accepted index of inflammatory reactivity, or a measurable prolongation of disease-free interval or of survival.
  • a reduction in tumor growth in 20 % of patients is considered efficacious (Frei III, E., The Cancer Journal 3:127-136 (1997)).
  • an effect of this magnitude is not considered to be a minimal requirement for the dose to be effective in accordance with this invention.
  • an effective dose is preferably 10-fold and more preferably 100-fold higher than the 50% effective dose (ED 50 ) of the compound in an in vivo assay as described herein.
  • the amount of active compound to be administered depends on the precise peptide or derivative selected, the disease or condition, the route of administration, the health and weight of the recipient, the existence of other concurrent treatment, if any, the frequency of treatment, the nature of the effect desired, for example, inhibition of tumor metastasis, and the judgment of the skilled practitioner.
  • a preferred dose for treating a subject, preferably mammalian, more preferably human, with a tumor is an amount of up to about 100 milligrams of active polypeptide-based compound per kilogram of body weight.
  • a typical single dosage of the peptide or peptidomimetic is between about 1 ng and about 100mg/kg body weight.
  • dosages in the range of about 0.01-20% concentration (by weight) of the compound, preferably 1-5% are suggested.
  • a total daily dosage in the range of about 0.1 milligrams to about 7 grams is preferred for intravenous administration.
  • Effective doses and optimal dose ranges may be determined in vitro using the methods described herein.
  • the anti-uPAR Ab (or other uPAR-binding ligand) should produce an inhibitory effect on CaSC self renewal/proliferation, migration, invasion/metastasis.
  • the methods are especially useful in producing an anti-tumor effect in a mammalian host, preferably human, harboring a tumor wherein the treatment results in reduction in size or growth rate of the tumor or destruction of the tumor.
  • the subject is a human.
  • a preferred disease or condition to be treated by the above method is tumor growth, invasion or metastasis of any of a number of types of cancer or tumor, including brain tumors.
  • brain tumors are astrocytoma, anaplastic astrocytoma, glioblastoma, glioblastoma multiformae, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma..
  • ATN-658 mAb described above is used to sort the cells obtained from a dispersed a human solid tumor biopsy into two populations: those expressing uPAR (uPAR+) and those lacking detectable surface uPAR. Both populations are injected SC into nude mice. Development of tumors at the site of inoculation is monitored.
  • the uPAR+ cells result in an increased incidence of tumors and increased growth rate compared to uPAR-negative cells.
  • the uPAR+ cells are further characterized in vitro and are shown to be resistant to chemotherapy compared to the uPAR-negative cells when grown in a clonogenic assay in agar.
  • These uPAR+ tumor cells also express stem cell markers and are characterized as follows: CD44 hl , CD24 10 , CDl 05+, CD20+, and CD 133+ and further exclude Hoechst dye.

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Abstract

Cancer or tumor stem cells (CaSCs) express urokinase-type plasminogen activator receptor (uPAR) on their surfaces. Ligands of uPAR, in particular antibodies are used to bind, detect, inactivate or purge uPAR-expressing cancer stem cells. Such antibodies or other ligands are also used in methods for diagnosing and treating cancer.

Description

ANTIBODIES TO UROKINASE-TYPE PLASMINOGEN ACTIVATOR RECEPTOR (uPAR) BIND CANCER STEM CELLS: USE IN DIAGNOSIS AND THERAPY
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention in the field of biochemistry, immunology and medicine relates to the use of antibodies ("Abs") or other ligands specific for the urokinase-type plasminogen activator receptor (uPAR) to bind, detect, inactivate or purge uPAR-expressing cancer stem cells. Such Abs or other non-Ab ligands are used in method for diagnosing and treating cancer.
Description of the Background Art
A significant body of evidence from studies in vitro and in vivo has established that the urokinase plasminogen activator (uPA) system is central to the process of metastasis, making it a promising target for cancer drug development (Mazar, AP et al. (1999) Angiogenesis 3: 15-32). In addition to uPA, its cell surface receptor (uPAR, also known as CD 87) is a suitable target for the design and development of cancer therapeutic and diagnostic agents (Mazar, AP (2001) Anti- Cancer Drugs 12: 397-400) because:
(a) uPAR is selectively expressed on metastatic tumor cells and angiogenic endothelial cells ("ECs"), but not on other cells;
(b) uPAR is an important participant in several extracellular and intracellular pathways required for metastasis that are currently the object of intense drug development efforts; and
(c) it is possible to interfere at several different points along the uPA pathway.
Thus, uPA and uPAR are promising targets for the development of diagnostics and therapeutics useful against many different types of tumors/cancers.
The uPA/uPAR System and Cancer
Metastasis and angiogenesis share many functional features that characterize invasive and migratory processes of tumor cells and of ECs. These features include (1) the up-regulation of protease and integrin expression, (2) the loss of cell-cell and cell-matrix contacts, (3) increased responsiveness to growth and differentiation factors, and (4) remodeling of extracellular matrix (ECM) and basement membrane (BasM). All of these contribute to tumor progression.
DC:50410124.1 Λ ATT-17 The uPA "system" which comprises the serine protease uPA, its receptor uPAR, and its specific serpin inhibitor, plasminogen activator inhibitor-type 1 (PAI-I) plays a central role in many of these activities which are responsible for:
(1) initiating cascades that result in the activation of plasminogen, activating several pro- metalloproteases (proMMPs),
(2) release and processing of latent growth factors such as fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGFβ),
(3) (a) interactions with components of the ECM such as vitronectin (Vn) and fibronectin (Fn), (b) direct interactions with several integrins including α5βl and αvβ3, and (c) remodeling the BasM and ECM to promote cell motility.
Further, the uPA system can also initiate localized fibrin turnover which may play a role in angiogenesis.
The expression of uPA and uPAR has been demonstrated in numerous tumor types including glioblastoma, prostate, breast, colon, hepatocellular, and renal cell carcinoma. (Mizukami IF et al.
(1994) Clin Immunol and Immunopathol 77:96-104; Hsu DW et al, (1995) Am J Pathol 147:114- 23; de Witte JH et al. (1999) Br J Cancer 79:1190-8). The expression of uPA and uPAR are typically greater in more aggressive forms of disease. On tumor cells, this expression is often highest at the invasive front of the tumor. (Buo, L et al., (1995) Human Pathol 26:1133-1138; Yamamoto M et al. (1994) Cancer Res 54:5016-5020). Strong immunohistochemical staining of uPAR in blood vessels is associated with the invasive front of breast, colon, and renal cell carcinomas (Bastholm L et al. Appl Immunohistochem MoI Morphol 7: 39-47; Nakata S et al. (1998) Int. J. Cancer 7P:179-186). In the colon carcinoma study, uPAR co-localized with VEGF. Tumor-associated macrophages from several tumor types express uPA and uPAR (Ohtani H et al.
(1995) Int J Cancer 62:691-6; Xu Y et al. (1997) Hum Pathol 28:206-13). uPA is chemotactic for monocytes and mediates both adhesion and migration of these cells. Adhesion and migration require only uPAR occupancy but not uPA catalytic activity. Thus, the uPA/uPAR system is believed to contribute to tumor progression by acting on multiple tumor-associated cell types.
The promise of targeting the uPA/uPAR system has yet to result in the development of agents suitable for the clinic. Small molecule approaches have been hampered by (1) the difficulty of potently inhibiting a protein-protein interaction (e.g., uPA-uPAR or uPAR-integrin), and (2) the
DC:50410124.1 9 ATT-17 lack of suitable leads or structural information amenable to medicinal chemistry efforts. Several identified peptide inhibitors of the uPA-uPAR interaction would suffer from the typically poor pharmacological properties of peptides and have not demonstrated the requisite levels of activity even in cell-based assays (Ploug M et al. (2001) Biochemistry 40:12157-68).
CANCER STEM CELLS (CaSCs)
It has recently become apparent that tumors include a rare cell population with stem cell-like properties, defined by self-renewal and proliferative capacity, which are responsible for growth and progression of the tumor. The first serious attempt to isolate a cancer stem cell was the system that John Dick and his colleagues developed. They transferred cells from patients with acute myelogenous leukemia (AML) successfully into immunodeficient mice (Bonnet D and Dick JE 1997, Nat Med 3 730-7). These leukemia-initiating cells shared part of the phenotype of normal hematopoietic stem cells (HSC), in that they were CD34+CD38lo/". True human HSC are also Thy- I+ and lack blood lineage markers. See, also, Soltysova, A et al., 2005, Neoplasma 52:435-40; Wang JC and Dick JE., 2005, Trends Cell Biol. 75:494-501; Warner JK et al., 2004, Oncogene 23:1164-11; Dick JE, 2003, Proc Natl Acad Sci USA 100:3541-49; Al-Hajj M et al., 2003, Proc NatlAcad Sci USA 700:3983-88; Bhatia R et al., 2003 Blood 101:4101-41;
Irving Weissman and colleagues sought to determine if the leukemia stem cells (LSC) were derived from HSC or progenitors (see, The Scientist, 2006, 20:35-36) using cells from AML patients that bore the AMLl /ETO chromosomal translocation. They were surprised to find that true HSC had the AML1/ETO chromosome, but lacked the potential to produce leukemia blast cells in culture, yielding only normal-looking myeloerythroid colonies. The Thyl" progenitors were the LSC. Miyamoto, T et al. Proc Natl Acad Sci, 2000, P7:7521-6. This finding was consistent with clinical data, and many of these treated patients with leukemia were healthy for as long as 150 months, yet their BM contained detectable normal HSC with the AML1/ETO chromosomal translocation. The authors concluded that this translocation was necessary but not sufficient for full AML.
Several independent events are required for the progression of chronic to acute leukemias in mice, and in mouse and human myelopoiesis, only HSC self-renew. Our interpretation (see figure, next page) is that most or all proto-oncogenic events short of acute leukemia occur in a succession of HSC clonal progeny; had such early events initially occurred in progenitors, they would be lost
DC:50410124.1 T. ATT-17 as e progenitor lifespan was completed. However, the emergence of the acute leukemic clone could occur at the HSC or progenitor level when the self-renewal pathway genes are activated. To give an example, in a particular group of patients (bearing bcr/abl translocations) the chronic phase leukemia is at the level of HSC, producing myeloid, erythroid, and B lymphoid cells. But when myeloid blast crisis emerges, it is progenitors that are responsible, mainly from the granulocyte- macrophage stage of hematopoiesis (Jamieson, CH et al. N Engl J Med, 2004, 351:657-67). This pattern is repeated in solid tumors in other tissues that are arranged in a cellular hierarchy, such as the brain (see below) and the breast. Breast cancers have a similar cellular hierarchy to the normal mammary gland from which they arose. In malignant breast tumors, a minority population of CD24"/loCD44+ cancer cells is self-renewing while most of the cancer cells are destined to stop proliferating (Reya, T. et al., Nature, 2004, 414:105-11). The finding that cancers arising in such diverse tissues as the blood, epithelium, and brain all contain cancer stem cells suggests that many, if not all, tumors fit this paradigm. It is possible that differences between malignant cells and their normal counterparts can be exploited to eliminate the cancer stem cells, resulting in improved outcome for patients.
As described by P.B. Dirks (The Scientist, 2006, 20:37), neural stem cell biology had its origins in the discovery that culturing mammalian brain cells in serum- free conditions (in the presence of epidermal growth factor (EGF) and bovine fibroblast growth factor (bFGF), yielded clonally derived colonies of undifferentiated neural cells and that cells in these colonies showed basic properties of stem cells: self-renewal and multi-lineage differentiation (Reynolds BA et al. , Science, 1992, 255:1707-10. Investigators began to question whether this approach could be used to isolate stem cells from brain tumors. Several groups showed that a minority of cells in a brain tumor could form colonies (clones) in vitro expressing neural precursor markers and demonstrated both self-renewal, in vitro differentiation to express mature neural-lineage markers (Ignatova, TN et al., GHa, 2002, JP:193-206; Singh, SK et al, Cancer Res, 2003, 65:5821-8; Hemmati, HD et al, Proc Natl Acad Sci, 2003,700:15178-83; Galli, R et al., Cancer Res, 2004, 64:7011-21). Thus, brain tumors contain a small population of cells with stem cell properties (as measured in vitro). The self-renewal and differentiation properties of the candidate brain tumor stem cells varied among tumors of different histologic types.
DC:50410124.1 Λ ATT-17 Clonogenic brain tumor cells were prospectively enriched by cell-sorting techniques employing the same cell-surface marker CD 133 (of unknown function) that was useful for enriching stem cells from normal human brain (Uchida, N et al., Proc Natl Acad Sci, 2000, 97: 14720-5). Human brain tumors of distinct types from adults and children were found to contain a subpopulation of CD133+ brain tumor cells with stem cell properties in vitro and that further defined a stem cell in vivo (Singh, SK et al, Nature, 2004, 452:396-401). Malignant brain tumors tended to have a relatively high frequency of CD133+ cells (up to 30%), whereas less aggressive tumors had fewer such cells (0.5-5.0%). As few as 100 malignant CD133+ cells could in some cases initiate growth of a serially transplantable brain tumor following intracerebral engraftment into immunodeficient (NOD/SCID) mice. Tumors that resulted from these CD133+ cell injections were a phenocopy of the original patient tumor. Moreover, injection of 105 CD133" cells survived when engrafted into the mouse brain, but these cells did not form brain tumor masses.
These above results demonstrated that brain tumors contain small numbers of cells that show a remarkable difference in tumorigenic potency compared to the bulk of the tumor. Nonetheless, the cell of origin for brain tumors remains uncertain. Recent evidence points to cells in subventricular zones, a normal site of ongoing neural stem cell activity, as a site of glial tumor initiation (Druckrey, H et al, Nature, 1996, 270:1378-9; Gil-Perotin, S et al., JNeurosci, 2006, 25:1107-16.
These various cancer stem cells are referred to (sometimes interchangeably) as cancer stem cells ("CaSC"), "cancer progenitor" cells, "tumor stem" cells (TuSC) or "tumor progenitor" cells. Increasing evidence supports "the CaSC hypothesis."
Normal stem cells in adult organisms are responsible for tissue renewal and repair of aged or damaged tissue. An important characteristic of stem cells is their ability for self-renewal without loss of proliferative capacity with each cell division. They can differentiate to form specific types of tissue under the influence of microenvironmental and other factors. Stem cells are believed to divide asymmetrically producing two daughter cells - one of which is a new stem cell and the second is more committed progenitor cell with the ability to differentiate and proliferate, but without the capacity for self-renewal. Cancer stem cells are in many aspects similar. Cells making up a tumor are heterogeneous and comprise (a) rare tumor initiating cells and (b) abundant non- tumor initiating cells. Tumor initiating cells - the CaSCs - can self-renew and proliferate, express typical stem cell markers, and are relatively resistant to drugs. It is not clear whether CaSCs
DC:50410124.1 « ATT-17 originate from normal stem cells as a consequence of genetic and epigenetic changes and/or whether they arise by de-differentiation of somatic tumor cells to a stem-like cell stage. It is likely that both mechanisms contribute to the origin of CaSCs. Dysregulation of stem cell self-renewal is a likely precursor to the development of cancer. CaSCs have been identified in, and isolated from, human tumors and in tumor cell lines (see references cited above). To date, the existence of CaSCs has been demonstrated in acute and chronic myeloid leukemia, breast cancer, brain tumors, lung cancer and gastrointestinal tumors.
The CaSC model is also consistent with certain clinical observations. Although standard chemotherapy kills most cells in a tumor, CaSCs survive. Despite their small number, they may be the cause of tumor recurrence, which sometimes takes place years after "successful" treatment of a primary tumor. Furthermore, growth of metastases at distinct sites of body and their cellular heterogeneity may be consequence of CaSC differentiation and/or dedifferentiation and asymmetric division. There is a recognized need in the art to increase our understanding of CaSCs in order to find approaches to destroying them, a step which may contribute significantly to the therapeutic management of malignant tumors. The present invention is directed to this objective. uPAR on Cancer Stem Cells
It was recently discovered that uPAR is expressed or upregulated on the surface of CaSCs (Gutova, M et al, Proc Amer Assoc Cancer Res 2006; 47: Abstr #3986, 2006, April). These investigators identified a small population of such cells in small cell lung cancer (SCLC). These CaSCs expressed high levels of the uPAR/CD87 gene, which is known to contribute to development of the most invasive phenotypes in several cancers, e.g. ovarian, prostate and brain ( Henic E et al, Gynecol Oncol, 2006, 707:28-39; Mamoune, A et al, Exp Cell Res., 2004, 70.91- 100; Mori T et al, J Neurooncol 2000, 46:115-23). These uPAR+ cells displayed high clonogenic potential in vitro and were able to generate a heterogeneous cell population similar to the parental tumor cell line both in vitro and in vivo. The investigators used a SCID mouse model to study human tumor growth and dissemination, inserting human fetal lung tissue under the kidney capsule of these mice and injecting primary and metastatic SCLC cells into these human fetal lung xenografts. Two month after the injection of SCLC cells, distant metastasis were detected in the host mouse lung and liver, whereas spleens, kidneys and brains of the host remained free of
DC:50410124.1 f. ATT-17 metastases. The metastatic human SCLC cells expressed high levels of uPAR, suggesting that uPAR+ cells were responsible for tumor progression.
Purging of Cancer Cells or CaSCs Normal Bone Marrow
Autologous bone marrow (BM) transplantation has been employed to induce or maintain a state of remission in patients with various malignancies, primarily leukemias. In this procedure, BM cells of a patient with a malignancy are removed during remission and stored for re-administration after the patient relapses into active disease (Gale, RP et al, 1987, Sem Hematol 24:40-54). This obviates the need for an allogeneic HLA-matched donor from whom BM transplants are burdened with other genetic incompatibilities. Despite its advantages, a significant drawback for autologous BM is that malignant cells, or CaSCs, may remain in the BM obtained during remission. Various approaches have been used to purge residual malignant cells from BM, but these attempts have been partially successful at best. (See, for example, Champlin R et al, 1987, Sem Hematol 24:55-67 and 74-80; Malik, Z et al, 1987, Brit J Cane 5(5:589-95). Malik et al, describe destruction of leukemic cells by photoactivation of endogenous porphyrins. In addition, certain amphipathic dyes have been reported to photosensitize leukemic cells while sparing normal mononuclear cells (Sieber, F. et al, 1986. Blood 68:32-6; Sieber, F. et al., 1987, Photochem Photobiol 46:11-6; Singer, CRJ et al, 1988, Brit J Hematol68:4\7 -22; Talpaz, M et al, 1988, Sem Hematol 25:62-73). US Pat. 5,087,636 describes methods to purge malignant cells from the bone marrow or other hemopoietic cell populations of leukemia patients with active disease (not in remission) or from patients with other (nonleukemic) malignancies who would benefit from such a purging procedure employing photoactivation of green porphyrins added to the hemopoietic cells.
U.S. Pat. 7,001,770 (February 21, 2006 ; to Atencio et al. describes a method for purging or ablating neoplastic cells that contaminate a population of normal cells ex vivo by administering a recombinant adenoviral vectors encoding p53. This includes ex vivo purging of autologous stem cell preparations (bone marrow purging) that comprise a population of hematopoietic, progenitor and stem cells capable of reconstituting long term hematopoietic function in patients being subjected to myeloablative therapy. Stem cell preparations are conventionally obtained by apheresis of mobilized or non-mobilized peripheral blood. This document discusses "3-log purges" {i.e. removal of approximately 99.9% of the tumor cells from the stem cell preparation) and more efficient "5-log purges" (removal of approximately 99.999% of tumor cells from the preparation.
DC:50410124.1 7 ATT-17 U.S. Pat. 6,331,175 (December 18, 2001 to M. David Goldenberg) discloses methods and compositions for detecting or treating normal, hypoplastic, ectopic or remnant tissue, an organ or cells in a mammal. The method involves parenteral administration to the subject, at a locus and by a route providing access to above-mentioned tissue or organ, of a composition comprising Abs or Ab fragments which specifically bind to targeted organ, tissue or cell. The antibody/fragment may be administered alone, or labeled or conjugated with an imaging agent, a therapeutic moiety, a cytoprotective agent or an activating agent.
Thus, the art recognizes the utility of removing selected populations of cells from a mixture, particularly malignant cells, before administering the remaining non-malignant and presumably therapeutic cells to the donor or a third party subject, as a therapeutic measure. The present invention is also directed to such an approach by using antibodies or other ligands to target uPAR on cancer cells and, more particularly, CaSCs, and remove them from a mixed population.
SUMMARY OF THE INVENTION
The present inventors have conceived of the use of anti-uPAR Abs, preferably monoclonal antibodies (mAbs) to identify CaSCs, positively select (isolate) such cells from mixed populations for study, diagnose the presence of CaSCs with these Abs, to treat primary or metastatic tumors by inhibiting the proliferation of or by killing such CaSCs. Moreover, the antibodies can be used to treat cell populations that are being administered to a subject, and that are suspected of comprising uPAR+ CaSCs ex vivo to purge such cells from the populations before the cells are infused or implanted in the subject. An example is to treat autologous bone marrow cells or enriched hematopoietic stem cells (HSCs) from cancer patients, primary leukemia patients, prior to re- infusion of these sources of HSCs. Treatment of such a cell population with uPAR Abs according to this invention may be performed in conjunction with other treatments that are intended to positively selected HSCs or negatively select remaining leukemic (or leukemic progenitor) cells.
The invention is therefore directed in part to a method for functionally inactivating or killing cancer stem cells (CaSCs), comprising providing to said CaSCs an effective amount of a ligand that binds to uPAR.
DC:50410124.1 O ATT-17 Also provided is method for treating a subject having or susceptible to a malignant disease characterized by the presence of CaSCs, comprising administering to the subject functionally- inactivating amount of a therapeutic composition that comprises a ligand that binds to uPAR.
In the above method, the ligand is preferably an antibody or an antigen binding fragment thereof.
In addition to using anti-uPAR Abs, preferably mAbs that are known in the art, and some of which are commercially available, the present inventors have produced a set of anti-uPAR mAbs with the property that they bind to uPA-uPAR complexes and that serve to inhibit the interactions of these proteins and downstream effects (such as further interactions with integrins). The action of such mAbs can inhibit tumor growth and metastasis by blocking angiogenesis, tumor invasion and proliferation. According to the present invention, such mAbs also act by interfering with self- renewal or differentiation of CaSCs.
In addition to acting directly on CaSCs these mAbs may be used to targeting therapeutic agents or diagnostic (imaging) agents to tumors, and via their binding to uPAR on CaSCs, to permit the identification, localization and/or elimination of these cells.
Several of these antibodies that target uPAR are effective in animal models of cancer growth (such as the A2780 ovarian cancer model and the A549 lung cancer model). The antibodies that are useful in the present invention preferably recognized peptide epitopes of uPAR as expressed on CaSCs.
Malignant tumor cells and CaSCs (as well as angiogenic ECs) gain a selective advantage in cell migration and invasiveness, resulting at least in part from the cells' expression of uPAR on their surface. These uPAR molecules are saturated by binding the endogenously produced ligand, uPA. Thus, mAbs (or other binding partners) that target and preferably inhibit uPA-uPAR interactions with downstream targets are useful in the treatment and/or diagnosis of cancer. Preferred downstream ligands of uPA-uPAR, or of uPAR alone, include integrins, low-density lipoprotein receptor-related protein (LRP) and others. Such downstream ligands mediate or play a role in cell signaling, migration and/or invasion. As disclosed in commonly assigned PCT Application PCT/US2005/018322, filed 25-May-2005 (published as WO 2005/116077 on 08-Dec- 2005), two mAbs, ATN-615 and ATN-658, specifically bind uPA-occupied uPAR and thus serve as exemplary molecules that can bind uPAR regardless of the presence of uPA ligand. The mAbs can detect both occupied and unoccupied uPAR in a tumor or other diseased tissue where the uPA
DC:50410124.1 Q ATT-17 system plays a role in the pathobiology. Preferred Abs or other non-Ab ligands for uPAR are those that do not bind to the uPA-binding site of uPAR.
The present inventors have identified the epitopes to which these Abs bind. Such peptides or natural or synthetic peptides or peptide derivatives that retain the 3D structure of these epitopes are useful as therapeutic and/or diagnostic agents.
The present invention is directed to use of uPAR-binding macromolecules, preferably Abs, antigen binding fragments such as single chain Abs =(scFv), non-Ab polypeptides and peptides, aptamers, etc., as well as small organic molecules to target CaSCs. These include Abs, etc., that bind to uPAR without inhibiting the binding of uPA. Some of these molecules interfere with downstream interactions of either uPA-uPAR or uPAR alone.
The above Ab may be a mAb, or an antigen binding fragment thereof. Preferred mAbs are humanized chimeric or human mAbs.
In a preferred embodiment, the above Ab or other uPAR ligand is (a) diagnostically labeled (with a detectable label); or (b) labeled with, conjugated to, or fused to (in the case of a polypeptide), a therapeutically active moiety, rendering the ligand therapeutically active.
Provided herein is a diagnostic composition comprising (a) the diagnostically labeled ligand, primarily an Ab, as above; and (b) a diagnostically acceptable carrier.
In the diagnostic composition the ligand is preferably labeled with a radionuclide, a PET- imageable agent, an MRI-imageable agent, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer. Preferred radionuclides are 3H, 14C, 35S,
123T I, 131T I, l l lτ In, 67, G-Λa, 9In Ru, 99mr Tτ,c, 67^ Cu, 57^ Co, 58^ Co, 51^ Cr, 59C Fe, 75C Se, 20Ir T^1l, and , 169Λ Yrib.
Preferred fluorescers or fluorogens are fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green and Texas Red.
The present invention provides a therapeutic anti-tumor pharmaceutical composition that inhibits tumor growth and/or tumor metastasis comprising (a) an effective amount of the therapeutically active ligand above, and (b) a pharmaceutically acceptable carrier. This composition is preferably in a form suitable for injection. The therapeutically active moiety may be conjugated directly to, or bound indirectly to, the ligand. A preferred therapeutic moiety is a chemotherapeutic drug, a toxin or a therapeutic radionuclide - described below.
DC:50410124.1 J Q ATT-17 In the above therapeutic composition, the therapeutically active moiety may be a peptide or polypeptide, e.g., a toxin, which is fused to the uPAR-binding Ab or other ligand.
This invention is directed to a method for inhibiting CaSC migration, invasion, proliferation or the process of tumor angiogenesis in which CaSCs play a role. Also included is a method for inducing apoptosis in uPAR+ CaSCs, comprising contacting these cells with an effective amount of a therapeutically active Ab or other uPAR-ligand, as above. Also included is a method for treating a subject having a disease, disorder or condition characterized by undesired tumor growth and/or tumor metastasis comprising administering to the subject an effective amount of the above therapeutic pharmaceutical composition that bind to tumor CaSCs.
One embodiment is an assay method for detecting uPAR-expressing cells in a cell mixture suspected of including CaSCs in a sample of tumor, tissue, organ, or cell population.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
This application incorporates by reference, in its entirety, commonly assigned PCT application PCT/US2005/018322, filed 25-MAY-2005, (published as WO 2005/116077 on 08-DEC-2005).
The present inventors have discovered that Abs, preferably mAbs, peptides or other chemical entities that target the uPAR can bind CaSCs and thereby be used to in the treatment and/or diagnosis of cancer or precancerous states.
The Antibody Approach
The present inventors generated a panel of mAbs targeting uPAR which is an ideal target for antibodies because it is expressed on the cell surface. In addition to the expression of uPAR on CaSCs, the focus, of the present invention, uPAR is also expressed at the tumor- vasculature interface (on invasive tumor cells, angiogenic endothelial cells, or tumor-associated macrophages). Thus, anti-uPAR Abs would be able to enter tumors, where CaSCs may be found, and serve as diagnostic agents or exert therapeutic effects. It is noteworthy that uPAR is not normally expressed on quiescent tissues, which should minimize the potential for toxicity when employing a therapeutic Ab and minimize non-specific signals (or false positives) when employing a diagnostic Ab.
As disclosed in WO 2005/116077, the present inventors and colleagues have raised mAbs against a fragment of the soluble form of uPAR (known as "suPAR") expressed in Drosophila S2 cells. In such cells, a minimally glycosylated isotype of suPAR is expressed. Use of this suPAR as
DC:50410124.1 \ \ ATT-17 an immunogen is expected to overcome heterogeneous binding to uPAR, a property associated with most other anti-uPAR mAbs. (For example, studies performed as part of a Leukocyte Antigen Workshop compared anti-uPAR antibodies available in 1995-1996 and found all of them to be specific for carbohydrate, not protein, epitopes (Manupello, J. et al, (1996) Tiss. Antigens 48: 368.).
Indeed, uPAR expressed in tumors is highly and heterogeneously glycosylated, and the glycosylation pattern and representation of different iso forms change in response to various signals (Stoppelli MP et al. (1985) Proc. Natl. Acad. Sci. USA 82 4939-4943). Thus, anti-uPAR antibodies raised against carbohydrate epitopes are unlikely to recognize all iso forms of uPAR and may cross- react undesirably with other proteins that comprise glycosylation structures similar to those present on uPAR. Use of S2 has led to the identification of mAbs that recognize the protein epitopes within suPAR. A mutant form of suPAR has been expressed in which all glycosylation sites have been mutated. The existing murine mAb clones may be humanized or primatized. The present inventors' ability to generate conformationally intact domain fragments of suPAR has allowed them to produce mAbs against isolated domains of suPAR (isolated Dl and isolated D2D3).
This invention is thus directed in part to methods using a mAb that binds to uPAR and is produced by a process comprising the initial step of immunizing a mammal, preferably a mouse, with
(a) a minimally glycosylated isotype of suPAR expressed in Drosophila cells, or
(b) a de-glycosylated mutant of suPAR in which 4 or 5 glycosylation sites have been mutated.
There are five ΛMinked glycosylation sites in wild-type uPAR: Asn52 (in Dl) Asn162 and Asn172 (in D2) and Asn and Asn (in D3). The latter four sites in D2 and D3 are preferably mutated to GIn to generate a preferred de-glycosylated suPAR immunogen for raising mAbs for use in the invention. Following immunization using standard protocols, conventional techniques are employed to generate hybridoma cell lines from the immunized animals and to generate mAbs having the desired properties.
It is frequently observed that tumors express uPAR that is cleaved by proteases expressed by these same tumors, leaving a residual D2D3 fragment still attached to the tumor (Sier CF et al. , Thromb Haemost., 2004, 91:403-11). If this is the case with CaSCs, successful targeting of these cells (including for in vivo imaging applications) require anti D2D3 antibodies. Various anti-uPAR
DC:50410124.1 yχ ATT-17 Abs, including anti-D2D3 Abs may be tested preferably in xenogeneic tumor models, two preferred examples of which are the A2780 and A549 models (described in more detail below).
According to the present invention, an Ab or mAb, has "essentially the same antigen- binding characteristics" as a reference mAb if it demonstrates a similar specificity profile (e.g., by rank order comparison), and has affinity for the relevant antigen (e.g., a uPAR epitope) within 1.5 orders of magnitude, more preferably within one order of magnitude, of the reference Ab.
The antibodies are evaluated for action on CaSCs by any of a number of assays. These may include functional assays, for example, inhibition of self-renewal or proliferation of CaSCs (including CaSC lines). This can be measured in clonogenic assays in vitro (see, for example Jacobs, P et al, Hematology, 2005, 70:321-6). Antibody internalization may also be measured.
The present invention comprises a method of ablating or killing CaSC by contacting them with a polypeptide molecule, preferably an anti-uPAR mAb, which, recognizes the uPAR on the surface of the CaSCs, and results in the binding, and, preferably the functional inactivation of the CaSC. "Functional inactivation" refers to a statistically significant reduction in the ability of the CaSC to (a) proliferate and undergo self-renewal, (b) differentiate and form colonies of tumor/cancer cells in vitro or in vivo, (c) migrate, extravasate, metastasize, and the like.
The Ab or uPAR-binding fragment thereof can be used alone, or can be combined with other natural or artificial agents that kill cells to which the Abs have bound, such as complement, or can be chemically linked or otherwise associated with another substance that is effective in killing or ablate the CaSCs after the Ab has bound. The Ab may, but is not required to, be internalized after binding cell surface uPAR.
The methods of the present invention are particularly useful in killing or ablating any type of CaSC that expresses uPAR on its surface. According to the present invention, every type of tumor has associated with it a CaSC, although these have been demonstrated definitively thus far in a limited number of cancer types. Thus, the present methods are particularly useful for binding to and functionally inactivating CaSCs associated with acute myeloid leukemia, chronic myeloid leukemia, breast cancer, various brain tumors, lung cancer and gastrointestinal cancer.
Another aspect of the present invention relates to a method of detecting the presence, or quantitating, CaSCs in a mixed cell population including a tumor/cancer cell population, whether in the form of a cell suspension or cell culture in vitro, or a solid tumor or a leukemia in a subject.
DC:50410124.1 \ 3 ATT-17 When contacted with CaSCs, the present Ab will bind to them, and if bound to a detectable label, will permit detection of these cells. Abs immobilized to a solid support, for example a resin or magnetic beads, can also be used to remove CaSCs from BM or whole blood (or any cell population). If bound to a therapeutic moiety, the Ab will bind to and inactivate or kill the target cells, for example, within a cancerous tissue.
In a particularly preferred embodiment, the method is directed to detecting CaSCs or inactivating/killing such cells in a tumor or cancerous tissue
Because the Abs of the present invention bind to living CaSCs therapeutic methods for eliminating or inactivating these cells are more effective than those which require that target cells be lysed or their proteins be denatured for Ab recognition and binding. For the same reasons, diagnostic and imaging methods which determine the location of living CaSCs are improved by employing the present Abs.
In addition, the present method permits the monitoring of the efficacy of a treatment regimen directed to eliminating or inactivating CaSCs in vivo by permitting identification and quantification of these cells in a subject after the therapy.
Antibodies Specific for uPAR and Binary and Ternary Complexes of uPA-uPAR
In the following description, reference will be made to various methodologies known to those of skill in the art of immunology, cell biology, and molecular biology. Publications and other materials setting forth such known methodologies to which reference is made are incorporated herein by reference in their entireties as though set forth in full. Standard reference works setting forth the general principles of immunology include Abbas, AK et al, Cellular and Molecular Immunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; Janeway, CA et al, Immunobiology. The Immune System in Health and Disease, 4th ed., Garland Publishing Co., New York, 1999; Roitt, I et al, Immunology, (current ed.) CV. Mosby Co., St. Louis, MO (1999); Klein, J, Immunology, Blackwell Scientific Publications, Inc., Cambridge, MA, (1990).
Monoclonal antibodies (mAbs) and methods for their production and use are described in Kohler and Milstein, Nature 256:495-491 (1975); U.S. Patent No. 4,376,110; Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum
DC:50410124.1 J4 ATT-17 Press, New York, NY (1980); H. Zola et al, in Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, 1982)).
Immunoassay methods are also described in Coligan, JE et al, eds., Current Protocols in Immunology, Wiley-Interscience, New York 1991 (or current edition); Butt, WR (ed.) Practical Immunoassay: The State of the Art, Dekker, New York, 1984; Bizollon, CA, ed., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, New York, 1984; Butler, JE, ELISA (Chapter 29), In: van Oss, CJ et al, (eds), IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, JE (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991; Weintraub, B, Principles of Radioimmunoassays, The Endocrine Society, March, 1986; Work, TS et al, Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, 1978; Dabbs, DJ, Diagnostic Immunohistochemistry, Churchill Livingstone, 2001.
Anti-idiotypic antibodies are described, for example, in Idiotypy in Biology and Medicine, Academic Press, New York, 1984; Immunological Reviews Vol. 79, 1984 and Vol. 90, 1986; Curr. Top. Microbiol, Immunol. Vol. 119, 1985; Bona, C. et al, CRC Crit. Rev. Immunol, pp. 33-81 (1981); Jerne, NK, Ann. Immunol. 725C:373-389 (1974); Urbain, J et al, Ann. Immunol. I33DΛ79- (1982); Rajewsky, K. et al, Ann. Rev. Immunol. 7:569-607 (1983).
The present invention provides Abs, both polyclonal and monoclonal, reactive with uPAR including those that bind uPA-uPAR complexes that inhibit interactions of uPAR with integrins or other downstream targets. The Abs may be xenogeneic, allogeneic, syngeneic, or modified forms thereof, such as humanized or chimeric Abs. Antiidiotypic Abs specific for the idiotype of, for example, an anti- uPAR Ab are also included.
The term "antibody" is meant to include both intact immunoglobulin (Ig) molecules as well as fragments and derivative thereof, that may be produced by proteolytic cleavage of Ig molecules or engineered genetically or chemically. Fragments include, for example, Fab, Fab', F(ab')2 and Fv, each of which is capable of binding antigen. These fragments lack the Fc fragment of intact Ab and have an additional advantage, if used therapeutically, of clearing more rapidly from the circulation and undergoing less non-specific tissue binding than intact Abs. Papain treatment of Ig's produces Fab fragments; pepsin treatment produces F(ab')2 fragments. These fragments may also produced by genetic or protein engineering using methods well known in the art. A Fab fragment is a multimeric protein consisting of the portion of an Ig molecule containing the immunologically active portions
DC:50410124.1 15 ATT-17 of an Ig heavy (H) chain and an Ig light (L) chain covalently coupled together and capable of specifically combining with antigen. Fab fragments are typically prepared by proteolytic digestion of substantially intact Ig molecules with papain using methods that are well known in the art. However, a Fab fragment may also be prepared by expressing in a suitable host cell the desired portions of Ig H chain and L chain using methods well known in the art. A (Fab')2 fragment is a tetramer that includes a fragment of two H and two L chains. The Fv fragment is a multimeric protein consisting of the immunologically active portions of an Ig H chain variable (V) region (VH) and an Ig L chain V region (VL) covalently coupled together and capable of specifically combining with antigen. Fv fragments are typically prepared by expressing in suitable host cell the desired portions of Ig VH region and VL region using methods well known in the art.
Single-chain antigen-binding protein or single chain Ab, also referred to as "scFv," is a polypeptide composed of an Ig VL amino acid sequence tethered to an Ig VH amino acid sequence by a peptide that links the C-terminus of the VL sequence to the N-terminus of the VH sequence.
Polyclonal Abs are obtained as sera from immunized animals such as rabbits, goats, rodents, etc. and may be used directly without further treatment or may be subjected to conventional enrichment or purification methods such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography.
An immunogen for generation of the anti-uPAR Abs of this invention may comprise uPAR, suPAR, uPA/uPAR or uPAR-integrin complexes/ or an epitope-bearing fragments or derivative thereof. Useful immunogens are produced in a variety of ways known in the art, e.g., expression of cloned genes using conventional recombinant methods, isolation from cells of origin, cell populations expressing high levels of e.g., uPA or uPAR, etc. In the case of shorter fragments, they may be chemically synthesized. A preferred immunogen is the D2D3 fragment of suPAR.
The mAbs may be produced using conventional hybridoma technology, such as the procedures introduced by Kohler and Milstein {Nature, 256:495-97 (1975)),-and modifications thereof (see above references). An animal, preferably a mouse is primed by immunization with an immunogen as above to elicit the desired Ab response in the primed animal.
B lymphocytes from the lymph nodes, spleens or peripheral blood of a primed, animal are fused with myeloma cells, generally in the presence of a fusion promoting agent such as polyethylene glycol (PEG). Any of a number of murine myeloma cell lines are available for such use: the P3-NSl/l-Ag4-l, P3-x63-k0Ag8.653, Sp2/0-Agl4, or HL1-653 myeloma lines (available
DC:50410124.1 \ β ATT-17 from the ATCC, Rockville, MD). Subsequent steps include growth in selective medium so that unfused parental myeloma cells and donor lymphocyte cells eventually die while only the hybridoma cells survive. These are cloned and grown and their supernatants screened for the presence of Ab of the desired specificity, e.g., by immunoassay techniques. Positive clones are subcloned, e.g., by limiting dilution, and the mAbs are isolated.
Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art (see generally Fink et ah, Prog. Clin. Pathol., 9:121-33 (1984)). Generally, the individual cell line is propagated in culture and the culture medium containing high concentrations of a single mAb can be harvested by decantation, filtration, or centrifugation.
Test Cells for Screening and Characterizing Antibodies
Pure uPAR immobilized onto plastic is preferred for the primary screening. Cells that overexpress uPAR (such as the HeLa line) may be used to demonstrate cell binding of an anti- uPAR mAb. Many tumor cell lines overexpressing uPAR are well-known and publicly available; these may be used for screening. Cells are generally plated in 96-well microplates. The cells may be fixed, e.g.,, with methanol/acetone (50/50), and the binding detected by immunofluorescence staining. Alternatively, the mAbs may be radiolabeled and binding detected by measurement of radioactivity. Hybridoma supernatants are preferably studied using methods employing immunofluorescence or ELISA.
In a preferred embodiment, the Ab is a mAb designated ATN-615 or ATN-658, both of which are IgGl Abs.
In another preferred embodiment, the Ab is a chimeric Ab that recognizes an epitope recognized by ATN-615 or ATN-658.
Chimeric Antibodies
The chimeric Abs of the invention comprise individual chimeric H and L Ig chains. The chimeric H chain comprises an antigen binding region derived from the H chain of a non-human Ab specific for e.g., uPA/uPAR or uPAR-integrin complex, for example, mAb ATN-615 or ATN-658, which is linked to at least a portion of a human CH region. A chimeric L chain comprises an antigen binding region derived from the L chain of a non-human Ab specific for the target antigen, such as the hybridoma ATN-615 or ATN-658, linked to at least a portion of a human CL region. As used herein,
DC:50410124.1 \ η ATT-17 the term "antigen binding region" refers to that portion of an Ab molecule which contains the amino acid residues that interact with an antigen and confer on the Ab its specificity and affinity for the antigen. The Ab region includes the "framework" amino acid residues necessary to maintain the proper conformation of the antigen-binding (or "contact") residues.
As used herein, the term "chimeric antibody" includes monovalent, divalent or polyvalent Igs. A monovalent chimeric Ab is an HL dimer formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain. A divalent chimeric Ab is tetramer H2L2 formed by two HL dimers associated through at least one disulfide bridge. A polyvalent chimeric Ab can also be produced, for example, by employing a CR region that aggregates (e.g., from an IgM H chain, termed the μ chain).
The invention also provides for "derivatives" of the mouse mAbs or the chimeric Abs, which term includes those proteins encoded by truncated or modified genes to yield molecular species functionally resembling the Ig fragments. The modifications include, but are not limited to, addition of genetic sequences coding for cytotoxic proteins such as plant and bacterial toxins. The fragments and derivatives can be produced from any of the hosts of this invention.
Abs, fragments or derivatives having chimeric H chains and L chains of the same or different V region binding specificity, can be prepared by appropriate association of the individual polypeptide chains, as taught, for example by Sears et al, Proc. Natl. Acad. ScL USA 72:353-357 (1975). With this approach, hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the Ig chains are separately recovered and then associated. Alternatively, the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled Ig, fragment or derivative.
The antigen binding region of the chimeric Ab (or a human mAb) of the present invention is derived preferably from a non-human Ab specific for e.g. , uPAR. Examples of preferred chimeric Abs (or human Abs) are those described in WO 2005/116077. Generally, the chimeric Abs of the present invention are produced by cloning DNA segments encoding the H and L chain antigen-binding regions of an Ab of the invention, preferably non-human, and joining these DNA segments to DNA segments encoding human CH and CL regions, respectively, to produce chimeric Ig-encoding genes.
Description of the molecular biological methods for producing such chimeric or other recombinant Abs are well-known in the art and are discussed in more detail in the present inventors' and their colleagues published application, WO 2005/116077. For in vivo use, particularly for injection into humans, it is desirable to decrease the immunogenicity of the mAb by
DC:50410124.1 J g ATT-17 making mouse-human (or rodent-human) chimeric Abs by humanizing the Abs using methods known in the art. The humanized Ab may be the product of an animal having transgenic human Ig Constant region genes (see for example WO90/10077 and WO90/04036). Alternatively, the Ab of interest may be genetically engineered to substitute the CHi, CH2, CH3, hinge domains, and/or the framework domain with the corresponding human sequence (see WO92/02190). Single Chain Antibodies
The Ab of the present invention may be produced as a single chain Ab or scFv instead of the normal multimeric structure. Single chain Abs include the hypervariable regions from an Ig of interest and recreate the antigen binding site of the native Ig while being a fraction of the size of the intact Ig (Skerra, A. et al (1988) Science, 240: 1038-1041; Pluckthun, A. et al (1989) Methods Enzymol 178: 497-515; Winter, G. et al (1991) Nature, 349: 293-299); Bird et al, (1988) Science 242:423; Huston et al (1988) Proc. Natl Acad. Sci. USA 85:5879; Jost CR et al,. J Biol Chem. 1994 2^:26267-26273; U.S. Patents No. 4,704,692, 4,853,871, 4,94,6778, 5,260,203, 5,455,030). DNA sequences encoding the V regions of the H chain and the L chain are ligated to a linker encoding at least about 4 amino acids (typically small neutral amino acids). The protein encoded by this fusion allows assembly of a functional variable region that retains the specificity and affinity of the original Ab.
One method of producing the single chain Abs of the present invention is to link two or more peptides or polypeptides together by protein chemistry techniques known in the art and described in more detail in WO 2005/116077.
Abs can be selected for particular desired properties. In the case of an Ab to be used in vivo, Ab screening procedures can include any of the in vitro or in vivo bioassays that measure binding to uPAR (or too uPA/uPAR or uPAR-integrin complexes), , to cells expressing the relevant polypeptide or peptide epitope. Moreover, the Abs may be screened in various of tumor models such as a xenogeneic mouse model in which a human tumor cell line expressing the antigen is grown in immunocompromised, e.g., nude, mice.
Diagnostically Labeled Antibody
The term "diagnostically labeled" means that the present Ab has attached to it a diagnostically detectable label. There are many different labels and methods of labeling known to those of ordinary skill in the art, described below. General classes of labels which can be used in
DC:50410124.1 \ 9 ATT-17 the present invention include radioactive isotopes, paramagnetic isotopes, and compounds which can be imaged by positron emission tomography (PET), fluorescent or colored compounds, etc. Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels. Useful radiolabels (radionuclides), which are detected simply by gamma counter, scintillation counter or autoradiography include 3H, 125I, 1311, 35S and 14C. 131I is also a useful therapeutic isotope (see below).
A number of U.S. patents, incorporated by reference herein, disclose methods and compositions for complexing metals to larger molecules, including description of useful chelating agents. The metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules. These documents include: U.S. Patents 5,627,286; 5,618,513; 5,567,408; 5,443,816; and 5,561,220.
Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The fluorophore, such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR., 1996). Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon Green™ and its derivatives, Rhodamine Green™ and Rhodol Green™, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups. Similarly, fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups. The long wavelength rhodamines, which are basically Rhodamine Green™ derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known. Their spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluoresceins for many biological applications. This group includes the tetramethylrhodamines, X-rhodamines and Texas Red™ derivatives. Other preferred fluorophores for derivatizing the peptide according to this invention are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives. Also included as labels are two related inorganic materials that have recently been described: semiconductor nanocrystals, comprising, for example, cadmium sulfate (Bruchez, M et al, Science 257:2013-2016 (1998), and quantum dots, e.g., zinc-sulfϊde-capped Cd selenide (Chan, WC et al, Science 281 :2016-2018 (1998)).
DC:50410124.1 20 ATT-17 In yet another approach, the amino group of the Ab is allowed to react with reagents that yield fluorescent products, for example, fluorescamine, dialdehydes such as o-phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate. 7-nitrobenz-2-oxa-l,3-diazole (NBD) derivatives, both chloride and fluoride, are useful to modify amines to yield fluorescent products.
The Ab of the invention can also be labeled for detection using fluorescence-emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the peptide using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA, see Example X, infra) or ethylenediaminetetraacetic acid (EDTA). DTPA, for example, is available as the anhydride, which can readily modify the NH2-containing peptides of this invention.
Radiolabeled antibodies labeled with γ-emitters, positron-emitters, x-ray emitters and fluorescence-emitters are suitable for localization and/or therapy, while β-emitters and α-emitters may also be used for therapy. Suitable radioisotopes for the methods of the present invention include: 211At, 123I, 125I, 126I, 131I, 133I, 212Bi, 77Br, 111In, 113In, 67Ga, 68, 95Ru, 97Ru, 103Ru, 105Ru, 107Hg, 203Hg, 186Re, 188Re, 121mTe, 122mTe, 125mTe, 165Tm, 167Tm, 168Tm, 99mTc, 18Fl, 111Ag, 197Pt, 109Pd, 67Cu, 32P, 33P, 90Y, 47Sc, 153Sm, 177Lu, 105Rh, 142Pr, 143Pr, 161Tb, 166No, 199Au, 57Co, 58Co, 51Cr, 59Fe, 75Se, 201Tl, and 169Yb. Preferably the radioisotope will emit in the 10-5,000 kev range, more preferably 50-1,500 kev, most preferably 50-500 kev.
Of the above isotopes, those preferred for diagnostic use include 123I, 131I, 111In, 67Ga, 97Ru,
99mr Tpc, 67/ C^u, 57/ C^o, 58^ Co, 51^ Cr, 59T F-e, 75C Se, 201r Tp,l, and , 169Λ Yrib.
Generally, the amount of labeled Ab needed for detectability in diagnostic use will vary depending on considerations such as age, condition, sex, and extent of disease in the patient, contraindications, if any, and other variables, and is to be adjusted by the individual physician or diagnostician. Dosage can vary from 0.001 mg/kg to 100 mg/kg.
The Ab can also be made detectable by coupling to a phosphorescent or a chemiluminescent compound. The presence of the chemiluminescent-tagged peptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescers are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. Likewise, a bioluminescent compound may be used to label the peptides. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence
DC:50410124.1 21 ATT-17 of a bio luminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
In yet another embodiment, colorimetric detection is used, based on chromogenic compounds which have, or result in, chromophores with high extinction coefficients.
In situ detection of the labeled peptide may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.
For diagnostic in vivo radioimaging, the type of detection instrument available is a major factor in selecting a radionuclide. The radionuclide chosen must have a type of decay which is detectable by a particular instrument. In general, any conventional method for visualizing diagnostic imaging can be utilized in accordance with this invention. Another factor in selecting a radionuclide for in vivo diagnosis is that its half-life be long enough so that the label is still detectable at the time of maximum uptake by the target tissue, but short enough so that deleterious irradiation of the host is minimized. In one preferred embodiment, a radionuclide used for in vivo imaging does not emit particles, but produces a large number of photons in a 140-200 keV range, which may be readily detected by conventional gamma cameras.
In vivo imaging may be used to detect occult metastases which are not observable by other methods. Imaging could be used, for example, to stage tumors non-invasively.
ASSAYS FOR EVALUATING BINDING AND ACTIVITY OF uPAR LIGANDS
Immunohistochemical Assays
One preferred assay for detecting the antigens in a tissue is by immunohistochemistry, using any conventional assay methods, with which the art is replete. A preferred assay is the one described in the Examples below. For a description of such methods, see, for example, Dabbs, DJ, Diagnostic Immunohistochemistry, Churchill Livingstone, 2001, which is incorporated by reference in its entirety.
Non-Histological Immunoassays
Preferred immunoassays are enzyme immunoassays (EIA' s) such as ELISA, which employ antigens or Abs immobilized to solid supports. For the present compositions and methods, the solid support is preferably any one of polystyrene, polypropylene, polyethylene, dextran, nylon,
DC:50410124.1 22 ATT-17 polyacrylamide, polyvinylidene difluoride, natural cellulose, modified cellulose, nitrocellulose, agarose and magnetic beads. In a preferred embodiment, the surface of polystyrene or other plastic multiwell plates serves as the solid support. In another embodiment, a solid support to which the Ab or antigen is affixed to the bottom or placed loosely in the wells of multiwell plates. Multiwell plates in which the bottoms of the wells comprise nitrocellulose or a similar membrane material and through which liquid can be moved under pressure or vacuum may also be used.
Typical, and preferred, immunoassays include "forward" assays in which the Ab immobilized to a solid support is first contacted with the sample being tested to bind or "extract" the antigen from the sample by formation of a binary immobilized Ab-antigen complex. After suitable incubation, the solid support is washed to remove the residue of the fluid sample including unbound antigen, if any, and then contacted with the solution containing an unknown quantity of labeled Ab (which functions as a "reporter molecule"). After a second incubation, that permits the labeled Ab to complex with the immobilized antigen through the unlabeled Ab, the solid support is washed a second time to remove the unreacted labeled Ab and the immobilized label is measured. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the amount of immobilized labeled Ab with the amount immobilized when a standard sample containing a known quantity of antigen is used.
So called "simultaneous" and "reverse" sandwich assays may also be used. A simultaneous assay involves a single incubation step as the immobilized Ab and labeled Ab are added simultaneously to the sample. After appropriate incubation, the solid support is washed to remove residue of the sample and uncomplexed labeled Ab. The presence or amount of labeled Ab associated with the solid support is then determined as in the above conventional "forward" sandwich assay.
In a "reverse" assay, a solution of labeled Ab is added to the sample after a suitable incubation period followed by addition of immobilized unlabeled Ab. After a second incubation, the solid phase material is washed in conventional fashion to free it of the residue of the sample and unreacted labeled Ab. The determination of immobilized Ab associated with the solid support is then determined as in the "simultaneous" and "forward" assays.
DC:50410124.1 23 ATT-17 Assay for Antibody Binding to uPAR on Whole Cells
The uPAR-targeting Ab and/or conjugate thereof is readily tested for binding to uPAR, preferably by measuring inhibition of the binding of [125I]DFP-uPA to uPAR in a competitive ligand-binding assay or by directly labeling the Ab with [125I] . The assay may employ whole cells that express uPAR, for example cells lines such as A2780 or HeLa. A preferred assay is conducted as follows. Cells (about 5 x 104/well) are plated in medium (e.g., MEM with Earle's salts/10% FBS + antibiotics) in 24-well plates, then incubated in a humid 5% CO2 atmosphere until the cells reach 70% confluence. Catalytically inactivated high molecular weight uPA (DFP-uPA) is radioiodinated using Iodo-gen® (Pierce) to a specific activity of about 250,000 cpm/μg. The cell-containing plates are then chilled on ice and the cells are washed twice (5 minutes each) with cold PBS/ 0.05% Tween-80. Test Abs and/or conjugates thereof are serially diluted in cold PBS/ 0.1 % BSA/ 0.01% Tween-80 and added to each well to a final volume of 0.3mL 10 minutes prior to the addition of the [125I]DFP-uPA. Each well then receives 9500 cpm of [125I]DFP-uPA at a final concentration of 0.2 nM). The plates are then incubated at 4°C for 2 hrs, after which time the cells are washed 3x (5 minutes each) with cold PBS/ 0.05% Tween-80. NaOH (IN) is added to each well in 0.5 mL to lyse the cells, and the plate is incubated for 5 minutes at room temperature or until all the cells in each well are lysed as determined by microscopic examination. The contents of each well are then aspirated and the total counts in each well determined using a gamma counter. Each compound is tested in triplicate and the results are expressed as a percentage of the total radioactivity measured in wells containing [125I]DFP-uPA alone, which is taken to represent maximum (100 %) binding.
The inhibition of binding of [125I]DFP-uPA to uPAR is usually dose-related, such that the concentration of the test compound necessary to produce a 50% inhibition of binding (the IC50 value), which is expected to fall in the linear part of the curve, is easily determined. In general, Abs and/or conjugates thereof have IC50 values of less than about 10"5 M. Preferably, Abs and/or conjugates thereof have IC50 values of less than about 10"6 M, more preferably, less than about 10"7M.
Assays of Biological Activity of Anti-uPAR Antibodies or other Ligands
Those of skill in the art will appreciate that the in vitro and in vivo assays useful for measuring the activity of the Abs or other uPAR-binding ligands of the invention or of conjugates thereof, as described herein, are intended to be illustrative and neither comprehensive nor limiting.
DC:50410124.1 24 ATT-17 Assay for EC migration
For EC migration studies, transwells are coated with type I collagen (50 μg/mL) by adding 200 μL of the collagen solution per transwell, then incubating overnight at 37°C. The transwells are assembled in a 24-well plate and a chemoattractant (e.g. , FGF -2) is added to the bottom chamber in a total volume of 0.8 mL media. ECs, such as human umbilical vein endothelial cells (HUVEC), which have been detached from monolayer culture using trypsin, are diluted to a final concentration of about 106 cells/mL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell. Inhibitors to be tested may be added to both the upper and lower chambers and the migration is allowed to proceed for 5 hrs in a humidified atmosphere at 37°C. The transwells are removed from the plate stained using DiffQuik®. Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (40Ox) to determine the number of cells migrated.
Biological Assay of Anti-Invasive Activity
The ability of cells such as ECs or tumor cells (e.g., PC-3 human prostatic carcinoma cells) to invade through a reconstituted basement membrane (Matrigel®) in an assay known as a Matrigel® invasion assay system is well known (Kleinman et al, Biochemistry 1986, 25: 312-318; Parish et al, 1992, Int. J. Cancer 52:378-383). Matrigel® is a reconstituted basement membrane containing type IV collagen, laminin, heparan sulfate proteoglycans such as perlecan (which bind to and localize bFGF), vitronectin as well as transforming growth factor-β (TGFβ), urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA) and the serpin known as plasminogen activator inhibitor type 1 (PAI-I) (Chambers et al., Cane. Res. 1995, 55:1578-1585). It is accepted in the art that results obtained in this type of assay for Abs and/or conjugates thereof or other ligands which target extracellular receptors or enzymes are predictive of the efficacy of these Abs and/or conjugates thereof in vivo (Rabbani et al., Int. J. Cancer 1995, 63: 840-845).
Such assays employ transwell tissue culture inserts. Invasive cells are defined as cells which traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom of the membrane. Transwells (e.g., from Costar) containing polycarbonate membranes (8.0 μm pore size) are coated with Matrigel® (e.g., from Collaborative Research), which has been diluted in sterile PBS to a final concentration of about 75 μg/mL (e.g., 60 μL of diluted Matrigel®
DC:50410124.1 25 ATT-17 per insert), and placed in the wells of a 24-well plate. The membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 μL of medium, e.g., DMEM, supplemented with antibiotics for 1 hour on a shaker table. The DMEM is removed from each insert by aspiration and 0.8 mL of complete DMEM (+/10 % FBS and antibiotics) is added to each well of the 24-well plate such that it surrounds the outside of the transwell ("lower chamber"). Fresh DMEM with antibiotics (lOOμL), human Glu-plasminogen (5 μg/mL), and any inhibitors to be tested are added to the top, inside of the transwell ("upper chamber"). The cells which are to be tested are trypsinized and resuspended in DMEM+antibiotics and added to the top chamber of the transwell at a final concentration of about 8x105 cells/mL. The final volume of the upper chamber is adjusted to 200 μL. The assembled plate is then incubated in a humid 5% CO2 atmosphere for about 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane. The membranes are detached from the transwell using an X-acto® blade, mounted on slides using Permount® and coverslips, then counted under a microscope using high power (e.g., 40Ox). A mean number of invading cells from 5-10 counted fields is calculated and plotted as a function of inhibitor concentration.
Tube-Formation Assays of Anti- Angiogenic Activity
ECs, for example, human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HMVEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 x 105 cells/mL with fibrinogen (5mg/mL in phosphate buffered saline (PBS) in a 1 :1 (v/v) ratio. Thrombin is added (5 units/ mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well). The fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound. The cells are incubated at 37°C in 5% CO2 for 4 days at which time the cells in each well are counted and classified as either rounded, elongated with no branches, elongated with one branch, or elongated with 2 or more branches. Results are expressed as the average of 5 different wells for each concentration of compound. Typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch). This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min et al, Cancer Res. 1996, 56: 2428-2433).
DC:50410124.1 26 ATT-17 In an alternate assay, EC tube formation is observed when ECs are cultured on Matrigel® (Schnaper HW et α/., J. Cell Physiol. 1995, 755:107-118). 104 EC /well are transferred onto Matrigel®-coated 24-well plates, and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the time of adding the ECs or at various time points thereafter. Tube formation can also be stimulated by adding (a) an angiogenic growth factor such as bFGF or VEGF, (b) a differentiation stimulating agent {e.g., PMA) or (c) a combination of these.
While not wishing to be bound by theory, this assay models angiogenesis by presenting to the ECs a particular type of basement membrane, namely the layer of matrix which migrating and differentiating ECs would be expected to encounter first. In addition to bound growth factors, the matrix components found in Matrigel® (and in basement membranes in situ), or proteolytic products thereof, may also be stimulatory for EC tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood, CH et ah, Biochim. Biophys. Acta 1990, 7052:89-118; Odedra, R et al., Pharmac. Ther. 1991, 4P:111-124).
Assays for Inhibition of Cell Proliferation
The ability of the Abs and/or conjugates of this invention to inhibit the proliferation of ECs may be determined in a 96-well format. Type I collagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x using PBS), 3-6 x 103 cells are plated per well and allowed to attach for 4 hrs (37°C/5% CO2) in Endothelial Growth Medium (EGM; Clonetics ) or M199 medium supplemented with 0.1-2% FBS. The medium and any unattached cells are removed at the end of 4 hrs and fresh medium supplemented with bFGF (1-10 ng/mL) or VEGF (1-10 ng/niL) is added to each well. Antibodies and/or conjugates to be tested are added last, and the plate is allowed to incubate (37°C/5% CO2) for 24-48 hrs. The chromogenic compound MTS (Promega) is added to each well and allowed to incubate from 1-4 hrs. The color developing in each well is directly proportional to the cell number, thereby serving as a surrogate for counting cells. Absorbance read at 490nm is used to determine the differences in cell numbers, i.e., proliferation, between control wells and those containing test Abs and/or conjugates.
A similar assay employing cultured adherent tumor cells may also be used. However, collagen may be omitted in this format. Tumor cells {e.g., 3-10 x 10 /well) are plated and allowed to adhere overnight. Serum-free medium is then added, and the cells forced to synchronize for 24 hrs. Medium + 10% FBS is then added to each well to stimulate proliferation. Antibodies and/or
DC:50410124.1 27 ATT-17 conjugates to be tested are included in some of the wells. After 24 hrs, MTS is added to the plate and the assay developed and read as above.
Assays of Cytotoxicity
The anti-pro liferative and cytotoxic effects of Abs and/or conjugates thereof may be determined for various cell types including tumor cells, ECs, fibroblasts and macrophages. This is especially useful when testing a Ab which has been conjugated to a therapeutic moiety such as a radiotherapeutic or a toxin. For example, a conjugate of one of the Abs of the invention with Bolton-Hunter reagent which has been iodinated with 131I would be expected to inhibit the proliferation of cells expressing uPAR (most likely by inducing apoptosis). Anti-pro liferative effects would be expected against tumor cells and stimulated endothelial cells but, under some circumstances not quiescent endothelial cells or normal human dermal fibroblasts. Any anti-proliferative or cytotoxic effects observed in the normal cells may represent non-specific toxicity of the conjugate.
A typical assay would involve plating cells at a density of 5-10,000 cells per well in a 96-well plate. The compound to be tested is added at a concentration 10x the IC50 measured in a binding assay (this will vary depending on the conjugate) and allowed to incubate with the cells for 30 minutes. The cells are washed 3X with media, then fresh media containing [3H]thymidine (1 μCi/mL) is added to the cells and they are allowed to incubate at 37°C in 5% CO2 for 24 and 48 hours. Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a β-counter. Proliferation may be measured non-radioactively using MTS reagent or CyQuant® to measure total cell number. For cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS (Genzyme).
Assay of Caspase-3 Activity
The ability of the Abs and/or conjugates to promote apoptosis of EC's may be determined by measuring activation of caspase-3. Type I collagen (gelatin) is used to coat a PlOO plate and 5xlO5 ECs are seeded in EGM + 10% FBS. After 24 hours (at 37°C/5% CO2) the medium is replaced by EGM + 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hrs, cell lysates prepared in 1% Triton X-IOO detergent, and the lysates assayed using the EnzChek®Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures' instructions.
DC:50410124.1 28 ATT-17 Corneal Angiogenesis Model
The protocol used is essentially identical to that described by Volpert, OV et al., J. Clin. Invest. 1996, 98:611-619. Briefly, female Fischer rats (120-140 gms) are anesthetized and pellets (5 μl) comprised of Hydron®, bFGF (150 nM), and the Abs and/or conjugates thereof to be tested are implanted into tiny incisions made in the cornea 1.0-1.5 mm from the limbus. Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay
This assay is performed essentially as described by Nguyen et al. , Microvascular Res. 1994, 47:31-40. A mesh containing either angiogenic factors (bFGF) or tumor cells plus a test compound, here the anti—uPAR Abs or conjugates, placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation of the sample. Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain visible blood vessels.
Matrigel® Plug Assay
This assay is performed essentially as described by Passaniti, A et al., 1992, Lab Invest. (57:519-528. Ice-cold Matrigel® (e.g., 500 μL) (Collaborative Biomedical Products, Inc., Bedford, MA) is mixed with heparin (e.g., 50 μg/ml), FGF-2 (e.g., 400 ng/ml) and the compound to be tested. In some assays, bFGF may be substituted with tumor cells as the angiogenic stimulus. The Matrigel® mixture is injected subcutaneously (s.c.) into 4-8 week-old athymic nude mice at sites near the abdominal midline, preferably 3 injections per mouse. The injected Matrigel® forms a palpable solid gel. Injection sites are chosen such that each animal receives a positive control plug (such as FGF2 + heparin), a negative control plug (e.g., buffer +heparin) and a plug that includes the compound being tested for its effect on angiogenesis, e.g., (FGF-2 + heparin + compound). All treatments groups are preferably run in triplicate. Animals are sacrificed by cervical dislocation at about 7 days post injection or another time that may be optimal for observing angiogenesis. The mouse skin is detached along the abdominal midline, and the Matrigel® plugs are recovered and scanned microscopically immediately at high resolution. Plugs are then dispersed in water and incubated at 37°C overnight. Hemoglobin (Hb) levels in the plugs are determined using Drabkin's
DC:50410124.1 29 ATT-17 solution (e.g., from Sigma) according to the manufacturers' instructions. The amount of Hb in the plug is an indirect measure of angiogenesis as it reflects the amount of blood in the sample.
In addition, or alternatively, animals may be injected prior to sacrifice with a 0.1 ml buffer (preferably PBS) containing a high molecular weight dextran to which is conjugated a fluorophore. The amount of fluorescence in the dispersed plug, determined fluorimetrically, also serves as a measure of angiogenesis in the plug. Staining with mAb anti-CD31 (CD31 is "platelet-endothelial cell adhesion molecule", "PECAM") may also be used to confirm neovessel formation and microvessel density in the plugs.
In Vivo Assessment of Angiogenesis Inhibition and Anti-Tumor Effects Using the Matrigel® Plug Assay with Tumor Cells
In this assay, tumor cells, for example 1-5 x 106 cells of the 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described above. A mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days. The anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug. Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran- fluorophore conjugate injected prior to sacrifice). To measure Hb or fluorescence, the plugs are first homogenized with a tissue homogenizer.
Xenograft Models of Subcutaneous Tumor Growth
Human Ovarian Carcinoma
A2780 human ovarian cancer line was established from tumor tissue from an untreated patient. The A2780 cells are maintained as a monolayer in RPMI 1640 medium supplemented with 2 mM glutamine, 0.01 mg/mL bovine insulin, and 10% FBS. (Hamilton, TC et al, Sem. Oncol. 1984; 77:285-293; Behrens, BC et al, Cancer Res. 1987; 47:414-418). Two million A2780 are inoculated in the right flank of nude Balb/c female mice. The A2780 tumor is staged to 50 to 200 mm range before treatment is. The IgG control Ab as well as the anti-D2D3 uPAR mAbs are administered by the intraperitoneal route at 10 mg/kg twice weekly on Monday and Friday. The cisplatin treatment group was staged to 1000 mm3; animals received 6 mg/kg once a week. Tumor volumes were measured twice a week. At the time of sacrifice, plasma is obtained and the tumor excised from each animal. Half of the tumor is snap frozen for biochemical assessment and the rest is placed in Zinc fixative for histological assessment.
DC:50410124.1 3Q ATT-17 Human Lung Carcinoma
A549, human lung carcinoma (ATCC Catalog No. CCL-185) cell line, was established through explant culture of lung carcinomatous tissue from a 58-year-old Caucasian male (Giard, DJ et al., J. Natl. Cancer Inst. 57:1417-23 (1973)). A549 cells are maintained in Ham's F12K medium supplemented with 2 mM L-glutamine, 0.15% NaHCO3, and 10 % FBS.
About 106 A549 carcinoma cells are inoculated in the right flank of C.B-17/Sys (scid/scid) Severe Combined Immunodeficient (SCID) female mice. Treatment is preferably initiated the day after tumor inoculation. The IgG control Ab (and the PBS control) as well as the anti-D2D3 uPAR mAb ATN-658 are administered intraperitoneally 10 mg/kg twice weekly on Monday and Friday. Initially tumor volumes are measured once a week. When the volume in any treatment group exceeds 300 mm3, measurements are obtained twice a week.
At the time of sacrifice, plasma is obtained and the tumor excised from each animal. Half of the tumor is snap frozen for biochemical assessment and the rest is placed in Zinc fixative for histological assessment. Xenograft Model of Metastasis
The Abs and/or conjugates are tested for inhibition of late metastasis using an experimental metastasis model such as that of Crowley et al., Proc. Natl. Acad. ScL USA 1993, 90 5021-5025). Late metastasis involves the steps wherein tumor cells attach and extravasate, invade locally, seed, proliferate and induce angiogenesis. Human prostatic carcinoma cells (PC-3) transfected with a reporter gene, preferably the green fluorescent protein (GFP) gene, but as an alternative with a gene encoding the enzymes chloramphenicol acetyl-transferase (CAT), luciferase or LacZ, are inoculated into nude mice. This approach permits utilization of either of these markers (fluorescence detection of GFP or histochemical colorimetric detection of the various enzymes) to follow the fate of these cells. Cells are injected, preferably iv, and metastases identified after about 14 days, particularly in the lungs but also in regional lymph nodes, femurs and brain. This mimics the organ tropism of naturally occurring metastases in human prostate cancer. For example, GFP-expressing PC-3 cells (106 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at lOOμg/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay of the detectable label.
DC:50410124.1 3 \ ATT-17 Pharmaceutical and Therapeutic Compositions and Their Administration
The compounds that may be employed in the pharmaceutical compositions of the invention include all uPAR ligands, typically polypeptide molecules, preferably Abs, described above, as well as pharmaceutically acceptable salts of various of these compounds. Pharmaceutically acceptable acid addition salts of the compounds of the invention containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art. Exemplary of the acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.
Pharmaceutically acceptable base addition salts of compounds of the invention containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hy droxymethy l)methy lamine .
As stated above, the compounds of the invention possess the ability to inhibit EC proliferation, motility, or invasiveness and angiogenesis, properties that are exploited in the treatment of cancer, in particular metastatic cancer. A composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to the active form.
Therapeutically Labeled Compositions
In a preferred embodiment, the mAbs describe herein are "therapeutically conjugated" or "therapeutically labeled" (terms which are intended to be interchangeable) and used to deliver a therapeutic agent to the site to which the compounds home and bind, such as sites of tumor metastasis or foci of infection/inflammation, restenosis or fibrosis. The term "therapeutically conjugated" means that the modified mAb is conjugated to another therapeutic agent that is directed either to the underlying cause or to a "component" of tumor invasion, angiogenesis, inflammation or other pathology. A therapeutically labeled polypeptide carries a suitable therapeutic "label" also referred to herein as a "therapeutic moiety." A therapeutic moiety is an atom, a molecule, a compound or any chemical component added to the peptide that renders it active in treating a target disease or condition, primarily one a associated with undesired angiogenesis. The therapeutic moiety may be bound directly or indirectly to the mAb. The therapeutically labeled mAb is
DC:50410124.1 32 ATT-17 administered as pharmaceutical composition which comprises a pharmaceutically acceptable carrier or excipient, and is preferably in a form suitable for injection.
Useful isotopes were discussed more fully above. Those isotopes preferred for therapeutic use include: 125I, 131I, 186Re, 188Re, 111Ag, 197Pt, 109Pd, 67Cu, 32P, 33P, 90Y, 47Sc, 53Sm, 177Lu, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, and 199Au. These atoms can be conjugated to the peptide directly, indirectly as part of a chelate, or, in the case of iodine, indirectly as part of an iodinated Bolton- Hunter group. The radioiodine can be introduced either before or after this group is coupled to the peptide compound.
Preferred doses of the radionuclide conjugates are a function of the specific radioactivity to be delivered to the target site which varies with tumor type, tumor location and vascularization, kinetics and biodistribution of the peptide carrier, energy of radioactive emission by the nuclide, etc. Those skilled in the art of radiotherapy can readily adjust the dose of the peptide in conjunction with the dose of the particular nuclide to effect the desired therapeutic benefit without undue experimentation.
Another therapeutic approach included here is the use of boron neutron capture therapy, where a boronated peptide is delivered to a desired target site, such as a tumor, most preferably an intracranial tumor (Barth, RF, Cancer Invest. 74:534-550 (1996); Mishima, Y (ed.), Cancer Neutron Capture Therapy, New York: Plenum Publishing Corp., 1996; Soloway, AH et al, (eds), J. Neuro-Oncol. 33:1-188 (1997). The stable isotope 10B is irradiated with low energy (<0.025eV) thermal neutrons, and the resulting nuclear capture yields α-particles and 7Li nuclei which have high linear energy transfer and respective path lengths of about 9 and 5 μm. This method is predicated on 10B accumulation in the tumor with lower levels in blood, endothelial cells and normal tissue {e.g., brain). Such delivery has been accomplished using epidermal growth factor (Yang. W et al, Cancer Res 57:4333-4339 (1997).
Other therapeutic agents which can be coupled to the mAbs according to the method of the invention are drugs, prodrugs, enzymes for activating pro-drugs, photosensitizing agents, nucleic acid therapeutics, antisense vectors, viral vectors, lectins and other toxins. The present invention contemplates dyes used, for example, in photodynamic therapy, conjugated to anti-uPAR Abs and fragments, and used in conjunction with appropriate nonionizing radiation (U.S. Pat. 6,331,175). Many drugs and toxins with known cytotoxic and apoptotic effects on cells can be found in compendia or textbooks of drugs and toxins, such as the Merck Index (most recent edition),
DC:50410124.1 33 ATT-17 Goodman & Gilman 's The Pharmacological Basis of Therapeutics, Brunton, LL et al, eds, 11th edition, McGraw-Hill Professional, New York, 2005, and the like, and in other references cited herein. Any such drug can be conjugated/linked to, or loaded onto, an anti-uPAR Ab by conventional means well know in the art. Such agents include taxol, nitrogen mustards, such as, mechlorethamine, cyclophosphamide, melphalan, uracil mustard and chlorambucil; ethylenimine derivatives, such as, thiotepa; alkyl sulfonates, such as, busulfan; nitrosoureas, such as, carmustine, lomustine, semustine and streptozotocin; triazenes, such as, dacarbazine; folic acid analogs, such as, methotrexate; pyrimidine analogs, such as, fluorouracil, cytarabine and azaribine; purine analogs, such as, mercaptopurine and thioguanine; vinca alkaloids, such as, vinblastine and vincristine; antibiotics, such as, dactinomycin, daunorubicin, doxorubicin, bleomycin, mithramycin and mitomycin; enzymes, such as, L-asparaginase; platinum coordination complexes, such as, cisplatin; substituted urea, such as hydroxyurea; methylhydrazine derivatives, such as, procarbazine; adrenocortical suppressants, such as, mitotane; hormones and antagonists, such as glucorticoids or mimics thereof (prednisone), progestins (hydroxyprogesterone caproate, medroprogesterone acetate and megestrol acetate), estrogens (diethylstilbestrol and ethinyl estradiol), antiestrogens (tamoxifen), and androgens (testosterone propionate and fluoxymesterone).
Lectins are proteins, commonly derived from plants, that bind to carbohydrates. Among other activities, some lectins are toxic. Some of the most cytotoxic substances known are protein toxins of bacterial and plant origin (Frankel, AE et ah, Ann. Rev. Med. 57:125-142 (1986)). These molecules binding the cell surface and inhibit cellular protein synthesis. The most commonly used plant toxins are ricin and abrin; the most commonly used bacterial toxins are diphtheria toxin and Pseudomonas exotoxin A. In ricin and abrin, the binding and toxic functions are contained in two separate protein subunits, the A and B chains. The ricin B chain binds to the cell surface carbohydrates and promotes the uptake of the A chain into the cell. Once inside the cell, the ricin A chain inhibits protein synthesis by inactivating the 60S subunit of the eukaryotic ribosome Endo, Y. et al, J. Biol. Chem. 262: 5908-5912 (1987)). Other plant derived toxins, which are single chain ribosomal inhibitory proteins, include pokeweed antiviral protein, wheat germ protein, gelonin, dianthins, momorcharins, trichosanthin, and many others (Strip, F. et al, FEBS Lett. 7P5: 1-8 (1986)). Diphtheria toxin and Pseudomonas exotoxin A are also single chain proteins, and their binding and toxicity functions reside in separate domains of the same protein Pseudomonas exotoxin A has the same catalytic activity as diphtheria toxin. Ricin has been used therapeutically by binding its toxic α-chain, to
DC:50410124.1 34 ATT-17 targeting molecules such as Abs to enable site-specific delivery of the toxic effect. Bacterial toxins have also been used as anti-tumor conjugates. As intended herein, a toxic peptide chain or domain is conjugated to a compound of this invention and delivered in a site-specific manner to a target site where the toxic activity is desired, such as a metastatic focus. Conjugation of toxins to protein such as Abs or other ligands are known in the art (Olsnes, S. et al., Immunol. Today 70:291-295 (1989); Vitetta, ES et al, Ann. Rev. Immunol. 5:197-212 (1985)).
The compounds of the invention, as well as the pharmaceutically acceptable salts thereof, may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or are more preferably used as injectable preparations. Solid or liquid pharmaceutically acceptable carriers may be employed.
Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid. Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like. Similarly, the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. When a liquid carrier is used, the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid {e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension. A summary of such pharmaceutical compositions may be found, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Easton Pennsylvania (Gennaro 18th ed. 1990).
The pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration. The pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
The present invention may be used in the diagnosis or treatment of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine. Thus, the pharmaceutical compositions can be used to treat domestic and commercial animals, including birds and more preferably mammals, as well as humans.
DC:50410124.1 35 ATT-17 The term "systemic administration" refers to administration of a composition or agent such as the polypeptide, described herein, in a manner that results in the introduction of the composition into the subject's circulatory system or otherwise permits its spread throughout the body, such as intravenous (i.v.) injection or infusion. "Regional" administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. Examples include intravaginal, intrapenile, intranasal, intrabronchial(or lung instillation), intracranial, intra-aural or intraocular. The term "local administration" refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass, subcutaneous (s.c.) injections, intramuscular (i.m.) injections. One of skill in the art would understand that local administration or regional administration often also result in entry of a composition into the circulatory system, i.e.,, so that s.c. or i.m. are also routes for systemic administration. Injectables or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions. Though the preferred routes of administration are systemic, such as i.v., the pharmaceutical composition may be administered topically or transdermally, e.g. , as an ointment, cream or gel; orally; rectally; e.g. , as a suppository.
For topical application, the compound may be incorporated into topically applied vehicles such as a salve or ointment. The carrier for the active ingredient may be either in sprayable or nonsprayable form. Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water. Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like. If desired, these may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like. Preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
Also suitable for topic application as well as for lung instillation are sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous
DC:50410124.1 3g ATT-17 propellant. The aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
For the preferred topical applications, especially for humans, it is preferred to administer an effective amount of the compound to an affected area, e.g., skin surface, mucous membrane, eyes, etc. This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, the severity of the symptoms, and the nature of the topical vehicle employed.
Other pharmaceutically acceptable carriers for polypeptide compositions of the present invention are liposomes, pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The active polypeptide is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension. The hydrophobic layer, or lipidic layer, generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature. Those skilled in the art will appreciate other suitable embodiments of the present liposomal formulations.
Therapeutic compositions for treating tumors and cancer may comprise, in addition to the peptide, one or more additional anti-tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase, topoisomerase inhibitors such as etoposide; or biological response modifiers, e.g., interferons or interleukins. In fact, pharmaceutical compositions comprising any known cancer therapeutic in combination with the peptides disclosed herein are within the scope of this invention. The pharmaceutical composition may also comprise one or more other medicaments to treat additional symptoms for which the target patients are at risk, for example, anti-infectives including antibacterial, anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.
DC:50410124.1 37 ATT-17 The therapeutic dosage administered is an amount which is therapeutically effective, as is known to or readily ascertainable by those skilled in the art. The dose is also dependent upon the age, health, and weight of the recipient, kind of concurrent treatment(s), if any, the frequency of treatment, and the nature of the effect desired, such as, for example, anti-inflammatory effects or anti-bacterial effect. Therapeutic Methods
The methods of this invention may be used to inhibit tumor growth and invasion in a subject or to suppress angiogenesis induced by tumors by inhibiting endothelial cell growth and migration. By inhibiting the growth or invasion of a tumor or angiogenesis, the methods result in inhibition of tumor metastasis. A vertebrate subject, preferably a mammal, more preferably a human, is administered an amount of the compound effective to inhibit tumor growth, invasion or angiogenesis. The compound or pharmaceutically acceptable salt thereof is preferably administered in the form of a pharmaceutical composition as described above.
Doses of the proteins (including Abs), peptides, peptide multimers, etc., preferably include pharmaceutical dosage units comprising an effective amount of the peptide. Dosage unit form refers to physically discrete units suited as unitary dosages for a mammalian subject; each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of, and sensitivity of, individual subjects
By an effective amount is meant an amount sufficient to achieve a steady state concentration in vivo which results in a measurable reduction in any relevant parameter of disease and may include growth of primary or metastatic tumor, any accepted index of inflammatory reactivity, or a measurable prolongation of disease-free interval or of survival. For example, a reduction in tumor growth in 20 % of patients is considered efficacious (Frei III, E., The Cancer Journal 3:127-136 (1997)). However, an effect of this magnitude is not considered to be a minimal requirement for the dose to be effective in accordance with this invention.
In one embodiment, an effective dose is preferably 10-fold and more preferably 100-fold higher than the 50% effective dose (ED50) of the compound in an in vivo assay as described herein.
DC:50410124.1 3g ATT-17 The amount of active compound to be administered depends on the precise peptide or derivative selected, the disease or condition, the route of administration, the health and weight of the recipient, the existence of other concurrent treatment, if any, the frequency of treatment, the nature of the effect desired, for example, inhibition of tumor metastasis, and the judgment of the skilled practitioner.
A preferred dose for treating a subject, preferably mammalian, more preferably human, with a tumor is an amount of up to about 100 milligrams of active polypeptide-based compound per kilogram of body weight. A typical single dosage of the peptide or peptidomimetic is between about 1 ng and about 100mg/kg body weight. For topical administration, dosages in the range of about 0.01-20% concentration (by weight) of the compound, preferably 1-5%, are suggested. A total daily dosage in the range of about 0.1 milligrams to about 7 grams is preferred for intravenous administration. The foregoing ranges are, however, suggestive, as the number of variables in an individual treatment regime is large, and considerable excursions from these preferred values are expected. Effective doses and optimal dose ranges may be determined in vitro using the methods described herein.
In the methods of this invention, the anti-uPAR Ab (or other uPAR-binding ligand) should produce an inhibitory effect on CaSC self renewal/proliferation, migration, invasion/metastasis. The methods are especially useful in producing an anti-tumor effect in a mammalian host, preferably human, harboring a tumor wherein the treatment results in reduction in size or growth rate of the tumor or destruction of the tumor. Preferably, the subject is a human.
A preferred disease or condition to be treated by the above method is tumor growth, invasion or metastasis of any of a number of types of cancer or tumor, including brain tumors. Examples of such brain tumors are astrocytoma, anaplastic astrocytoma, glioblastoma, glioblastoma multiformae, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, anaplastic ependymoma, myxopapillary ependymoma, subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma..
DC:50410124.1 39 ATT-17 Having now generally described the invention, the same will be more readily understood through reference to the following example which is provided by way of illustration, and is not intended to be limiting of the present invention, unless specified.
EXAMPLE I
Fractionation and Activity of uPAR+ Tumor Stem Cells In Vivo
ATN-658 mAb described above is used to sort the cells obtained from a dispersed a human solid tumor biopsy into two populations: those expressing uPAR (uPAR+) and those lacking detectable surface uPAR. Both populations are injected SC into nude mice. Development of tumors at the site of inoculation is monitored.
The uPAR+ cells result in an increased incidence of tumors and increased growth rate compared to uPAR-negative cells.
The uPAR+ cells are further characterized in vitro and are shown to be resistant to chemotherapy compared to the uPAR-negative cells when grown in a clonogenic assay in agar. These uPAR+ tumor cells also express stem cell markers and are characterized as follows: CD44hl, CD2410, CDl 05+, CD20+, and CD 133+ and further exclude Hoechst dye.
All the references cited above are incorporated herein by reference in their entirety, whether specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
DC:50410124.1 4Q ATT-17

Claims

WHAT IS CLAIMED IS:
1. A method for functionally inactivating or killing cancer stem cells (CaSCs), comprising providing to said CaSCs an effective amount of a ligand that binds to uPAR.
2. A method for treating a subject having or susceptible to a malignant disease characterized by the presence of CaSCs, comprising administering to the subject functionally- inactivating amount of a therapeutic composition that comprises a ligand that binds to uPAR.
3. The method of claim 1 or 2 wherein the ligand is an antibody or antigen binding fragment thereof.
4. The method of claim 3 wherein the antibody is a mAb.
5. An immunological method of ablating a CaSC in a mammalian subject, comprising administering to said subject requiring such ablation, an effective amount of an antibody or antigen binding fragment thereof specific for uPAR, wherein the antibody or fragment is conjugated to a therapeutically active moiety as an ablation agent.
6. The method of claim 3 wherein the antibody is a mAb.
7 A method of ablating or killing CaSCs in a subject in need thereof, comprising providing to the subject an effective amount of an antibody or antigen-binding fragment thereof that is specific for uPAR, and which, when contacted with a uPAR-expressing CaSC, binds to uPAR, which antibody or fragment is linked or conjugated to an therapeutically active moiety effective for killing or ablating the CaSCs after the antibody binds to said cells.
8. The method of claim 7 wherein the antibody is a mAb.
9. The method of any of claims 1-8 wherein said ligand or antibody is conjugated to a therapeutically active moiety.
10. The method of claim 9 wherein the therapeutically activity moiety is a drug, a toxin or a therapeutic radionuclide.
DC:50410124.1 4 \ ATT-17
11. The method of claim 10, wherein the radionuclide is selected from the group consisting of 125I, 131I, 186Re, 188Re, 111Ag, 197Pt, 109Pd, 67Cu, 32P, 33P, 90Y, 47Sc, 53Sm, 177Lu, 105Rh, 142Pr, 143Pr, 161Tb, 166Ho, and 199Au.
12. The method of claim 9 wherein a therapeutically active moiety is a peptide or polypeptide fused to said ligand.
13. The method of claim 12 wherein the fused peptide or polypeptide is a toxin.
14. A method for identifying the presence or determining the number of CaSCs in a cell population providing contacting the cell population with a detectably labeled ligand that binds to uPAR, and detecting the presence or enumerating CaSCs to which said ligand is bound.
15. The method for claim 14 wherein the ligand is labeled with a radionuclide, a PET- imageable agent, an MRI-imageable agent, a fluorescer, a fluorogen, a chromophore, a chromogen, a phosphorescer, a chemiluminescer or a bioluminescer.
16. The method of claim 15, wherein the label is a radionuclide selected from the group consi •s +ti i•ng o fr 3τ Hτ, 14r C>, 35C S, 123T I, 131T I, 11 lτ In, 67/ G-ιa, 9In Ru, 99mr Tτ,c, 67^ Cu, 57^ Co, 58^ Co, 51^ Cr, 59C Fe, 75C Se, 20Ir T^1l, and 169Yb.
17. The method of claim 15 wherein the fluorescer or fluorogen selected from the group consisting of fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green and Texas Red.
18. The method of any of claims 14-17 the ligand is a uP AR- specific antibody or antigen binding fragment thereof.
19. The method of claim 18 wherein the antibody is a mAb.
20. A method for purging CaSCs from a population of cells, comprising contacting the cell population with an amount of a ligand that binds to uPAR sufficient to destroy the CaSCs.
21. The method of claim 20 wherein the cell population is one that comprises hemopoietic stem cells.
DC:50410124.1 42 ATT-17
22. The method of claim 21 wherein the cell population is a bone marrow cell population
23 The method of any of claims 20-22 wherein the ligand is an antibody or antigen binding fragment thereof.
24. The method of claim 23, wherein the antibody is a mAb.
25. A method of treating a subject with a disease or condition requiring transplantation of hemopoietic stem cells, comprising administering to said subject an effective number of said stem cells which have been purged of CaSCs by the method of any of claims 20-24.
DC:50410124.1 43 ATT-17
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010028820A3 (en) * 2008-09-10 2010-06-17 Life & Brain Gmbh Peripheral zone tumor cells, methods for their preparation and use
WO2013167130A1 (en) * 2012-05-08 2013-11-14 Rigshospitalet 177-Lu LABELED PEPTIDE FOR SITE-SPECIFIC uPAR-TARGETING

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5985276A (en) * 1996-09-03 1999-11-16 Gsf Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Destruction of contaminating tumor cells in stem cell transplants using bispecific antibodies
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
US7115360B2 (en) * 2000-08-03 2006-10-03 Regents Of The University Of Michigan Isolation and use of solid tumor stem cells
US7361336B1 (en) * 1997-09-18 2008-04-22 Ivan Bergstein Methods of cancer therapy targeted against a cancer stem line

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6113897A (en) * 1989-04-07 2000-09-05 Cancerforskiningsfonden Af 1989 Antibodies and their use
US5985276A (en) * 1996-09-03 1999-11-16 Gsf Forschungszentrum Fur Umwelt Und Gesundheit Gmbh Destruction of contaminating tumor cells in stem cell transplants using bispecific antibodies
US7361336B1 (en) * 1997-09-18 2008-04-22 Ivan Bergstein Methods of cancer therapy targeted against a cancer stem line
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases
US7115360B2 (en) * 2000-08-03 2006-10-03 Regents Of The University Of Michigan Isolation and use of solid tumor stem cells

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010028820A3 (en) * 2008-09-10 2010-06-17 Life & Brain Gmbh Peripheral zone tumor cells, methods for their preparation and use
US9103819B2 (en) 2008-09-10 2015-08-11 Rheinische Friedrich-Wilhelms-Universität Bonn Peripheral zone tumor cells, methods for their preparation and use
WO2013167130A1 (en) * 2012-05-08 2013-11-14 Rigshospitalet 177-Lu LABELED PEPTIDE FOR SITE-SPECIFIC uPAR-TARGETING
US10994032B2 (en) 2012-05-08 2021-05-04 Trt Innovations Aps 177-Lu labeled peptide for site-specific uPAR-targeting
US20210236666A1 (en) * 2012-05-08 2021-08-05 Rigshospitalet 177-Lu LABELED PEPTIDE FOR SITE-SPECIFIC uPAR-TARGETING

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