# Patent

## Title
Methods for inhibiting the binding of endosialin to ligands

## Abstract

The invention provides methods for inhibiting the interaction of endosialin with endosialin ligands. The inhibition is effectuated on the genetic level, by inhibiting endosialin gene expression, and on the protein level, by blocking the interaction of cell-surface expressed endosialin with ligands such as fibronectin and collagen. The invention provides methods for identifying inhibitors of the interaction of endosialin with endosialin ligands. Also provided are methods for inhibiting angiogenesis and neovascularization in vivo and in vitro.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 60/910,362, filed Apr. 5, 2007, and of U.S. Provisional Application No. 60/980,026, filed Oct. 15, 2007, both of which are incorporated by reference herein.


FIELD
The invention relates generally to the field of immunotherapeutics. More specifically, the invention relates to compositions and methods for the disruption of endosialin interaction with its substrates to inhibit cellular functions, including angiogenesis and cell motility.
BACKGROUND
Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety and for all purposes.
Angiogenesis is a regulated process involving the formation of new blood vessels. It plays an essential role in normal growth, embryonic development, wound healing, and other physiological processes (Yancopoulos et al. (2000) Nature, 407:242-8). Within the developing capillary, extracellular matrix (ECM) proteins serve as a structural scaffold for proliferating endothelial and tumor tissues and provide support for the growth of tumor cells. De novo angiogenesis is involved in several disease states including cancer, where the formation of new “embryonic-like” blood vessels (referred to as neovascularization herein) appear that differ from normal vasculature with regards to structure and function (Hanahan et al. (2000) Cell, 100:57-70). A number of in vivo and in vitro studies have demonstrated biological differences between normal and disease-associated vasculature using various model systems of angiogenesis, thereby raising the possibility of novel anti-angiogenic compounds that can selectively inhibit vessel formation of the embryonic-type, tumor-associated endothelial cells for therapy of neovascular disease. In light of these opportunities for therapy, an intense search for potential targets that can specifically inhibit tumor and other neovascular disease-associated endothelial or stromal (fibroblasts, pericytes, etc.) cell growth and function is ongoing.
In an attempt to identify such targets, strategies have been designed to identify cell surface antigens of tumor stroma as well as isolate specific proteins or RNA that are expressed in tumor stromal cells (Rettig et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10832-6; St. Croix et al. (2000) Science, 289:1197-1202). These strategies have identified a cell surface protein that appears to be specifically expressed in tumor stromal cells referred to as endosialin (or tumor endothelial marker 1 (TEM1) or CD248).
Examination of gene expression patterns in normal and neoplastic tissue indicates upregulation of endosialin mRNA expression in tumor neovessels. (St Croix et al (2000) Science, 289:1197-1202). Similar endosialin expression levels were noted in human colorectal cancer (Rmali et al. (2005) World J. Gastroenterol., 11: 1283-1286), breast cancer tissues (Davies et al. (2004) Clin. Exp. Metastasis, 21:31-37), and histiocytomas (Dolznig et al. (2005) Cancer Immun., 5: 10). Human endosialin expression has been observed in highly invasive glioblastoma, anaplastic astrocytomas, and metastatic carcinomas, including melanomas (Brady et al. (2004) J. Neuropathol. Exp. Neurol., 63:1274-1283; Huber et al. (2006) J. Cutan. Pathol., 33:145-155).
The use of antibodies in immunohistochemistry studies have found robust expression of endosialin in a number of neovascular endothelial cells, fibroblasts and/or pericytes (Virgintino et al. (2007) Angiogenesis, 10:35-45) in malignant tissues, while expression in cell lines derived from embryonic-like endothelial cultures such as but not limited to HUVEC (Human Umbilical Vein Endothelial Cells) or HMVEC-(Neonatal Dermal Microvascular Endothelial Cells) is limited. Analysis of antibodies, polypeptides or non-protein ligands that can bind to endosialin have identified a subset of such molecules that can suppress the ability of endosialin to bind to its substrate and/or suppress intracellular activities leading to cell stasis or death.
Rettig et al. described monoclonal antibodies that recognize antigens on vessels within various cancer types (Rettig et al. (1992) Proc. Natl. Acad. Sci. USA, 89:10832-6). One of these was designated FB5 and was generated through immunization of mice with human embryonic fibroblasts. FB5 recognizes a  ̃100 kDa protein on the surface of a neuroblastoma cell line, LA1-5s (U.S. Pat. No. 5,342,757). FB5 is a murine antibody (IgG1) that binds to endosialin and has been shown to recognize endothelial cells associated with a variety of different cancer types. Structural evaluation has classified endosialin as a C-type lectin-like, integral membrane protein, comprised of five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats). The protein also contains a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. The protein appears to be a glycoprotein. Carbohydrate analysis shows that the endosialin core protein has an abundance of O-linked glycosylation (Christian et al. (2001) J. Biol. Chem., 276:48588-48595). Subsequent work combined the complementarity determining regions (CDRs) of the mouse FB5 into a human IgG1 backbone to create a humanized antibody that binds to vessels within malignant tissues as well as a subset of cells in HMVEC cultures.
Tem1 knockout mice develop normally and exhibit normal wound healing, suggesting that endosialin is not required for neovascularization during fetal development or wound repair. (Nanda et al. (2006) Proc. Natl. Acad. Sci. USA, 103:3351-3356). When colorectal cancer cells were implanted in the abdominal sites of Tem1 knockout mice, however, the loss of endosialin expression correlated with a reduction in tumor growth, invasion, and metastases as compared to parental animals. The absence of endosialin expression has been shown to reduce growth, invasion, and metastasis of human tumor xenografts in an endosialin knockout mouse. (Nanda et al. (2006) Proc. Natl. Acad. Sci. USA, 103:3351-3356). Additionally, lack of endosialin led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels.
Neovascularization is associated with a number of disease states. In cancer it is believed that neovascularization is important to supply tumors with blood. In non-oncology cancer or malignant diseases such as retinopathy and macular degeneration, uncontrolled neovascularization causes loss of sight (Wilkinson-Berka (2004) Curr. Pharm. Des., 10:3331-48; Das and McGuire (2003) Prog. Retin. Eye Res., 22:721-48). Moreover, several reports have identified a role of neovascularization in inflammatory disease (Paleolog and Miotla (1998) Angiogenesis, 2(4):295-307). Methods to better understand molecular pathways in embryonic-like endothelial and precursor cells as well as endothelial-associated cells (pericytes, fibroblasts, etc.) associated with these disease states will lead to the development of novel drugs to treat these diseases. Conversely, neovascularization is associated with wound healing (Galiano et al. (2004) Am. J. Pathol., 164:1935-47). Identification of molecular pathways that promote vascularization for wound healing can offer the ability to identify drugs and factors that can promote these processes for enhancing wound treatment associated with trauma, burns and infection.
A difficult problem in effective antiangiogenic and proangiogenic therapy is the nondefined nature of biological processes of molecules and associated pathways that are important for activating cellular processes associated with neovascularization (Bagley et al. (2003) Cancer Res., 63:5866-73). The ability to identify and elucidate molecules and their function in regulating a given pathway can lead to the isolation of effective compounds that have stimulatory or inhibitory activity in neovascular-associated diseases such as cancer, inflammation, ocular disease, cardiovascular disease, and wound healing. The ability to isolate and study these compounds via molecular-based assays would provide further utility for evaluating their effects to specifically suppress or stimulate the normal biology of cells involved in neovascularization in contrast to adult-like endothelial cells associated with vessels in normal adult tissue (Asahara and Kawamoto (2004) Am. J. Physiol. Cell Physiol., 287:C572-9).
SUMMARY
The invention features methods for inhibiting the interaction of an endosialin-expressing cell with a ligand for endosialin.
In one aspect, the methods comprise inhibiting expression of endosialin in an endosialin-expressing cell at the genetic level. Ligands for endosialin can be extracellular matrix proteins such as collagen or fibronectin. In some embodiments, the ligand is collagen I or collagen IV. In preferred embodiments, the cell is a mammalian cell. Regulation of endosialin expression at the genetic level can be effectuated by any means suitable in the art, such as antisense nucleic acid molecule, double stranded RNA, ribozymes, hammerhead ribozymes, decoy oligonucleotides, and the like. Regulation of endosialin expression can also be accomplished by knocking out the gene encoding endosialin.
In another aspect, the methods comprise physically obstructing endosialin expressed on the surface of an endosialin expressing cell, thereby inhibiting the interaction of the cell with an endosialin ligand. Ligands for endosialin can be extracellular matrix proteins such as collagen (e.g., collagen I or collagen IV) or fibronectin.
Obstruction of cell surface endosialin can be effectuated by any means suitable in the art, such as small molecule inhibitors, polypeptide inhibitors, antibodies that specifically bind to endosialin, antibodies that specifically bind to an endosialin ligand, and the like. In some embodiments, competitive inhibitors are employed to inhibit the interaction of endosialin or an endosialin-expressing cell with a ligand for endosialin. In some embodiments, the competitive inhibitors may be endosialin ligands, endosialin-binding fragments of endosialin ligands, for example, endosialin-binding fragments of collagen or fibronectin. Preferred competitive inhibitors are endosialin-binding fragments of collagen I, collagen IV, or fibronectin. Most preferred competitive inhibitors are the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, and the 30 kDa heparin binding fragment of fibronectin.
Suitable antibodies can be chimeric antibodies, humanized antibodies, fully human antibodies, antigen-binding fragments that specifically bind antigen, and the like. In some embodiments, the affinity of antibody for antigen is preferably less than about 1×10−7 M, more preferably less than about 1×10−8 M, even more preferably less than about 1×10−9 M, and most preferably less than about 1×10−10 M. In some preferred embodiments, the antibody is an anti-endosialin antibody or an antigen-binding fragment that specifically recognizes endosialin. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:28, 30, and 32, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 13, 15, and 17, respectively. In some embodiments, the antibodies or antigen-binding fragments can comprise a heavy chain comprising a variable domain of SEQ ID NO: 34 and a light chain comprising a variable domain of SEQ ID NO: 19. In some embodiments, the antibodies or antigen-binding fragments can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:22 or 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 11. Antibodies M4 and M4.1 are humanized antibodies to human endosialin. While antibodies M4 and M4.1 share a light chain sequence, they differ in their heavy chain by a single amino acid sequence shown, for example, at residue 429 of SEQ ID NO:20 relative to residue 429 of SEQ ID NO:24. The amino acid change is the result of a single nucleotide alteration shown, for example, at nucleotide 1286 of SEQ ID NO: 19 relative to nucleotide 1286 of SEQ ID NO:23. In some embodiments, the antibodies that can be used in accordance with the invention are produced by cells having ATCC Access. No. PTA-7554 or ATCC Access. No. PTA-9017.
Inhibition of endosialin interaction with ligands for endosialin can affect pathways, cascades, and downstream effects brought about by the normal interaction. For example, obstructing or inhibiting the interaction of an endosialin-expressing cell with an endosialin ligand can inhibit the activation of integrins, the activation of matrix metalloproteases, and/or the expression of matrix metalloproteases. Cell motility can be inhibited. Most preferably, angiogenesis or neovascularization is inhibited.
In some embodiments, inhibition of the interaction of the endosialin-expressing cell with its ligand inhibits the activation of integrin β1, β2, or β3. In some embodiments, inhibition of the interaction of the endosialin-expressing cell with its ligand inhibits migration of the cell. In some embodiments, inhibition of the interaction of the endosialin-expressing cell with its ligand inhibits the activation or expression of a matrix metalloprotease. In preferred embodiments, the matrix metalloprotease is MMP-9.
The invention also features methods for inhibiting angiogenesis or neovascularization. The methods include in vitro and in vivo inhibition of angiogenesis or neovascularization. In some aspects, the methods comprise administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment that specifically binds endosialin or composition that obstructs endosialin expressed on the surface of a cell such that the interaction of the cell with an endosialin ligand is inhibited. This inhibition suppresses angiogenesis and/or neovascularization of a tissue, organ, or neoplasm in the subject to which the composition is administered. Ligands for endosialin can be extracellular matrix proteins such as collagen (e.g., collagen I or collagen IV) or fibronectin. The composition can comprise any molecule, such as those described and exemplified herein, that can physically obstruct the interaction of cell surface endosialin with at least one endosialin ligand. Examples of such molecules include, without limitation, small molecule inhibitors, polypeptide inhibitors, antibodies that specifically bind to endosialin, antibodies that specifically bind to an endosialin ligand, antigen-binding fragments, and the like. Suitable antibodies can be chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, and the like.
Also featured are assays and methods for identifying agents that enhance (“agonists”) or reduce (“antagonists”) the interaction of endosialin with an endosialin ligand. In one aspect of methods for identifying such antagonists, the methods comprise contacting endosialin with a test compound, thereby forming an endosialin-test compound complex, contacting the endosialin-test compound complex with a ligand for endosialin, and quantifiably measuring the interaction of endosialin with the ligand in the presence and in the absence of the test compound, wherein a decrease in the level of interaction of endosialin with the ligand in the presence of the test compound indicates that the test compound is an antagonist of the interaction of endosialin with the ligand. In one embodiment, the methods for identifying agonists or antagonists of the interaction of endosialin with a ligand for endosialin comprises contacting endosialin with a ligand for endosialin in the presence and absence of a test compound and quantifiably measuring the interaction of endosialin with the ligand, wherein an increase or a decrease in the level of interaction of endosialin with the ligand in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of the interaction of endosialin with the ligand. In the inventive assays, the endosialin can be bound to a cell membrane, a cell membrane fragment, an artificial lipid bilayer, or a solid support. In some aspects, the ligand can be bound to a solid support. Ligands for endosialin can be extracellular matrix proteins such as collagen or fibronectin.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A and 1B show immunohistochemical analysis of endosialin-positive cells of malignant tissue. Tumors were isolated from patients with colorectal cancer and flash frozen in liquid nitrogen. Samples were thin-sectioned and stained with anti-endosialin or isotype control antibody. As shown, vessels in the tumor (FIG. 1A) stained positive for endosialin while isotype control antibody stained serial section was negative (FIG. 1B).
FIGS. 2A and 2B show immunohistochemical analysis of endosialin-positive cells of normal tissue. Normal tissues were isolated from patients by biopsy and flash frozen in liquid nitrogen. Samples were thin-sectioned and stained with anti-endosialin or isotype control antibody. As shown, normal tissue contained few EPCs (arrow, FIG. 2A) while isotype control antibody stained serial section were negative (FIG. 2B). Many normal tissues tested had a few EPCs as determined by in situ or antibody staining.
FIG. 3 shows isolation of endosialin-positive cells from primary endothelial cultures. HMVEC cultures were enriched for EPCs by panning. Endosialin-panned cultures were then compared to HMVEC parental cultures for percentage of endosialin-expressing cells. As shown, the panned culture had a much higher number of endosialin-positive cells as compared to the non-panned parental culture as determined by immunostaining via anti-endosialin antibody followed by a fluorescent conjugated secondary antibody. Cell number of each field was determined by light microscopy.
FIG. 4 shows that recombinant endosialin (Fc-TEM1) binds to extracellular matrix proteins (EMPs). ELISA plates, precoated with EMP fibronectin (FN), collagen (COL; including collagen type I (COLI) and collagen type IV (COL IV)), vitronectin (VN), laminin (LN), or gelatin (Gel), were blocked with ELISA assay buffer prior to addition of purified Fc-TEM1 protein at increasing concentrations. Following a two hour incubation, the plates were washed and assayed for binding using a HRP-linked goat-anti-mouse secondary mAb specific for the Fc tail using standard ELISA conditions. Plates were washed and developed and then assayed using a plate reader at OD 450 nm. As shown in FIG. 4A, the Fc-TEM1 bound to FN and COL robustly while weak binding was observed with LM, VN, or Gel. For FIG. 4B, an ELISA plate was pre-coated overnight with the following antigens: Staphylococcus enterotoxin B (STEB), ovalbumin (OVA), bovine gamma globulin (BGG), tumor-associated 90-kD glycoprotein antigen expressed on most melanoma cells (TA90), hen egg lysozyme (HEL), tetanus toxoid (TT), 1% BSA, human mesothelin, human GM-CSF, goat IgG, and mouse IgG. Fc-TEM1 was added at increasing amounts (5, 10, 50 ug/ml) and allowed to adhere for 2 hours. Plates were then washed and HRP-conjugated goat-anti-mouse antibody was added to detect bound Fc-TEM1.
FIG. 5 shows mapping of fibronectin (FN) binding domains to endosialin. Proteolytic and recombinant fragments derived from fibronectin (FN) were assessed for the ability to support TEM-1 binding. FN fragments evaluated include: the N-terminal 70 kDa fragment (Sigma Cat. No. F0287) (obtained by cathepsin D digestion of fibronectin); the 30 kDa heparin binding fragment (Sigma Cat. No. F9911); the 45 kDa gelatin binding fragment (Sigma Cat. No. F0162) (both obtained from trypsin digestion of the 70 kDa fragment); the 120 kDa fragment containing the cell attachment domain (“the 120 kDa fragment”); and two recombinant fragments: Fn2, which contains the first 7 FN type III domains, and Fn4, which contains the site of interchain disulfide bonds and α4β1 integrin binding domain. The diagram of FN structure was adapted from Wierzbicka-Patynowski et al. (2003) J. Cell Sci., 116:3269-76.
FIG. 6 shows recombinant Fc-TEM1 binding to EMP and fibronectin in the presence of inhibitors. M4 is a humanized antibody to human endosialin, while rbtTEM1 is a rabbit antibody to human endosialin. The assay was performed as described in FIG. 4, except that antibodies were added to measure the ability to perturb or block Fc-TEM1 to bind to FN. As shown in this figure, M4 was able to inhibit Fc-TEM1 binding to FN while a non-specific control (HuIgG) was not.
FIG. 7 shows endosialin binding to EMP fragments and inhibition thereof by endosialin-EMP inhibitor compounds. The fibronectin fragments are illustrated in FIG. 5. FIG. 7A shows binding to protein fragments derived from native FN. FIG. 7B shows binding to protein fragments derived from denatured FN. For FIGS. 7A and 7B, FN fragment was coated on an ELISA plate at the indicated concentrations. An anti-FN polyclonal antibody followed by addition of HRP-conjugated goat-anti-rabbit secondary antibody was used to detect intact bound proteins. Fc-TEM1 (1.25 ug/ml) was added and allowed to bind for 2 hours. Plates were then washed and HRP-conjugated goat-anti-mouse antibody was added to detect bound Fc-TEM1. The hatched bar (Fc-TEM1-native) in FIG. 7B represents Fc-TEM1 binding to nondenatured FN. As shown, endosialin binds to the N-terminal region of fibronectin, as little or no binding was detected for binding to fragments FN-2, FN-4, or 120 kDa. Polyclonal antibodies to fibronectin bound to all fragments. FIGS. 7C and 7D show binding of Fc-TEM1 to the 70 kDa fragment of FN and inhibition of the interaction by endosialin-EMP inhibitor compounds. Whole FN and the 70 kDa FN protein were coated onto an ELISA plate at a fixed concentration of  ̃15 nmol/well for both proteins. Fc-TEM1 (1.25 ug/ml) was preincubated at 4° C. for 1 hour with increasing amounts of anti-endosialin antibody M4 or isotype control IgG. Fc-TEM1/M4 (FIG. 7C) or Fc-TEM1/IgG (FIG. 7D) complexes were added to FN- and 70 kDa-coated wells and incubated for 2 hours at room temperature. Bound Fc-TEM1 protein was detected by the addition of HRP-conjugated goat-anti-mouse secondary antibody.
FIG. 8 illustrates a change in cell morphology on a gelatinous protein mixture sold under the trade name MATRIGEL (BD Biosciences) upon expression of endosialin. 8E4 cells of either CHO-K1 or CHO-TEM1 were seeded onto a 96-well-plate coated with MATRIGEL and incubated at 37° C. Following overnight incubation, the cells were photographed for macroscopic examination of tubule formation.
FIG. 9 shows endosialin-mediated cellular binding to EMP fragments. CHO cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (CHOTEM1) while those transfected with mock (CHOK1) did not. For FIG. 9A, Chinese Hamster Ovary (CHO) cells were added to a pre-coated 96-well plate containing various ECM proteins. The cells were allowed to adhere for 1 hour at 37° C. and wells were washed extensively to remove any loosely bound cells. The number of attached cells was determined using the CELLTITER-GLO Luminescent Cell Viability Assay. Abbreviations: Col, Collagen; FN, fibronectin; LN, laminin; TN, tenascin; VN, vitronectin; Neg, bovine serum albumin. For FIG. 9B, Chinese Hamster Ovary (CHO) cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed by FACS analysis to express cell surface endosialin (CHOTEM1) while those transfected with mock (CHOK1) did not. Cells were then tested for the ability to bind EMP fibronectin alone or in combination with anti-endosialin antibody M4 or control IgG. FIG. 9B demonstrates that anti-endosialin antibody M4 reduces endosialin-mediated cell adhesion to FN. Cells (1.5E5) were preincubated for 1 hour at 4° C. with antibody M4 (100 ug/ml) or an IgG isotype control antibody (100 ug/ml). For FIG. 9C, cells were tested for the ability to bind full length FN or fibronectin fragments. As shown in FIG. 9A, the number of adherent CHO-TEM 1 cells was 6-fold higher than the number of parental CHO-K1 cells in wells coated with FN. No significant differences in adhesion between CHO-K1 and CHO-TEM1 on surfaces coated with laminin or vitronectin were observed, while adhesion to collagens and tenascin was too weak to assess any valuable differences (FIG. 9A). Pretreatment of CHO-TEM1 cells with M4 antibody resulted in 50% reduction of TEM 1-FN-dependent cell adhesion, while IgG control antibody had no effect (FIG. 9B). M4 antibody treatment did not affect FN-dependent, endosialin-independent cell adhesion (baseline adhesion) of parental CHO-K1 cells. CHO-TEM1 cells showed a 3- to 5-fold increased adhesion to FN, 70 kDa, and 30 kDa fragments compared to parental CHO-K1 cells, whereas no significant adhesion was seen to 45 kDa or Fn2 fragments. CHO-TEM1 cells bound MATRIGEL five times better than CHO-K1 (FIG. 9C).
FIG. 10 shows identification of endosialin-EMP collagen inhibitor compounds. CHO cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (CHOTEM1) while those transfected with mock (CHOK1) did not. Cells were then tested for the ability to bind EMP Collagen Type I (COL I) alone or in combination with anti-endosialin antibody M4 or control IgG. As shown, over-expression of endosialin results in increased cell binding to COL I which can be blocked by endosialin inhibitors such as M4 in contrast to control molecule (IgG). RbtTEM1 also suppressed Fc-TEM1 binding to COL I (data not shown).
FIG. 11 shows endosialin-mediated cellular binding to EMP collagen. CHO cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (CHOTEM1) while those transfected with mock (CHOK1) did not. Cells were then tested for the ability to bind EMP collagen type I. As shown, over-expression of endosialin results in increased cell binding to COL I.
FIG. 12 shows mediation of cell migration by endosialin and inhibition thereof by anti-endosialin antibody M4. The ability of M4 to inhibit the migration of CHO-TEM1 and CHO-K1 cells through MATRIGEL- (FIG. 12A) or FN- (FIG. 12B) coated membranes was determined. Cells were added to the top chamber and allowed to migrate for 48 hours at 37° C. The membrane was removed and the number of migrated cells was determined using the CELLTITER-GLO Luminescence Cell Viability Assay. Where indicated, cells were treated with M4 or IgG isotype control for the duration of experiment. As shown in FIG. 12A, CHO-K1 cells exhibited modest cell migration, whereas CHO-TEM1 cells showed >10-fold enhanced migration. M4 antibody treatment, but not control IgG, abolished CHO-TEM1 cell migration. Similar results were observed in migration experiments using transwell chambers coated with FN (FIG. 12B).
FIG. 13 shows endosialin enhancement of cellular pathways. CHO cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (CHOTEM1) while those transfected with mock (CHOK1) did not. Cells were then tested for the ability to upregulate cellular pathways. One such pathway is the MMP9 pathway, which plays a role in cellular migration. As shown, over-expression of endosialin results in increased MMP-9 activity in contrast to control cells.
FIG. 14 shows the effect of blocking endosialin on β integrin activation. Human embryonic kidney 293 (HEK293) cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (293TEM1) while those transfected with mock (293T) did not. Cells were then tested for the ability to upregulate cellular pathways. One such pathway is the integrin pathway which plays a role in cellular migration. As shown, over-expression of endosialin results in increased integrin β1 activity (FIG. 14B) in contrast to control cells while direct effect on cell surface β1 expression is not changed (FIG. 14A). Treatment of cells with the endosialin inhibitor M4 resulted in suppressed integrin activity while no effect on cell surface levels were observed (FIG. 14B). These data show the ability to use endosialin inhibitors to suppress integrin function in endosialin-expressing cells.
FIG. 15 illustrates that antibody M4.1 recognizes unreduced human TEM-1 in CHO-TEM-1 cells and human primary pericytes but not murine TEM-1 in mouse 2H11 cells. Rabbit polyclonal against human TEM-1 (rabPAb TEM-1) recognizes human TEM-1 in CHO-TEM-1 cells and human pericytes, but also murine TEM-1 in mouse 2H11 cells. Neither M4.1 nor rabPAb TEM-1 reacted against lysates from parental CHO-K1 cells or mouse NS0 and MS1 cells due to lack of TEM-1 expression in these cells. Only rabPAb TEM-1 reacted with reduced human TEM-1, albeit to a lesser extent when compared to unreduced TEM-1.



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Various terms relating to the methods and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein.
It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
Each range recited herein includes all combinations and sub-combinations of ranges, as well as specific numerals contained therein.
The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
“Endosialin specific assay” (ESA) refers to assays that can be used to identify compounds that can directly or indirectly perturb endosialin expression or biological activity that results in modified direct or indirect binding of endosialin-expressing cells or endosialin to EMPs via endosialin or integrin-mediated mechanisms as well as modify cellular endogenous pathways such as but not limited to matrix metalloprotease (MMPs) and/or cellular proliferation or survival.
“Polynucleotide,” synonymously referred to as “nucleic acid” or “nucleic acid molecule,” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.
A “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
“Polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Polypeptides of the invention include conservatively modified variants. One of skill will recognize that substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alter, add or delete a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The term “express,” “expressed,” or “expression” of a nucleic acid molecule refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. For example, but not by way of limitation, a regulatory gene such as an antisense nucleic acid or interfering nucleic acid can be expressed by transcription as antisense RNA or RNAi or shRNA. The term also encompasses translation of RNA into one or more polypeptides, and encompasses all naturally occurring post-transcriptional and post-translational modifications.
A cell has been “transformed” or “transfected” by exogenous or heterologous nucleic acids such as DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. With respect to eukaryotic cells, a stably transformed cell, or “stable cell” is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
As used herein, “test compound” refers to any purified molecule, substantially purified molecule, molecules that are one or more components of a mixture of compounds, or a mixture of a compound with any other material that can be utilized in the methods of the present invention. Test compounds can be organic or inorganic chemicals, or biomolecules, and all fragments, analogs, homologs, conjugates, and derivatives thereof. “Biomolecules” include proteins, polypeptides, nucleic acids, lipids, monosaccharides, polysaccharides, and all fragments, analogs, homologs, conjugates, and derivatives thereof. Test compounds can be of natural or synthetic origin, and can be isolated or purified from their naturally occurring sources, or can be synthesized de novo. Test compounds can be defined in terms of structure or composition, or can be undefined. The compound can be an isolated product of unknown structure, a mixture of several known products, or an undefined composition comprising one or more compounds. Examples of undefined compositions include cell and tissue extracts, growth medium in which prokaryotic, eukaryotic, and archaebacterial cells have been cultured, fermentation broths, protein expression libraries, and the like.
“Knockdown” refers to a cell or organism having reduced expression of one or more genes. As will be appreciated by those skilled in the art, a knockdown will exhibit at least about a 50% reduction in expression, preferably will exhibit at least about a 67% reduction in expression, and more preferably will exhibit at least about a 75% reduction in expression, although higher reductions are possible, including at least about a 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more reduction in expression.
“Inhibit” or “inhibition” or “interfere” means to reduce, decrease, block, prevent, delay, suppress, inactivate, desensitize, stop, or downregulate the biological activity or expression of a gene, gene product (e.g., polypeptide), or pathway of interest. In some preferred embodiments of the invention, the inhibition of the expression or biological activity of a protein or pathway of interest, for example, endosialin or cell migration pathway, refers to a decrease (inhibition or downregulation) of greater than from about 50% to about 99%, and more specifically, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. The inhibition may be direct, i.e., operate on the molecule or pathway of interest itself, or indirect, i.e., operate on a molecule or pathway that affects the molecule or pathway of interest.
“Recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
“Effective amount” and “therapeutically effective amount” are used interchangeably herein, and refer to an amount of an antibody, antigen-binding fragment, or composition, as described herein, effective to achieve a particular biological result such as, but not limited to, biological results disclosed, described, or exemplified herein. Such results may include, but are not limited to, the treatment of disease associated with angiogenesis or neovascularization, as determined by any means suitable in the art.
As used herein, “measure” or “determine” refers to any qualitative or quantitative determinations.
“Endosialin ligand” refers to any chemical, biomolecule, complex, or analog, homolog, or derivative thereof that can bind to, interact with, stimulate, and/or alter expression of endosialin.
Except when noted, “subject” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, dogs, cats, rats, mice, and other animals. Accordingly, “subject” or “patient” as used herein means any mammalian patient or subject to which the compositions of the invention can be administered.
“Angiogenesis” refers to the formation of new blood vessels.
“Neovascularization” refers to a pathological proliferation of new blood vessels in a tissue(s) or organ(s) that normally do(es) not contain blood vessels, or a pathological proliferation of blood vessels of a different type or quantity than normal for a particular tissue or organ.
“Epitope” refers to an immunological determinant of an antigen that serves as an antibody-binding site. As used herein, the term “conformational epitope” refers to a discontinuous epitope formed by a spatial relationship between amino acids of an antigen other than an unbroken series of amino acids.
“Isolated” means altered “by the hand of man” from the natural state. If a molecule or composition occurs in nature, it has been “isolated” if it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living plant or animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated” as the term is employed herein.
“Substantially the same” with respect to nucleic acid or amino acid sequences, means at least about 65% identity between two or more sequences. Preferably, the term refers to at least about 70% identity between two or more sequences, more preferably at least about 75% identity, more preferably at least about 80% identity, more preferably at least about 85% identity, more preferably at least about 90% identity, more preferably at least about 91% identity, more preferably at least about 92% identity, more preferably at least about 93% identity, more preferably at least about 94% identity, more preferably at least about 95% identity, more preferably at least about 96% identity, more preferably at least about 97% identity, more preferably at least about 98% identity, and more preferably at least about 99% or greater identity.
It has been discovered in accordance with the present invention that endosialin specifically interacts with extracellular matrix proteins, including fibronectin or collagen. It has also been discovered that this interaction promotes cell migration and further promotes and facilitates angiogenesis. Further to these observations, it has been discovered that disruption of the interaction between endosialin and extracellular matrix proteins can suppress cell migration and can suppress angiogenesis. Accordingly, the invention features methods for inhibiting the interaction of endosialin with endosialin ligands.
In one aspect, the invention features methods for inhibiting the interaction of endosialin expressed by an endosialin-expressing cell with an endosialin ligand. In some preferred embodiments, the methods comprise inhibiting endosialin expression by the cell. Inhibiting the expression of endosialin can occur at the gene level or the protein level. For example, inhibiting the expression of endosialin can comprise targeting the DNA encoding endosialin, or targeting the mRNA transcript of the endosialin gene.
Methods of gene regulation are known and readily practiced in the art, and are all suitable for use in the inventive methods. For example, in cells specifically engineered to express a transgene encoding endosialin (e.g., SEQ ID NO: 1, 3, or 5), the transgene can be placed under control of an inducible promoter. Inducible promoters suitable for use in this invention will be known to those of skill in the art.
In some preferred embodiments, genes encoding endosialin can be inhibited through the use of a variety of other post-transcriptional gene silencing (RNA silencing) techniques. RNA silencing involves the processing of double-stranded RNA (dsRNA) into small 21-28 nucleotide fragments by an RNase H-based enzyme (“Dicer” or “Dicer-like”). The cleavage products, which are siRNA (small interfering RNA) or miRNA (micro-RNA) are incorporated into protein effector complexes that regulate gene expression in a sequence-specific manner.
RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA), which is distinct from antisense and ribozyme-based approaches (see Jain, Pharmacogenomics (2004) 5:239-42, for a review of RNAi and siRNA). RNA interference is useful in a method for inhibiting the expression of endosialin in a cell or in an animal such as a human by transforming the cell, or by administering to the animal a nucleic acid (e.g., dsRNA) that hybridizes under stringent conditions to a gene encoding endosialin, and attenuates expression of the target gene. RNA interference provides shRNA or siRNA that comprise multiple sequences that target one or more regions of the endosialin gene. Double stranded RNA (dsRNA) molecules (shRNA or siRNA) are believed to direct sequence-specific degradation of mRNA in cells of various types after first undergoing processing by an RNase III-like enzyme called DICER (Bernstein E et al. (2001) Nature 409:363-366) into smaller dsRNA molecules comprised of two 21 nt strands, each of which has a 5′ phosphate group and a 3′ hydroxyl, and includes a 19 nt region precisely complementary with the other strand, so that there is a 19 nt duplex region flanked by 2 nt-3′ overhangs. RNAi is thus mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3′ overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. In mammalian cells, dsRNA longer than approximately 30 nucleotides typically induces nonspecific mRNA degradation via the interferon response. However, the presence of siRNA in mammalian cells, rather than inducing the interferon response, results in sequence-specific gene silencing.
Viral vectors or DNA vectors encode short hairpin RNA (shRNA), which are processed in the cell cytoplasm to short interfering RNA (siRNA). In general, a short, interfering RNA (siRNA) comprises an RNA duplex that is preferably approximately 19 basepairs long and optionally further comprises one or two single-stranded overhangs or loops. An siRNA may comprise two RNA strands hybridized together, or may alternatively comprise a single RNA strand that includes a self-hybridizing portion. siRNAs may include one or more free strand ends, which may include phosphate and/or hydroxyl groups. siRNAs typically include a portion that hybridizes under stringent conditions with a target transcript. One strand of the siRNA (or, the self-hybridizing portion of the siRNA) is typically precisely complementary with a region of the target transcript, meaning that the siRNA hybridizes to the target transcript without a single mismatch. In certain embodiments of the invention in which perfect complementarity is not achieved, it is generally preferred that any mismatches be located at or near the siRNA termini.
siRNAs have been shown to downregulate gene expression when transferred into mammalian cells by such methods as transfection, electroporation, cationic liposome-mediated transfection, or microinjection, or when expressed in cells via any of a variety of plasmid-based approaches. RNA interference using siRNA is reviewed in, e.g., Tuschl (2002) Nat. Biotechnol. 20:446-8; Yu J-Y et al. (2002) Proc. Natl. Acad. Sci. USA, 99:6047-52; Sui G et al. (2002) Proc. Natl. Acad. Sci. USA, 99:5515-20; Paddison et al. (2002) Genes and Dev., 16:948-58; Brummelkamp et al. (2002) Science, 296:550-3, 2002; Miyagashi et al. (2002) Nat. Biotechnol., 20:497-500; and, Paul et al. (2002) Nat. Biotechnol., 20:505-8. As described in these and other references, the siRNA may consist of two individual nucleic acid strands or of a single strand with a self-complementary region capable of forming a hairpin (stem-loop) structure. A number of variations in structure, length, number of mismatches, size of loop, identity of nucleotides in overhangs, etc., are consistent with effective siRNA-triggered gene silencing. While not wishing to be bound by any theory, it is thought that intracellular processing (e.g., by DICER) of a variety of different precursors results in production of siRNA capable of effectively mediating gene silencing. Generally it is preferred to target exons rather than introns, and it may also be preferable to select sequences complementary to regions within the 3′ portion of the target transcript. Generally it is preferred to select sequences that contain approximately equimolar ratio of the different nucleotides and to avoid stretches in which a single residue is repeated multiple times.
siRNAs may thus comprise RNA molecules having a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3′ overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides. As used herein, siRNAs also include various RNA structures that may be processed in vivo to generate such molecules. Such structures include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, preferably a 3′ overhang. Preferably, the stem is approximately 19 bp long, the loop is about 1-20, more preferably about 4-10, and most preferably about 6-8 nt long and/or the overhang is about 1-20, and more preferably about 2-15 nt long. In certain embodiments of the invention the stem is minimally 19 nucleotides in length and may be up to approximately 29 nucleotides in length. Loops of 4 nucleotides or greater are less likely subject to steric constraints than are shorter loops and therefore may be preferred. The overhang may include a 5′ phosphate and a 3′ hydroxyl. The overhang may, but need not comprise a plurality of U residues, e.g., between 1 and 5 U residues. Classical siRNAs as described above trigger degradation of mRNAs to which they are targeted, thereby also reducing the rate of protein synthesis. In addition to siRNAs that act via the classical pathway, certain siRNAs that bind to the 3′ UTR of a template transcript may inhibit expression of a protein encoded by the template transcript by a mechanism related to but distinct from classic RNA interference, e.g., by reducing translation of the transcript rather than decreasing its stability. Such RNAs are referred to as microRNAs (miRNAs) and are typically between approximately 20 and 26 nucleotides in length, e.g., 22 nt in length. It is believed that they are derived from larger precursors known as small temporal RNAs (stRNAs) or mRNA precursors, which are typically approximately 70 nt long with an approximately 4-15 nt loop (Grishok et al. (2001) Cell, 106:23-4; Hutvagner et al. (2001) Science, 293:834-8; Ketting et al. (2001) Genes Dev., 15:2654-9). Endogenous RNAs of this type have been identified in a number of organisms including mammals, suggesting that this mechanism of post-transcriptional gene silencing may be widespread (Lagos-Quintana et al. (2001) Science, 294:853-8, 2001; Pasquinelli (2002) Trends Gen., 18:171-3). MicroRNAs have been shown to block translation of target transcripts containing target sites in mammalian cells (Zeng et al. (2002) Mol. Cell, 9: 1327-33).
siRNAs such as naturally occurring or artificial (i.e., designed by humans) mRNAs that bind within the 3′ UTR (or elsewhere in a target transcript) and inhibit translation may tolerate a larger number of mismatches in the siRNA/template duplex, and particularly may tolerate mismatches within the central region of the duplex. In fact, there is evidence that some mismatches may be desirable or required as naturally occurring stRNAs frequently exhibit such mismatches as do mRNAs that have been shown to inhibit translation in vitro. For example, when hybridized with the target transcript such siRNAs frequently include two stretches of perfect complementarity separated by a region of mismatch. A variety of structures are possible. For example, the mRNA may include multiple areas of nonidentity (mismatch). The areas of nonidentity (mismatch) need not be symmetrical in the sense that both the target and the mRNA include nonpaired nucleotides. Typically the stretches of perfect complementarity are at least 5 nucleotides in length, e.g., 6, 7, or more nucleotides in length, while the regions of mismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.
Hairpin structures designed to mimic siRNAs and mRNA precursors are processed intracellularly into molecules capable of reducing or inhibiting expression of target transcripts (McManus et al. (2002) RNA 8:842-50). These hairpin structures, which are based on classical siRNAs consisting of two RNA strands forming a 19 bp duplex structure are classified as class I or class II hairpins. Class I hairpins incorporate a loop at the 5′ or 3′ end of the antisense siRNA strand (i.e., the strand complementary to the target transcript whose inhibition is desired) but are otherwise identical to classical siRNAs. Class II hairpins resemble mRNA precursors in that they include a 19 nt duplex region and a loop at either the 3′ or 5′ end of the antisense strand of the duplex in addition to one or more nucleotide mismatches in the stem. These molecules are processed intracellularly into small RNA duplex structures capable of mediating silencing. They appear to exert their effects through degradation of the target mRNA rather than through translational repression as is thought to be the case for naturally occurring mRNAs and siRNAs.
Thus, it is evident that a diverse set of RNA molecules containing duplex structures is able to mediate silencing through various mechanisms. For the purposes of the present invention, any such RNA, one portion of which binds to a target transcript and reduces its expression, whether by triggering degradation, by inhibiting translation, or by other means, is considered to be an siRNA, and any structure that generates such an siRNA (i.e., serves as a precursor to the RNA) is useful in the practice of the present invention.
A further method of RNA interference for use in the present invention is the use of short hairpin RNAs (shRNA). A plasmid containing a DNA sequence encoding for a particular desired siRNA sequence is delivered into a target cell via transfection or virally-mediated infection. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to transfected siRNA molecules and are used by the cell to mediate RNAi of the desired protein. The use of shRNA has an advantage over siRNA transfection as the former can lead to stable, long-term inhibition of protein expression. Inhibition of protein expression by transfected siRNAs is a transient phenomenon that does not occur for times periods longer than several days. In some cases, this may be preferable and desired. In cases where longer periods of protein inhibition are necessary, shRNA mediated inhibition is preferable. The use of shRNA is particularly preferred. Typically, siRNA-encoding vectors are constructs comprising a promoter, a sequence of the target gene to be silenced in the “sense” orientation, a spacer, the antisense of the target gene sequence, and a terminator.
Inhibition of the expression of endosialin can also be effectuated by other means that are known and readily practiced in the art. For example, antisense nucleic acids can be used. Antisense RNA transcripts have a base sequence complementary to part or all of any other RNA transcript in the same cell. Such transcripts have been shown to modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA (Denhardt (1992) Ann. N Y Acad. Sci., 660:70-6, 1992; Nellen et al. (1993) Trends Biochem. Sci., 18:419-23; and, Baker et al. (1999) Biochim. Biophys. Acta., 1489: 3-18). Accordingly, in certain embodiments of the invention, inhibition of endosialin in a cell is accomplished by expressing an antisense nucleic acid molecule in the cell.
Antisense nucleic acids are generally single-stranded nucleic acids (DNA, RNA, modified DNA, or modified RNA) complementary to a portion of a target nucleic acid (e.g., an mRNA transcript) and therefore able to bind to the target to form a duplex. Typically, they are oligonucleotides that range from 15 to 35 nucleotides in length but may range from 10 up to approximately 50 nucleotides in length. Binding typically reduces or inhibits the function of the target nucleic acid, such as a gene encoding endosialin. For example, antisense oligonucleotides may block transcription when bound to genomic DNA, inhibit translation when bound to mRNA, and/or lead to degradation of the nucleic acid. Inhibition of the expression of endosialin can be achieved by the administration of antisense nucleic acids or peptide nucleic acids comprising sequences complementary to those of the mRNA that encodes the endosialin protein. Antisense technology and its applications are well known in the art and are described in Phillips (ed.) Antisense Technology, Methods Enzymol., 2000, Volumes 313 and 314, Academic Press, San Diego, and references mentioned therein. See also Crooke (ed.) “ANTISENSE DRUG TECHNOLOGY: PRINCIPLES, STRATEGIES, AND APPLICATIONS” (1st Edition) Marcel Dekker and references cited therein.
Antisense oligonucleotides can be synthesized with a base sequence that is complementary to a portion of any RNA transcript in the cell. Antisense oligonucleotides can modulate gene expression through a variety of mechanisms including the modulation of RNA splicing, the modulation of RNA transport and the modulation of the translation of mRNA. Various properties of antisense oligonucleotides including stability, toxicity, tissue distribution, and cellular uptake and binding affinity may be altered through chemical modifications including (i) replacement of the phosphodiester backbone (e.g., peptide nucleic acid, phosphorothioate oligonucleotides, and phosphoramidate oligonucleotides), (ii) modification of the sugar base (e.g., 2′-O-propylribose and 2′-methoxyethoxyribose), and (iii) modification of the nucleoside (e.g., C-5 propynyl U, C-5 thiazole U, and phenoxazine C) (Wagner (1995) Nat. Medicine, 1:1116-8; Varga et al. (1999) Immun. Lett., 69:217-24; Neilsen (1999) Curr. Opin. Biotech., 10:71-5; and Woolf (1990) Nucleic Acids Res., 18:1763-9).
Inhibition of endosialin gene expression can also be effectuated by use of ribozymes. Certain nucleic acid molecules referred to as ribozymes or deoxyribozymes have been shown to catalyze the sequence-specific cleavage of RNA molecules. The cleavage site is determined by complementary pairing of nucleotides in the RNA or DNA enzyme with nucleotides in the target RNA. Thus, RNA and DNA enzymes can be designed to cleave to any RNA molecule, thereby increasing its rate of degradation (Cotten et al. (1989) EMBO J., 8:861-6; and, Usman et al. (1996) Curr. Opin. Struct. Biol., 1:527-33). Hammerhead ribozymes are also routinely used in gene regulation (Lyngstadaas (2001) Crit. Rev. Oral Biol. Med., 12:469-78).
In preferred aspects of the invention, the cells targeted by the inventive methods can be specifically transformed with transcription-silencing nucleic acids such as shRNA or siRNA, or can be transformed with vectors encoding such nucleic acids such that the cell expresses the inhibitory nucleic acid molecules. Transformation of the cells can be carried out according to any means suitable in the art, including those described and exemplified herein.
Decoy oligonucleotides are also suitable for regulating the expression of endosialin-encoding genes. Recent clinical trials have tested the ability of decoy oligonucleotides to sequester pathogenic proteins. Decoy oligonucleotides generally contain an enhancer element that can penetrate cells, and once inside cells, the decoy oligonucleotides bind to sequence-specific DNA-binding proteins and interfere with transcription (Fichou et al. (2006) Trends Biotechnol., 24:563-70; Nakamura et al. (2002) In Vivo, 16:45-8; Tomita et al. (1997) Exp. Nephrol., 5:429-34).
Genetic regulation of endosialin expression can also be effectuated by knockdown of the gene encoding endosialin. As will be appreciated by those of skill in the art, the sequence of the endosialin gene (from any organism of interest), for example, SEQ ID NO: 1, 3, or 5, can be used to generate nucleic acid molecules and vectors for knockdown expression of the endosialin gene. Considered in terms of their sequences, the nucleic acid molecules that encode regulatory, particularly inhibitory, sequences derived from SEQ ID NOs: 1, 3, and 5, include allelic variants, homologs, and natural mutants of SEQ ID NOs: 1, 3, and 5. Because such variants and homologs are expected to possess certain differences in nucleotide sequence, this invention provides isolated polynucleotides that have at least about 60%, preferably at least about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% or 70%, more preferably at least about 71%, 72%, 73%, 74%, 75%, 76%, 77%. 78%, 79%, or 80%, even more preferably 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even more preferably 90%, 91%, 92%, 93%, 94%, 95%, and most preferably 96%, 97%, 98% and 99% or more identity with any knockdown nucleic acid derived from SEQ ID NOs: 1, 3, or 5. Because of the natural sequence variation likely to exist among genes encoding these regulatory sequences in different organisms, one skilled in the art would expect to find this level of variation, while still maintaining the unique properties of the knockdown polynucleotides. Accordingly, such variants and homologs are considered substantially the same as one another and are included within the scope of the present invention.
Knockdown nucleic acid molecules can be prepared by two general methods: (1) they may be synthesized from appropriate nucleotide triphosphates, or (2) they may be isolated from biological sources. Both methods utilize protocols well known in the art.
The availability of nucleotide sequence information such as the entire nucleic acid sequence of endosialin, for example, SEQ ID NOs: 1, 3, and 5, enables preparation of an isolated nucleic acid molecule of the invention by oligonucleotide synthesis. Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices. The resultant construct may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC). A synthetic DNA molecule so constructed may then be cloned and amplified in an appropriate vector.
Knockdown nucleic acids may be maintained as DNA in any convenient cloning vector. In some preferred aspects, clones are maintained in plasmid cloning/expression vector, either of which can be propagated in a suitable prokaryotic or eukaryotic host cell.
Knockdown nucleic acid molecules include cDNA, genomic DNA, RNA, and fragments thereof which may be single-, double-, or even triple-stranded. Thus, this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule of the present invention, in particular, SEQ ID NOs: 1, 3, or 5. Such oligonucleotides are useful as probes for detecting genes encoding endosialin, or for the positive or negative regulation of expression of genes encoding endosialin at or before translation of the mRNA into the protein. Methods in which oligonucleotides or polynucleotides may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR) and ligase chain reaction (LCR).
Also featured in accordance with the present invention are vectors and kits for producing transgenic host cells that comprise a polynucleotide encoding a regulatory sequence for inhibiting the expression of endosialin, or homolog, analog or variant thereof in a sense or antisense orientation, or a construct under control of cell or tissue-specific promoters and/or other regulatory sequences. Such vectors are suitable for modulating, and preferably inhibiting, the expression of endosialin.
Suitable host cells include, but are not limited to, plant cells, bacterial cells, yeast and other fungal cells, insect cells and mammalian cells that express endosialin. The cells can be neoplastically transformed. More preferred are human cells.
Vectors for transforming a wide variety of these host cells are well known to those of skill in the art. They include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.
The coding region of a regulatory sequence can be placed under a powerful constitutive promoter, such as the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin, human myosin, human hemoglobin, human muscle creatine, and others. In addition, many viral promoters function constitutively in eukaryotic cells and are suitable for use in the present invention. Such viral promoters include without limitation, Cytomegalovirus (CMV) immediate early promoter, the early and late promoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, the long terminal repeats (LTRs) of Maloney leukemia virus, Human Immunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV), and other retroviruses, and the thymidine kinase promoter of Herpes Simplex Virus. Other promoters are known to those of ordinary skill in the art. In one embodiment, the coding region of the regulatory sequence is placed under an inducible promoter such as the metallothionein promoter, tetracycline-inducible promoter, doxycycline-inducible promoter, promoters that contain one or more interferon-stimulated response elements (ISRE) such as protein kinase R 2′,5′-oligoadenylate synthetases, Mx genes, ADAR1, and the like. Other suitable inducible promoters will be known to those of skill in the art.
Knockdown vectors can be used to transform various endosialin-expressing cells with regulatory nucleic acid sequences. Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used to construct recombinant cells for purposes of carrying out the inventive methods, in accordance with the various embodiments of the invention. The technique used should provide for the stable transfer of the heterologous gene sequence to the host cell, such that the heterologous gene sequence is heritable and expressible by the cell progeny, and so that the necessary development and physiological functions of the recipient cells are not disrupted. Techniques which may be used include but are not limited to chromosome transfer (e.g., cell fusion, chromosome-mediated gene transfer, micro cell-mediated gene transfer), physical methods (e.g., transfection, spheroplast fusion, microinjection, electroporation, liposome carrier), viral vector transfer (e.g., recombinant DNA viruses, recombinant RNA viruses) and the like (described in Cline (1985) Pharmac. Ther., 29:69-92).
Knockdown cells with inhibited expression of endosialin can be created by inhibiting the translation of mRNA encoding the transport protein by “post-transcriptional gene silencing.” The gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein. Full-length antisense molecules can be used for this purpose. Alternatively, antisense oligonucleotides targeted to specific regions of the mRNA that are critical for translation may be utilized. Antisense molecules may be provided in situ by transforming cells with a DNA construct which, upon transcription, produces the antisense RNA sequences. Such constructs can be designed to produce full-length or partial antisense sequences. This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example, see Waterhouse et al. (1998) Proc. Natl. Acad. Sci. U.S.A., 95:13959-64). In this regard, dsRNA containing sequences that correspond to part or all of at least one intron have been found particularly effective. In one embodiment, part or all of the coding sequence antisense strand is expressed by a transgene. In another embodiment, hybridizing sense and antisense strands of part or all of the coding sequence for one endosialin are transgenically expressed.
Cells that can be targeted by or otherwise used in the methods of the invention include endosialin-expressing cells that naturally express endosialin or cells transfected with a recombinant plasmid expressing endosialin. Primary endosialin-expressing cells of the invention can be isolated from tissues or purchased from vendors selling endothelial cells, such as but not limited to HUVEC or HMVEC as well as primary and cultured fibroblasts. Transfected cells of the invention include any insect expression cell line known, such as for example, Spodoptera frugiperda cells. The expression cell lines may also be yeast cell lines, such as, for example, Saccharomyces cerevisiae and Schizosaccharomyces pombe cells. The expression cells may also be mammalian cells such as, for example Chinese Hamster Ovary, baby hamster kidney cells, human embryonic kidney line 293, normal dog kidney cell lines, normal cat kidney cell lines, monkey kidney cells, African green monkey kidney cells, COS cells, and non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines, myeloma cell lines, mouse NIH/3T3 cells, LMTK cells, mouse sertoli cells, human cervical carcinoma cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, TR1 cells, MRC 5 cells, and FS4 cells.
Inhibiting the expression of endosialin inhibits the interaction of endosialin with any endosialin ligand. Endosialin ligands include extracellular matrix proteins such as fibronectin and collagen.
Also featured in accordance with the present invention are methods for inhibiting the interaction of endosialin expressed by an endosialin expressing cell with an endosialin ligand that comprise blocking or obstructing the endosialin expression by the cell. Thus, for example, a physical barrier serves to inhibit, impede, or otherwise hinder the interaction of expressed endosialin with an endosialin ligand. Any chemical or biomolecule can serve to obstruct this interaction. For example, small molecules, polypeptides, antibodies, and antigen-binding fragments thereof that specifically bind to endosialin, or in the alternative, specifically bind to an endosialin ligand can be used in this methods.
In some preferred embodiments, inhibiting the interaction of an endosialin-expressing cell with a ligand for endosialin comprises inhibiting the binding of the endosialin ligand to the expressed endosialin. For example, the interaction between endosialin and its ligand is hindered, blocked, impeded, or otherwise obstructed with a molecular barrier. In this way, access to the cell by the ligand is thus hindered, inhibited, blocked, impeded, obstructed or prevented. Obstruction of the endosialin can occur by any means suitable in the art, such as with a chemical or biomolecule.
For example, chemicals suitable for use in the inventive methods include, but are not limited to, amino acid structures, steroids, cyclines, anthracenes, heavy metals, quinilone, terpenes, phenolics, glycosides, alkyloids, lipids, etc. or mixtures thereof that can exert a biological effect on endosialin-expressing cells. Chemicals can be generated by chemical synthesis or derived from biocatalysis or derived from biological fluids. Chemicals can be derived from human, non-human mammalian, plant, yeast, fungi and/or prokaryotic sources.
In some embodiments, competitive inhibitors are employed to inhibit the interaction of endosialin or an endosialin-expressing cell with a ligand for endosialin. A “competitive inhibitor” competes with the ligand for the binding site to endosialin. In some embodiments, the competitive inhibitors are endosialin ligands, for example, collagen (e.g., collagen I or IV) or fibronectin, or endosialin-binding fragments thereof. Most preferred competitive inhibitors are the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, and the 30 kDa heparin binding fragment of fibronectin.
In highly preferred embodiments, antibodies or antigen-binding fragments thereof are used to obstruct the interaction of expressed endosialin with endosialin ligands. The antibodies or antigen-binding fragments can be specific for an epitope on endosialin, or can be specific for an epitope on an endosialin ligand. Antibodies and antigen-binding fragments thereof specific for endosialin are more preferred. Antibodies to ligands such as fibronectin, collagen, and the like are commercially available.
Suitable antibodies can be polyclonal or monoclonal, or can be derivatives or fragments of antibodies that retain specificity for endosialin or an endosialin ligand. The antibodies can be from any of the five classes of antibodies, i.e., the IgA, IgD, IgE, IgG and IgM isotypes. Suitable antibodies also include the IgY isotype generally found in hen or turkey serum and hen or turkey egg yolk.
Antibody derivatives and antigen-binding fragments are suitable for use in the inventive methods, and such derivatives comprise portions of intact antibodies that retain antigen-binding specificity of the parent antibody molecule. For example, derivatives can comprise at least one variable region (either a heavy chain or light chain variable region). Examples of suitable antibody derivatives and fragments include, without limitation antibodies with polyepitopic specificity, bispecific antibodies, diabodies, and single-chain molecules, as well as Fab, F(ab′)2, Fd, Fabc, and Fv molecules, single chain (Sc) antibodies, individual antibody light chains, individual antibody heavy chains, chimeric fusions between antibody chains and other molecules, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. All antibody isotypes can be used to produce antibody derivatives and fragments. Antibody derivatives and fragments can be recombinantly produced.
Antibodies suitable for use in the inventive methods can be derived from any species. For example, the antibodies can be mouse, rat, goat, horse, swine, bovine, chicken, rabbit, donkey, human, and the like. For use in methods of treatment, or for administration to humans, non-human derived antibodies can be structurally altered to be less antigenic upon administration to a human patient.
Thus, in some embodiments of the invention, the antibodies used in the inventive methods are chimeric antibodies. Chimeric antibodies and methods to produce them are well known and established in the art. For example, a chimeric antibody may comprise a mouse antigen binding domain with a human Fc or other such structural domain.
In some embodiments, the antibodies are humanized antibodies. Humanized antibodies can be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FWR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FWR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. (1986) Nature, 321:522-5; Reichmann et al. (1988) Nature, 332:323-9; and Presta (1992) Curr. Op. Struct. Biol., 2:593-6.
In preferred aspects of the invention, the antibodies are fully human. This means that the antibody is solely from human origin, or otherwise consists of an amino acid sequence identical to a human form of the antibody.
The antibodies of the invention can be labeled or otherwise conjugated to various chemical or biomolecule moieties, for example, for therapeutic or diagnostic applications. The moieties can be cytotoxic, for example, bacterial toxins, viral toxins, radioisotopes, and the like. The moieties can be detectable labels, for example, fluorescent labels, radiolabels, biotin, and the like. Additional moieties include, but are not limited to glycosylation, acetylation, pegylation, phosphorylation, and amidation. The antibodies useful in the methods of the invention may themselves by derivatized by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other proteins, and the like.
Those of skill in the art will recognize that antibody specificity is primarily determined by the six CDR regions, especially H chain CDR3 (Kala et al. (2002) J. Biochem., 132:535-41; Morea et al. (1998) J. Mol. Biol., 275:269-94; and, Chothia et al. (1987) J. Mol. Biol., 196:901-17). Antibody framework regions, however, can play a role in antigen-antibody interactions (Panka et al. (1988) Proc. Natl. Acad. Sci. USA, 85:3080-4), particularly with respect to their role in conformation of CDR loops (Foote et al. (1992) J. Mol. Biol., 224:487-99). Thus, antibodies suitable for use in the inventive methods can comprise any combination of H or L chain CDR or FWR regions that confer antibody specificity for endosialin or endosialin ligands.
In some embodiments, the invention contemplates the use of isolated human antibodies and antigen-binding fragments thereof that specifically bind to endosialin. In some embodiments, suitable antibodies or antigen-binding fragments can comprise a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:28, 30, and 32, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 13, 15, and 17, respectively. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO:27, 29, and 31, respectively. In some embodiments, the light chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO: 12, 14, and 16, respectively. In some embodiments, the antibodies or antigen-binding fragments can comprise a heavy chain comprising a variable domain of SEQ ID NO: 34 and a light chain comprising a variable domain of SEQ ID NO: 19. In some embodiments, the heavy chain variable domain is encoded by the nucleotide sequence of SEQ ID NO:33. In some embodiments, the light chain variable domain is encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the antibodies or antigen-binding fragments can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:22 or 26 and a light chain comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO:21 or 25 and the light chain is encoded by the nucleotide sequence of SEQ ID NO: 10. In some embodiments, the antibodies or antigen-binding fragments comprise a heavy chain comprising SEQ ID NO:20 or 24 and a light chain comprising SEQ ID NO:9. In some embodiments, the antibodies comprise a heavy chain encoded by the nucleic acid sequence of SEQ ID NO: 8 or 23. Antibodies or antigen-binding fragments can comprise a light chain encoded by the nucleic acid sequence that comprises SEQ ID NO:7.
Antibody-producing cells producing antibodies that can be used in accordance with the invention have been placed with the Amer. Type Cult. Coll. (10801 University Blvd., Manassas, Va. 20110-2209) on Apr. 24, 2006 and on Mar. 11, 2008 and have been assigned Access. Nos. PTA-7554 and PTA-9017, respectively.
It is to be understood that, because of the natural sequence variation likely to exist among heavy and light chains and the genes encoding them, one skilled in the art would expect to find some level of variation within the amino acid sequences or the genes encoding them, while still maintaining the unique binding properties (e.g., specificity and affinity) of the antibodies of the present invention. Such an expectation is due in part to the degeneracy of the genetic code, as well as to the known evolutionary success of conservative amino acid sequence variations, which do not appreciably alter the nature of the encoded protein. Accordingly, such variants and homologs are considered substantially the same as one another and are included within the scope of the present invention.
Variants having single or multiple amino acid substitutions, deletions, additions, or replacements that retain the biological properties (e.g., binding affinity or immune effector activity) of the antibodies described herein are contemplated for use in the invention. The skilled person can produce variants having single or multiple amino acid substitutions, deletions, additions or replacements. These variants may include, for example: (a) variants in which one or more amino acid residues are substituted with conservative or nonconservative amino acids, (b) variants in which one or more amino acids are added to or deleted from the polypeptide, (c) variants in which one or more amino acids include a substituent group, and (d) variants in which the polypeptide is fused with another peptide or polypeptide such as a fusion partner, a protein tag or other chemical moiety, that may confer useful properties to the polypeptide, such as, for example, an epitope for an antibody, a polyhistidine sequence, a biotin moiety and the like. Antibodies of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or nonconserved positions. In other embodiments, amino acid residues at nonconserved positions are substituted with conservative or nonconservative residues. The techniques for obtaining these variants, including genetic (suppressions, deletions, mutations, etc.), chemical, and enzymatic techniques, are known to the person having ordinary skill in the art.
The present invention contemplates antibodies, or antigen-binding fragments thereof, having amino acid sequences that are substantially the same as the previously described amino acid sequences. For example, such antibodies or antigen-binding fragments may include those wherein the heavy chain CDR1, CDR2, and CDR3 are at least 90% identical to SEQ ID NO:28, 30, and 32, respectively, and/or wherein the light chain CDR1, CDR2, and CDR3 are at least 90% identical to SEQ ID NO: 13, 15, and 17, respectively. In some embodiments, such antibodies or antigen-binding fragments may include those wherein the heavy chain variable domain is at least 90% identical to SEQ ID NO: 34 and/or wherein the light chain variable domain is at least 90% identical to SEQ ID NO: 19. In some embodiments, the antibodies or antigen-binding fragments may include those wherein the heavy chain is at least 90% identical to SEQ ID NO:22 or 26 and/or wherein the light chain is at least 90% identical to SEQ ID NO: 11. In some embodiments, the antibodies or antigen-binding fragments may include those wherein the heavy chain is at least 90% identical to SEQ ID NO:20 or 24 and/or wherein the light chain is at least 90% identical to SEQ ID NO:9. For example, antibodies M4 and M4.1 are humanized antibodies to human endosialin. While antibodies M4 and M4.1 share a light chain sequence, they differ in their heavy chain by a single amino acid sequence shown, for example, at residue 429 of SEQ ID NO:20 relative to residue 429 of SEQ ID NO:24. The invention further contemplates antibodies, or antigen-binding fragments thereof, that compete for binding to endosialin with antibody M4 or M4.1. The invention further contemplates antibodies, or antigen-binding fragments thereof, that bind the same epitope of endosialin as antibody M4 or M4.1.
Antibodies suitable for use in the methods of the invention can have binding affinities for the target antigen, such as endosialin or an endosialin ligand, that include a dissociation constant (KD) of less than 1×10−2 M. In some embodiments, the KD is less than 1×10−3 M. In other embodiments, the KD is less than 1×10−4 M. In some embodiments, the KD is less than 1×10−5 M. In still other embodiments, the KD is less than 1×10−6 M. In other embodiments, the KD is less than 1×10−7 M. In other embodiments, the KD is less than 1×10−8 M. In other embodiments, the KD is less than 1×10−9 M. In other embodiments, the KD is less than 1×10−10 M. In still other embodiments, the KD is less than 1×10−11 M. In some embodiments, the KD is less than 1×10−12 M. In other embodiments, the KD is less than 1×10−13 M. In other embodiments, the KD is less than 1×10−14 M. In still other embodiments, the KD is less than 1×10−15 M.
Specificity and/or affinity of antibodies that bind to endosialin can optionally be optimized by directed evolution of the cells producing the antibody, by using a dominant negative allele of a mismatch repair gene such as PMS1, PMS2, PMS2-134, PMSR2, PMSR3, MLH1, MLH2, MLH3, MLH4, MLH5, MLH6, PMSL9, MSH1, and MSH2 introduced into the antibody-producing cells. Cells containing the dominant negative mutant will become hypermutable and accumulate mutations at a higher rate than untransfected control cells. A pool of the mutating cells may be screened for clones that produce higher affinity/specificity of antibodies or binding proteins, or that produce higher titers of antibodies or binding proteins, or that simply grow faster or better under certain conditions. The technique for generating hypermutable cells using dominant negative alleles of mismatch repair genes is described in U.S. Pat. No. 6,146,894. Alternatively, mismatch repair may be inhibited using the chemical inhibitors of mismatch repair described in WO 02/054856. The technique for enhancing antibodies using the dominant negative alleles of mismatch repair genes or chemical inhibitors of mismatch repair may be applied to mammalian, yeast, plant or prokaryotic expression cells expressing cloned immunoglobulin or protein genes as well. Cells expressing the dominant negative alleles or small molecule can be “cured” in that the dominant negative allele can be turned off, if inducible, eliminated from the cell while the small chemical can be removed from grow culture resulting in cells that are genetically stable once more and no longer accumulate mutations at the abnormally high rate.
Inhibiting the expression of endosialin inhibits the interaction of endosialin with any endosialin ligand. Endosialin ligands include extracellular matrix proteins, such as fibronectin and collagen. Any collagen subtype can serve as a ligand to endosialin. Collagen I and Collagen IV are more preferred.
Inhibiting the interaction of endosialin with endosialin ligands inhibits pathways and cascades that are upregulated or otherwise activated as a result of this interaction. For example, endosialin interaction with endosialin ligands can promote the expression and/or activation of adhesion molecules such as integrins, which mediate cell attachment to the extracellular matrix or to other cells, and which mediate cell signal pathways, among other things.
Integrins tend to exist as heterodimers containing two distinct chains, an α (alpha) and β (beta) subunit. There are approximately 18 α and 8 β subunits that have been characterized. In addition, a number of integrin subunits exist as variants via differential splicing. The various combinations of alpha and beta integrin subunits results in over 24 unique active integrin complexes (Hynes (2002) Cell, 110:673). Integrin subunits penetrate the plasma membrane, and in general contain short cytoplasmic domains of about 40-70 amino acids. Outside the cell plasma membrane, the alpha and beta chains lie in close proximity to each other along a length of about 23 nm. The amino-termini of each integrin chain are juxtaposed within 5 nm of each other to form a ligand-binding region for EMP interaction. Integrins are categorized using several criteria. Alpha chains are classified as such because a subset of the α chains have structural elements inserted near the amino-terminus called alpha-A domain because it has a similar structural motif as the A-domains within the von Willebrand factor. Integrins carrying this domain can either bind to collagens (integrin complexes α1β1 and α2β1), or act as cell-cell adhesion molecules with those complexes containing integrins of the β2 family. Two main functions of integrins are attachment of the cell to extracellular matrix proteins and signal transduction mediated from the EMP-integrin binding to the cell. In addition, integrins are also involved in a wide range of other biological activities including binding of viruses, such as adenovirus, Echo viruses, Hanta viruses, foot and mouth disease viruses as well as binding to cells involved in immune patrolling and cell-cell contact for cell migration. Integrins couple EMPs (which depends on the integrin complex) to the cytoskeleton within the cell. Several ligands of integrins have been identified. The more common ligands are fibronectin, vitronectin, collagen, and laminin. The interactions between integrin, an EMP and the microfilaments inside the cell are linked via scaffolding proteins including talin, paxillin and alpha-actinin. These interactions result in regulation of kinases like FAK (focal adhesion kinase) and Src kinase family members to phosphorylate substrates such as p130CAS thereby recruiting signaling adaptors such as Crk for mediating cellular responses including pathway activation, cellular proliferation and/or survival. Any of these integrin-associated functions can be assembled as a screening assay to monitor integrin activity as a function of endosialin activity for assays to identify pharmacologic agents or effective endosialin targeting molecules of this invention. Moreover, in light of the invention disclosed here, targeting integrins with pharmacologic agents to endosialin in endosialin-expressing cells have broad opportunities in suppressing integrin-mediated viral infection and other pathologies.
Thus, inhibiting the interaction of endosialin with an endosialin ligand inhibits the expression and/or activation of integrin molecules on the endosialin-expressing cell. In some preferred embodiments, the expression or activation of integrin β1, β2, or β3 is suppressed by the inhibition.
Other molecules and pathways whose expression and/or activation are affected by the inhibition of endosialin interaction with endosialin ligands include matrix metalloproteinases (MMPs). MMPs are zinc-dependent proteases that play a role in, among other things, degradation of extracellular matrix proteins, cell surface receptors, and the like. MMPs play a role in cell migration, proliferation, and angiogenesis, among other things. The MMP family of enzymes have a common zinc binding motif (HExxHxxGxxH) within their active site, and a conserved methionine following the active site. MMPs are classified by their homology to one another, substrate specificity and partly on their cellular localization. They are broadly grouped into 4 classes: collagenase, stromelysins, gelatinase, and the membrane type MMPs (MT-MMPs). Collagenase-type MMPs are capable of degrading triple-helical fibrillar collagens into distinctive fragments. These collagens are the major components of bone and cartilage, and this class of MMPs are the only known mammalian enzymes capable of degrading them. They include MMP-1 (Interstitial collagenase); MMP-8 (Neutrophil collagenase); MMP-13 (Collagenase 3); and MMP-18. Stromelysin-type MMP enzymes display a broad ability to cleave EMPs but are unable to cleave the triple-helical fibrillar collagens. This class includes MMP-3 (Stromelysin 1); MMP-10 (Stromelysin 2); MMP-11 (Stromelysin 3); MMP-12 (Macrophage metalloelastase); MMP-19 (RASI-1, also referred to as stromelysin-4); and MMP-20 (enamelysin); MMP-22 (C-MMP) and MMP-27. Gelatinase-type MMPs degrade mainly type IV collagen and gelatin, and are distinguished by the presence of an additional domain inserted into the catalytic domain. This gelatin-binding region is positioned immediately before the zinc binding motif, and forms a separate folding unit which does not disrupt the structure of the catalytic domain. This class includes MMP-2 (72 kDa gelatinase, gelatinase-A); MMP-9 (92 kDa gelatinase, gelatinase-B). Finally, the membrane-bound MMPs are those that are attached to the outer cellular membrane. They include: The type-II transmembrane cysteine array MMP-23; the glycosyl phosphatidylinositol-attached MMPs 17 and 25 (MT4-MMP and MT6-MMP respectively); and the type-I transmembrane MMPs 14, 15, 16, 24 (MT 1-MMP, MT2-MMP, MT3-MMP, and MT5-MMP respectively). All of these MMPs have a furin cleavage site in the pro-peptide, which is a feature also shared by MMP-11.
Thus, inhibiting the interaction of endosialin with an endosialin ligand inhibits the expression and/or activation of MMPs on the endosialin-expressing cell. In some preferred embodiments, the expression or activation of MMP-1, MMP-2, MMP-8, MMP-9, MMP-12, MMP-13, or MMP-18 is suppressed by the inhibition.
Without intending to be bound to any particular theory or mechanism of operation, it is believed that endosialin functions directly or indirectly in angiogenesis, particularly with respect to neovascularization and diseases such as cancer. Therefore, it is believed that disrupting the binding of endosialin or endosialin-expressing cells to endosialin ligands, or disrupting endosialin-mediated activation of integrins, expression of MMPs and/or cellular proliferation/survival can suppress vascularization associated with neovascular disease.
Accordingly, the invention also features methods for inhibiting angiogenesis. The methods can be carried out in vitro or in vivo. In one aspect, the methods for inhibiting angiogenesis comprise administering to a subject a therapeutically effective amount of a composition that obstructs endosialin expressed on the surface of a cell, wherein said obstruction inhibits the interaction of said cell with a ligand for endosialin, and wherein the inhibiting of said interaction of said cell with said ligand inhibits angiogenesis of a tissue, organ, or neoplasm in the subject.
In another aspect, the methods for inhibiting angiogenesis comprise contacting a cell, cell culture, tissue, or organ with a composition that obstructs endosialin expressed on the surface of a cell, wherein said obstruction inhibits the interaction of said cell with a ligand for endosialin, and wherein the inhibiting of said interaction of said cell with said ligand inhibits angiogenesis by said cell, cell culture, tissue, or organ.
In some preferred embodiments, the composition comprises at least one competitive inhibitor described herein. In some embodiments, the competitive inhibitors are endosialin ligands, for example, collagen, fibronectin, or endosialin-binding fragments thereof. Preferred competitive inhibitors are fragments of collagen I, collagen IV, or fibronectin. Most preferred competitive inhibitors are the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, and the 30 kDa heparin binding fragment of fibronectin.
In preferred embodiments, the composition comprises at least one antibody that specifically binds to endosialin. Such antibodies preferably have an affinity for endosialin that is less than about 1×10−7 M, more preferably less than about 1×10−8 M, more preferably less than about 1×10−9 M, and more preferably less than about 1×10−10 M. Antibodies that specifically bind to endosialin can include those antibodies whose characteristics are described and exemplified herein. For example, in some preferred aspects, the antibody that specifically binds to endosialin comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:28, 30, and 32, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 13, 15, and 17, respectively. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO:27, 29, and 31, respectively. In some embodiments, the light chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO:12, 14, and 16, respectively. In some embodiments, the antibodies can comprise a heavy chain comprising a variable domain of SEQ ID NO: 34 and a light chain comprising a variable domain of SEQ ID NO: 19. In some embodiments, the heavy chain variable domain is encoded by the nucleotide sequence of SEQ ID NO:33. In some embodiments, the light chain variable domain is encoded by the nucleotide sequence of SEQ ID NO:18. In some embodiments, the antibodies can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:22 or 26 and a light chain comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO:21 or 25 and the light chain is encoded by the nucleotide sequence of SEQ ID NO:10. In some embodiments, the antibodies comprise a heavy chain comprising SEQ ID NO:20 or 24 and a light chain comprising SEQ ID NO:9. In some embodiments, the antibodies comprise a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:8 or 23. Antibodies can comprise a light chain encoded by the nucleic acid sequence that comprises SEQ ID NO:7. Antibody-producing cells producing antibodies that can be used in accordance with the invention have been placed with the Amer. Type Cult. Coll. (10801 University Blvd., Manassas, Va. 20110-2209) on Apr. 24, 2006 and on Mar. 11, 2008 and have been assigned Access. Nos. PTA-7554 and PTA-9017, respectively. The antibodies can be polyclonal, monoclonal, antigen-binding fragments, chimeric, humanized, fully human, and the like as described herein.
Inhibiting the expression of endosialin inhibits the interaction of endosialin with any endosialin ligand. Endosialin ligands include extracellular matrix proteins such as fibronectin and collagen.
The invention also features methods for inhibiting neovascularization. The methods can be carried out in vitro or in vivo. In one aspect, the methods for inhibiting neovascularization comprise administering to a subject a therapeutically effective amount of a composition that obstructs endosialin expressed on the surface of a cell, wherein said obstruction inhibits the interaction of said cell with a ligand for endosialin, and wherein the inhibiting of said interaction of said cell with said ligand inhibits neovascularization of a tissue, organ, or neoplasm in the subject.
In another aspect, the methods for inhibiting neovascularization comprise contacting a cell, cell culture, tissue, or organ with a composition that obstructs endosialin expressed on the surface of a cell, wherein said obstruction inhibits the interaction of said cell with a ligand for endosialin, and wherein the inhibiting of said interaction of said cell with said ligand inhibits neovascularization of said cell, cell culture, tissue, or organ.
In some preferred embodiments, the composition comprises at least one competitive inhibitor described herein. In some embodiments, the competitive inhibitors are endosialin ligands, for example, collagen, fibronectin, or endosialin-binding fragments thereof. Preferred competitive inhibitors are fragments of collagen I, collagen IV, or fibronectin. Most preferred competitive inhibitors are the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, and the 30 kDa heparin binding fragment of fibronectin.
In preferred embodiments, the composition comprises at least one antibody that specifically binds to endosialin. Such antibodies preferably have an affinity for endosialin that is less than about 1×10−7 M, more preferably less than about 1×10−8 M, more preferably less than about 1×10−9 M, and more preferably less than about 1×10−10 M. Antibodies that specifically bind to endosialin can include those antibodies whose characteristics are described and exemplified herein. For example, in some preferred aspects, the antibody that specifically binds to endosialin comprises a heavy chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO:28, 30, and 32, respectively, and a light chain comprising CDR1, CDR2, and CDR3 of SEQ ID NO: 13, 15, and 17, respectively. In some embodiments, the heavy chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO:27, 29, and 31, respectively. In some embodiments, the light chain CDR1, CDR2, and CDR3 are encoded by nucleotide sequences of SEQ ID NO:12, 14, and 16, respectively. In some embodiments, the antibodies can comprise a heavy chain comprising a variable domain of SEQ ID NO: 34 and a light chain comprising a variable domain of SEQ ID NO: 19. In some embodiments, the heavy chain variable domain is encoded by the nucleotide sequence of SEQ ID NO:33. In some embodiments, the light chain variable domain is encoded by the nucleotide sequence of SEQ ID NO:18. In some embodiments, the antibodies can comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:22 or 26 and a light chain comprising the amino acid sequence of SEQ ID NO:11. In some embodiments, the heavy chain is encoded by the nucleotide sequence of SEQ ID NO:21 or 25 and the light chain is encoded by the nucleotide sequence of SEQ ID NO:10. In some embodiments, the antibodies comprise a heavy chain comprising SEQ ID NO:20 or 24 and a light chain comprising SEQ ID NO:9. In some embodiments, the antibodies comprise a heavy chain encoded by the nucleic acid sequence of SEQ ID NO:8 or 23. Antibodies can comprise a light chain encoded by the nucleic acid sequence that comprises SEQ ID NO:7. Antibody-producing cells producing antibodies that can be used in accordance with the invention have been placed with the Amer. Type Cult. Coll. (10801 University Blvd., Manassas, Va. 20110-2209) on Apr. 24, 2006 and Mar. 11, 2008 and have been assigned Access. No. PTA-7554 and Access. No. PTA-9017, respectively. The antibodies can be polyclonal, monoclonal, antigen-binding fragments, chimeric, humanized, fully human, and the like as described herein.
Inhibiting the expression of endosialin inhibits the interaction of endosialin with any endosialin ligand. Endosialin ligands include extracellular matrix proteins such as fibronectin, for example, human fibronectin (SEQ ID NO:35) and collagen.
The invention also features assays and methods for identifying agonists and antagonists of the interaction of endosialin with a ligand for endosialin. In some embodiments, the methods comprise contacting endosialin with a test compound, contacting the endosialin-test compound complex with a ligand for endosialin, and quantifiably measuring the interaction of endosialin with the ligand in the presence and in the absence of the test compound. An increase or decrease in the level of interaction of endosialin with ligand in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of the interaction of endosialin with the ligand.
In some embodiments, the methods comprise contacting an endosialin-expressing cell with a test compound, contacting the endosialin expressing cell with a ligand for endosialin, and quantifiably measuring the expression or activation of integrin molecules such as integrin β1, β2, or β3 on the cell in the presence and in the absence of the test compound. An increase or decrease in the level of expression or activation of the integrin molecules on the cell in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of the interaction of endosialin with said ligand.
In some embodiments, the methods comprise contacting an endosialin-expressing cell with a test compound, contacting the endosialin expressing cell with a ligand for endosialin, and quantifiably measuring the expression or activation of MMPs such as MMP-1, MMP-2, MMP-8, MMP-9, MMP-12, MMP-13, or MMP-18 on the cell in the presence and in the absence of the test compound. An increase or decrease in the level of expression or activation of the MMP molecules on the cell in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of the interaction of endosialin with said ligand.
In the inventive assays, the endosialin can be bound to a cell membrane, preferably a mammalian cell membrane, a cell membrane fragment, an artificial lipid bilayer, or to a suitable solid support. The endosialin ligand can be an extracellular matrix protein, including without limitation fibronectin or collagen.
One strategy for generating test compounds with potential biologic activity against endosialin or endosialin-expressing cells, i.e., potential interaction with endosialin, involves but is not limited to screening phage libraries producing phage coat peptides that can be screened to identify polypeptides in a library that can potentially serve to inhibit the interaction between endosialin and an endosialin ligand.
The following examples are provided to describe the invention in greater detail. They are intended to illustrate, not to limit, the invention.
Example 1
Immunohistochemistry Analysis of Endosialin Expression on Malignant Tissue
Use of antibodies to detect endosialin-expressing cells was shown by immunohistochemistry of malignant tissues. The anti-endosialin or normal IgG antibody was applied to freshly frozen human colorectal cancer tissues at two concentrations (0.5 μg/mL and 2.5 μg/mL). Phosphate-buffered saline [PBS (0.15M NaCl, pH 7.2)]+1% bovine serum albumin served as the diluent for the primary antibodies. Tissues were embedded in Tissue-Tek® O.C.T. medium, frozen on dry ice, and stored in sealed plastic bags below −70° C. Tissues were sectioned at approximately 5 μm, and fixed for 10 minutes in room temperature acetone. Slides were stored below −70° C. until staining. Just prior to staining, slides were fixed for 10 seconds in 10% neutral-buffered formalin.
Cryosections were rinsed twice in phosphate-buffered saline (PBS [0.15M NaCl, pH 7.2]). Endogenous peroxidase was blocked by incubating the slides with the peroxidase solution provided in the Dako EnVisionTM Kit for 5 minutes and rinsing twice in PBS (0.15M NaCl, pH 7.2). Next, the slides were treated with a protein block designed to reduce nonspecific binding for 20 minutes. The protein block was prepared as follows: PBS (0.15M NaCl, pH 7.2); 0.5% casein; 1% bovine serum albumin (BSA); and 1.5% normal goat serum. Following the protein block, the primary antibody (test article M4, negative control antibody, or none [buffer alone as the assay control]) was applied at room temperature for one hour. Next, the slides were rinsed two times with PBS (0.15M NaCl, pH 7.2), treated with the peroxidase-labeled goat anti-IgG polymer supplied in the Dako EnVisionTM Kit for 30 minutes (EnVisionTM polymer used at the concentration provided by manufacturer), rinsed two times with PBS (0.15M NaCl, pH 7.2), and treated with the substrate-chromogen (DAB) solution supplied in the Dako EnVisionTM Kit for 8 minutes. All slides were rinsed in water, counterstained with hematoxylin, dehydrated and coverslipped for interpretation. As shown, vessels in the tumor (FIG. 1A) stained positive for endosialin while isotype control antibody stained serial section was negative (FIG. 1B).
Example 2
Immunohistochemistry Analysis of Endosialin Expression on Healthy Tissue
Use of antibodies to detect endosialin-expressing cells was shown by immunohistochemistry of normal tissues. Briefly, normal tissue specimens were sectioned by cryostat and analyzed for endosialin expression as described above. Normal tissues contained very few fibroblast/dendritic-like cells that expressed endosialin albeit not as robustly or homogenously as was observed in the vessels within tumors (FIGS. 2A and B). These cells are useful to study the effects of neovascularization and can be isolated for gene expression to study profiles of cell growth, differentiation, migration or signature identification. They can be studied in vivo, ex vivo or in vitro using methods known by those skilled in the art as well as those listed below.
Example 3
Isolation and Enrichment of Endosialin-Expressing Cells
To demonstrate that proteins that can bind to endosialin serve as an effective way to enrich for endothelial or fibroblast-like endosialin-expressing cells, Human Microvascular Endothelial Cells (HMVECs) were panned using an antibody that can bind to endosialin to isolate an enriched population of endosialin-expressing cells from a starting pool containing 5-10% endosialin-expressing cells. Not wanting to be bound by the method or specific reagents below, this example demonstrates the use of endosialin antibodies that can isolate endosialin expressing cells.
Briefly, 96-well plates were coated in sterile conditions with goat anti human IgG Fcγ. Next, 20 μg/ml of a human anti-endosialin antibody M4 was added to the plates and three wells (A, B, C) as controls without the antibody and incubated for 1 hr at 4° C. HMVECs were harvested from 10 cm petri dish cultures with DPBS/EDTA rather than trypsin to avoid any damage to the cell membranes, thereby leaving the endosialin cell surface proteins intact. Pooled cells were plated at two different concentrations, either 100,000 cells/well or 50,000 cells/well in the 96-well plates after aspirating and washing off any unbound anti-endosialin antibody. Cells were incubated in plates for one hour at 4° C. Plates were then washed with DPBS/FBS four times (until control wells A, B, and C showed no cells within the wells). The 50,000 cells/well plate showed very few cells while the 100,000 cells/well wells contained a number of cells attached to plate. Cells were then incubated for three days in appropriate growth media. The control B and C wells were picked from the 100,000 cells/well plates for immunostaining. Calcein, AM dye was used to stain the cells for visualization using a Nikon® Eclipse TS100 Fluorescence Microscope. Positive panned cultures were expanded for growth and further analysis for homogeneous endosialin expression as described below.
To further determine the ability to isolate endosialin-expressing cells, anti-endosialin-antibody panned HMVEC cells and non-panned HMVEC cultures were prepared for immunostaining using a fluorescent anti-endosialin antibody or fluorescent α-β1-integrin antibody as control. As expected, greater than 90% of the cells stained positive for α-β1-integrin from panned and non-panned cultures (not shown), while greater than 90% of cells stained positive for endosialin from panned cultures while only 5-7% of cells stained positive for endosialin in unpanned HMVEC cultures. These data demonstrate the ability to isolate and enrich viable endosialin-expressing cells using endosialin-binding proteins such as antibodies.
As shown in FIG. 3, the panned culture had a much higher number of endosialin-positive cells as compared to the non-panned parental culture as determined by immunostaining via anti-endosialin antibody followed by a fluorescent conjugated secondary antibody. Cell number of each field was determined by light microscopy.
Example 4
Endosialin Interaction with Extracellular Matrix Proteins
Construction of TEM1 and Fc-TEM-1 expression plasmids. PCR was used to amplify a DNA fragment representing amino acids 1-685 of the TEM1 open reading frame (GenBank # AF279142) from LA1-5S genomic DNA. The resulting amplicons were digested with EcoRI and XbaI and ligated into pEF6-V5-HisA (Invitrogen). To generate Fc-TEM-1, the extracellular region of TEM1 was fused to monomeric murine IgG2b Fcγ domain and ligated into the derivative pEF6-EK-Mm-IgG2b-Fcγ-ND vector which contains an enterokinase recognition region (DDDD) followed by a modified murine IgG2b Fcγ(hinge through CH3) domain. To prevent dimerization, the four cysteine residues responsible for inter-heavy chain disulfide bonding were changed to serine. The resulting monomeric, secreted fusion protein consists of the full-length TEM1 extracellular domain and the murine IgG2b Fcγ. The integrity of all plasmid sequences was verified using Beckman® DTCS chemistry (Beckman Coulter, Fullerton, Calif.). Raw data were acquired with a CEQ 8000 DNA sequencer and analyzed using Vector NTI® software (Invitrogen).
Purification of Fc-TEM-1. CHO-TEM1-Fcγ cells were cultured at 25 L scale in IS-CHO-CD medium (Irvine Scientific, Santa Ana, Calif.), supplemented with 2 mM L-glutamine, 1× Penicillin/Streptomycin, 6 g/L soybean hydrolysate and 2.2 g/L sodium bicarbonate (Irvine Scientific), on a Wave20/50EH platform fitted with a Cellbag50® (GE Healthcare, Piscataway, N.J.), until the viability of the culture reached 50-70%. Conditioned extra-cellular medium was clarified using a Flexstand® Benchtop Pilot Hollow Fiber System (GE Healthcare) equipped with a 0.2 μm hollow fiber cartridge (GE Healthcare) until 0.5 L of culture remained in the holding vessel. At this point, the concentrated cell mass was washed with 4 L of phosphate-buffered saline (PBS, 20 mM K Phosphate, 150 mM NaCl pH 7.2), to recover remaining extra-cellular fluid. The 4 L wash was pooled with the clarified feedstock. The clarified culture medium was then concentrated twelve-fold (29 L to 2.5 L); using a Prep/Scale® Spiral Wound 2.5 ft2 100 k ultrafiltration cartridge set in a Prep/Scale® holder (Millipore, Billerica, Mass.), and driven by a peristaltic pump at an inlet pressure of 20 PSI, and a recirculation rate of approximately 400 mL/min. The resulting concentrated feedstock was filter-sterilized through bottle top filters equipped with a 0.2 μm membrane (Nalgene). TEM1-Fcγ was captured by protein A affinity chromatography, over a 10×100 mm ProSep-vA® (Millipore) column, and eluted by addition of 5 column volumes of elution buffer (100 mM citrate/10 mM acetate pH 3.0). Eluted material was dialyzed against buffer QA (20 mM Tris-Cl pH 8.0), and further purified by ion-exchange chromatography over a 5 mL HiTrap® Q-FF column (GE Healthcare). Bound proteins were washed with 15% buffer QB (20 mM Tris-Cl, 1 M NaCl pH 8.0), followed by elution of bound Fc-TEM-1 using 35% buffer QB. Eluted proteins were concentrated by ultra-filtration in a Model 8400 positive pressure ultra-filtration module (Millipore) fitted with a 100 kDa MWCO membrane (Millipore), to a final volume of approximately 5 mL. Concentrated Fc-TEM-1 was purified by preparative size exclusion chromatography on a Sephacryl® S-300HR 26×60 column (GE Healthcare), equilibrated with PBS. Fractions containing purified Fc-TEM-1 were pooled, concentrated to a nominal range between 0.1-1 mg/mL by ultra-filtration using a 100 kDa MWCO membrane and stored in single-use aliquots at −80° C.
Purified Fc-TEM-1 (2.9 μg) was loaded onto a 4-12% Bis-Tris gel (Invitrogen, Inc.), and electrophoresed in MOPS Running Buffer (50 mM MOPS, 50 mM Tris, 3.5 mM SDS, 1 mM EDTA pH 7.7), for 40 minutes. For staining, the gel was fixed for 15 minutes in Fix solution (50% methanol, 10% acetic acid), washed twice for 10 minutes in deionized water, and stained for at least one hour using GelCode Blue colloidal Coomassie Blue stain (Pierce). The gel was de-stained by repeated washing with deionized water.
Pre-coated Fibronectin (FN), Collagen I (Col I), Collagen IV (Col IV), Laminin (LN) (BD Biosciences, San Diego Calif.), Vitronectin (VN), or Gelatin (Gel) (Chemicon Intl.) 96-well plates were used to assess Fc-TEM-1 binding. The binding of TEM 1 to Col I, Col IV, and FN was not due to trace contaminants in the purified protein from human plasma since neither Col I nor Col IV was detected in FN by anti-Col antibody ELISA, nor was FN detected in Col (data not shown). All plates were blocked with assay buffer (0.5% BSA, 0.5% Tween-20 in PBS) for 2 h prior to the addition of Fc-TEM-1 fusion protein, a soluble endosialin generated by fusion of the N-terminal leader sequence and the entire extracellular domain of endosialin to a murine gamma heavy chain. Following 1 h incubation at room temperature, the plates were washed and the HRP-goat anti-human IgG (H+L) antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) was added for 1 h. Color development was assessed using the SureBlueTM TMB Peroxidase Substrate (KPL, Gaithersburg, Md.). Both BSA and a human isotype control antibody were used as negative controls. Fc-TEM-1 did not bind BSA, nor did the human isotype bind any of the ECM proteins (data not shown).
As shown in FIG. 4A, the Fc-TEM1 bound to fibronectin and collagen I and IV in a dose dependent manner while no binding was observed within the entire dose range to LN or VN. Interestingly, while Fc-TEM1 bound collagen, no detectable binding to gelatin (heat-denatured collagen) was observed. None of four murine isotype IgG antibodies tested could bind to any of the ECM proteins, ruling out the possibility that the interactions were mediated by murine Fc backbone of the Fc-TEM 1 fusion protein (data not shown). A fusion protein containing the murine gamma heavy chain and only the lectin domain of endosialin also binds FN (data not shown).
To confirm selectivity of the Fc-TEM1 and ECM protein interaction, Fc-TEM1 was applied to ELISA plates coated with different purified proteins. Antigen-specific ELISAs were performed by coating TP Immunomini ELISA plates with 1 ug/ml STEB (Staphylococcus enterotoxin B vaccine), 2 ug/ml bovine gamma globulin, 2 ug/ml tumor-associated 90 kD glycoprotein antigen expressed on most melanoma cells (TA90), 2 ug/ml hen egg lysozyme, 1:500 dilution of tetanus toxoid, 1% BSA, 0.2 ug/ml human mesothelin, 2 ug/ml ovalbumin (OVA), 1 ug/ml human GM-CSF, 2 ug/ml goat IgG, 2 ug/ml mouse IgG dissolved in bicarbonate coating buffer (pH 9.6) (Sigma) overnight at 4° C. The plates were washed three times with washing buffer (containing 0.5% TWEEN-20) blocked with 1× assay buffer for 2 hours at room temperature and ELISA was performed as described above. As shown in FIG. 4B, Fc-TEM1 did not bind to any of the proteins tested except for the anti-mouse Fc used as a positive control.
Example 5
Inhibition of TEM1 Binding to Human Plasma Fibronectin
96-well plates were pre-coated with Fibronectin (FN), and the ability of anti-TEM-1 antibodies to block Fc-TEM-1 mediated adhesion was assessed by ELISA. Briefly, the FN-coated plate was blocked with assay buffer (0.5% BSA, 0.5% Tween-20 in PBS) for 2 h prior to the addition of fusion proteins. Fc-TEM-1 was pre-incubated for 1 h at 4° C. with the antibodies M4 (a humanized anti-endosialin antibody described as ES1 in U.S. Pat. Publication No. 20060239911), human isotype (HuIgG), or anti-TEM-1 antibody raised in rabbits (RbtTEM1). M4 does not bind to species homologs of endosialin with the exception of nonhuman primates. The binding epitope for M4 has been mapped to the extracellular lectin domain of endosialin. The protein/antibody complex was added to the FN-coated plate and allowed to adhere for 1 h at room temperature at which time the plates were washed and the HRP-goat anti-human IgG (H+L) antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) was added for 1 h. Color development was assessed using the SureBlueTM TMB Peroxidase Substrate (KPL, Gaithersburg, Md.). As shown in FIG. 6, M4 suppressed Fc-TEM1 binding to fibronectin, whereas a non-specific control (HuIgG) did not suppress binding. RbtTEM1 also suppressed Fc-TEM1 binding to fibronectin (data not shown).
Example 6
Endosialin Mediates Adhesion to Fibronectin
CHO-TEM1 cells stably expressing endosialin (verified by FACS with M4 antibody; data not shown) were generated. CHO-K1 cells were maintained in RPMI supplemented with L-glutamine, 1% minimal essential amino acids, Sodium pyruvate, Non-Essential amino acids, and 10% heat-inactivated FBS (Invitrogen, Carlsbad, Calif.). CHO-K1 cells (3E6) (ATCC, Manassas, Va.) were electroporated with 10 ug linearized plasmid DNA in a 0.4 mm electroporation cuvette. A pulse of 170V/1000 uF was delivered using a GENE PULSER (BioRad, Hercules, Calif.). Electroporated cells were allowed to recover for 24 hours after which Blasticidin (5 ug/ml)-resistant clones were selected. Endosialin expression was verified by FACS and cells were sorted for high expression.
Cells (1.5×105 cells/well) were washed and suspended in PBS containing Mg2+/Ca2+, and added in quadruplicate to a 96-well-plate coated with Fibronectin and allowed to adhere for 1 h. Where indicated, cells were pre-incubated with antibody (100 ug/mL) M4 or human isotype (IgG) for 1 h prior to the start of the assays. After the cells were allowed to adhere the plate was washed 5 times with PBS and viability was measured using CellTiter-GloTM (Promega, Madison, Wis.). FIG. 9B shows that over-expression of endosialin results in increased cell binding to fibronectin, which can be blocked by endosialin inhibitors such as antibody M4, in contrast to controls such as nonspecific IgG.
Example 7
Endosialin Binding to Fibronectin and Fibronectin Fragments
Fibronectin is a large complex glycoprotein that exists as a dimer covalently linked by a disulfide bridge at the C-terminus of the protein (Ruoslahti et al. (1981) J. Biol. Chem., 256:7277-7281; Wierzbicka-Patynowski & Schwarzbauer (2003) J. Cell Sci., 116:3269-3276; Magnusson & Mosher (1998) Arterioscler. Thromb. Vasc. Biol., 18:1363-1370). Fibronectin fragments either derived from enzymatic degradation or alternative splicing have been reported to be associated with certain disease states and possess distinct biological functions (Magnusson & Mosher (1998) Arterioscler. Thromb. Vasc. Biol., 18:1363-1370; Labat-Robert (2002) Semin. Cancer Biol., 12:187-195; Homandberg (1999) Front Biosci., 4:D713-730).
The ability of Fc-TEM-1 to bind to different fibronectin fragments was assessed. Equimolar amounts of proteins were diluted in coating buffer (50 mM Carbonate-bicarbonate, pH9.4), added to an ELISA plate (Greiner Bio-one, Monroe, N.C.) and incubated overnight at 4° C. All plates were blocked with assay buffer (0.5% BSA, 0.5% Tween-20 in PBS) for 2 h prior to the addition of Fc-TEM-1. Following 1 h incubation at room temperature, the plates were washed and the HRP-goat anti-human IgG (H+L) antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) was added for 1 h. Color development was assessed using the SureBlueTM TMB Peroxidase Substrate (KPL, Gaithersburg, Md.). To assess the integrity of coated fibronectin proteins, the rabbit polyclonal antibody directed against fibronectin (FN Ab) was used to detect that all FN fragments were recognizable and coated evenly.
The full length human plasma purified Fibronectin and 120 kDa cell attachment FN-fragment proteins were purchased from Chemicon Intl. (Temucula, Calif.), proteolytic 30 kDa, 45 kDa, 70 kDa fragments from Sigma (St. Louis, Mo.), and the recombinant human fibronectin fragments 2 and 4 (FN2 and FN4, respectively) from R&D Systems (Minneapolis, Minn.).
As shown in FIG. 7A, Fc-TEM1 binds the amino terminal 70 kDa fragment of FN and its proteolytic cleavage products (45 kDa and 30 kDa fragments). The extent of binding varied among the different fragments and was less than that seen with whole FN. In contrast, Fc-TEM1 did not bind the 120 kDa FN fragment or the recombinant fragments Fn2 or Fn4. This lack of binding was unlikely due to uneven coating or degradation since all FN fragments were strongly detected by an anti-FN polyclonal antibody. This is evidence that the FN domain involved in interaction with endosialin resides within the 70 kDa amino terminal portion. To determine whether the reduced binding capacity upon further digestion of the 70 kDa fragment indicates that Fc-TEM1 binds to a region located in close proximity to the proteolytic cleavage site or that TEM1 recognizes conformationally dependent epitopes within the amino terminus of FN that is altered after further digestion, the ability of Fc-TEM 1 to bind reduced forms of FN proteins was examined. While anti-FN antibody was able to recognize reduced FN, indicating equivalent coating, the binding of Fc-TEM1 was completely ablated as shown in FIG. 7B. Similar to whole FN, anti-endosialin antibody M4 blocked Fc-TEM 1 binding to the 70 kDa fragment in a dose-dependent manner (FIG. 7C), while an isotype control antibody had no effect (FIG. 7D). These results indicate that Fc-TEM1 recognizes conformationally dependent epitopes located within the amino terminus of FN that can be impaired upon further proteolytic degradation.
Example 8
Association of Cell Surface Fibronectin with Endosialin
CHO-TEM1 cells stably expressing endosialin (verified by FACS with M4 antibody; data not shown) were generated. CHO-K1 cells were maintained in RPMI supplemented with L-glutamine, 1% minimal essential amino acids, Sodium pyruvate, Non-Essential amino acids, and 10% heat-inactivated FBS (Invitrogen, Carlsbad, Calif.). CHO-K1 cells (3E6) (ATCC, Manassas, Va.) were electroporated with 10 ug linearized plasmid DNA in a 0.4 mm electroporation cuvette. A pulse of 170V/1000 uF was delivered using a GENE PULSER (BioRad, Hercules, Calif.). Electroporated cells were allowed to recover for 24 hours after which Blasticidin (5 ug/ml)-resistant clones were selected. Endosialin expression was verified by FACS and cells were sorted for high expression.
The level of cell surface FN was examined by flow cytometry in parental CHO-K1 and CHO-TEM1 cells using a polyclonal anti-FN antibody. Cells were harvested in Cell Dissociation Buffer (Invitrogen, Carlsbad, Calif.), washed, and resuspended in ice-cold PBS+1% FBS. Cells were incubated for 1 hour on ice with primary antibody, M4 (10 ug/ml), washed, and incubated with FITC-conjugated goat-anti-human secondary antibody (Southern Biotech, Birmingham, Ala.) and analyzed on an EASYCYTE Flow Cytometer (Guava Technologies, Hayward, Calif.). 15-20% higher levels of surface FN in CHO-TEM1 cells compared to CHO-K1 cells were observed constantly (data not shown). Association of cell surface FN with endosialin was examined.
Using an anti-FN antibody, FN was immunoprecipitated from both CHO-K1 and CHO-TEM1 lysates, followed by Western blot using the same antibody or an anti-TEM1 antibody (M4). Cells (10E7) were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS]) supplemented with Complete Mini Protease Inhibitor Cocktail (Roche Diagnostics, Indianapolis, Ind.) and centrifuged at 13,000 rpm for 15 min to remove debris. Protein G Sepharose 6 Fast Flow Beads (Amersham Biosciences, Piscataway, N.J.) were washed 3 times with PBS and anti-FN antibody (1 ug) was captured by gentle rocking at 4° C. Equal amounts of protein per sample were pre-cleared by the addition of unbound Protein G. After 2 hours of incubation, the Protein G was removed and the supernatant was added to the antibody-Sepharose complex and incubated overnight at 4° C. After extensive washing with RIPA buffer, the bound protein was removed by boiling for 10 minutes in NuPAGE® LDS sample buffer (Invitrogen) containing 5% β-mercaptoethanol. Proteins were separated using SDS-polyacrylamide gel electrophoresis on a 4-12% Bis-Tris gel (Invitrogen) and transferred to PVDF membrane. Immunoblotting was conducted using rabbit polyclonal antibodies specific for fibronectin (Abcam, Cambridge, Mass.) or endosialin (Morphotek, Inc., Exton, Pa.), detected with a goat-anti-rabbit HRP-conjugated antibody, and visualized using SUPERSIGNAL West Pico chemiluminescent substrate (Pierce, Rockford, Ill.). The integrity and purity of soluble Fc-TEM 1 was also monitored by Western blot. Protein (5 ug) was boiled for 5 min in 4×NUPAGE LDS Sample Buffer (Invitrogen) containing 5% β-mercaptoethanol, subjected to electrophoresis on a NUPAGE 4-12% Bis-Tris gel (Invitrogen), and transferred to PVDF membrane and immunoblotting was performed as described above.
It was found that FN can immunoprecipitate endosialin from CHO-TEM1 lysates (data not shown). In contrast, in cell lysates immunoprecipitated with a normal IgG that did not pull down FN, no endosialin could be detected (data not shown). At least two different approaches (ELISA and coimmunoprecipitation) provide strong evidence of FN and endosialin interaction. Similar results were obtained using HEK-293T cells ectopically expressing endosialin (data not shown).
Example 9
Cells Expressing Endosialin Cultured on MATRIGEL Form Web-Like Structures
While no differences in growth or survival were observed between parental CHO-K1 and CHO-TEM1 cells cultured on plastic surface, a drastically different morphology was observed when these cells were cultured on MATRIGEL. Parental CHO-K1 cells grew into isolated cell clusters with minimal protrusions after 2 days of culturing (FIG. 8, top panels), while CHO-TEM1 cells grew into clusters forming a web-like network (FIG. 8, bottom left panel). In addition, CHO-TEM 1 cells within the cluster exhibited protrusions reaching out to other clusters (FIG. 8, bottom right panel). Over time, CHO-TEM1 cells but not CHO-K1 cells moved closer to each other to form larger clusters (data not shown).
Example 10
Endosialin Expression Increases Cell Adhesion to Fibronectin, and the 70 kD or 30 kD N-Terminus of Fibronectin
To assess adhesion to FN fragments, equimolar amounts of protein fragments were pre-coated overnight and then blocked for 2 h with PBS containing 10 mg/mL BSA. MATRIGEL served as a positive control. CHO-K1 or CHO-TEM1 cells (1.5×105 cells/well) harvested in cell dissociation buffer were washed and suspended in PBS containing Mg2+/Ca2+, and added in quadruplicate to an ECM Cell Adhesion Array Kit (Millipore) or plated on individually coated FN, LN, Gel, and Col I plates (BD Biosciences) and allowed to adhere for 1 h. Following incubation, each well was washed 5 times with PBS and viability was measured using CellTiter-Glo®. Where indicated, cells were pre-incubated with antibody for 1 hour prior to the start of the assay. As shown in FIG. 9A, the number of adherent CHO-TEM 1 cells was 6-fold higher than the number of parental CHO-K1 cells in wells coated with FN. No significant differences in adhesion between CHO-K1 and CHO-TEM1 on surfaces coated with laminin or vitronectin were observed, while adhesion to collagens and tenascin was too weak to assess any valuable differences (FIG. 9A). Pretreatment of CHO-TEM1 cells with M4 antibody resulted in 50% reduction of TEM1-FN-dependent cell adhesion, while IgG control antibody had no effect (FIG. 9B). M4 antibody treatment did not affect FN-dependent, endosialin-independent cell adhesion (baseline adhesion) of parental CHO-K1 cells.
Plates were precoated with equimolar amounts of whole FN, FN proteolytic fragments, and MATRIGEL. CHO-TEM1 cells showed a 3- to 5-fold increased adhesion to FN, 70 kDa, and 30 kDa fragments compared to parental CHO-K1 cells, whereas no significant adhesion was seen to 45 kDa or Fn2 fragments. CHO-TEM1 cells bound MATRIGEL five times better than CHO-K1 (FIG. 9C). These data indicate that endosialin enhances the adhesion of cells to extracellular matrices and that the amino terminus of FN is involved with these interactions.
Example 11
Endosialin Binds to Collagen I and M4 Inhibits This Binding
A 96-well plate pre-coated with Collagen I was used to assess M4 ability to block Fc-TEM-1 binding. The plate was blocked with assay buffer (0.5% BSA, 0.5% Tween-20 in PBS) for 2 h prior to the addition of protein at the indicated concentration (μg/mL). Fc-TEM-1 was pre-incubated for 1 h at 4 C with the antibodies M4 or human isotype (Human IgG). The protein/antibody complex was then added to the Col I-coated plate and allowed to adhere for 1 h at room temperature at which time the plates were washed and the HRP-goat anti-human IgG (H+L) antibody (Jackson Immunoresearch Laboratories, West Grove, Pa.) was added for 1 h. Color development was assessed using the SureBlueTM TMB Peroxidase Substrate (KPL, Gaithersburg, Md.). As shown in FIG. 10, over-expression of endosialin results in increased cell binding to COL I which can be blocked by endosialin inhibitors such as M4, in contrast to controls such as nonspecific IgG. RbtTEM1 also suppressed Fc-TEM1 binding to Col I (data not shown).
Example 12
Endosialin Increases Cell Adhesion to Collagen
CHO-K1 or CHO-TEM1 cells (1.5×105 cells/well) were washed and suspended in PBS containing Mg2+/Ca2+, and added in quadruplicate to a 96-well plate coated with Collagen I and allowed to adhere for the indicated time points. After the cells were allowed to adhere the plate was washed 5 times with PBS and viability was measured using CellTiter-Glo®. As shown in FIG. 11, over-expression of endosialin results in increased cell binding to COL I.
Example 13
Cell Migration Assay
The BD BioCoat Tumor Invasion SystemTM (BD Bioscience) and Human Fibronectin Cell Culture inserts (BD Bioscience) were used to assess TEM 1-mediated migration of cells. Cells were harvested with nonenzymatic cell dissociation buffer and diluted to a concentration of 4E5 cells/ml in growth media supplemented with 2% fetal bovine serum (FBS), and 500 ul of cell suspension was added to the top chamber of the membrane insert. To create a gradient, growth media containing 20% FBS was added to the bottom chamber. Cells were incubated for 48 hours, after which the insert was removed and cells that had migrated through the coated membrane were counted using CELLTITER-GLO (Promega). Cells were pretreated with antibody as indicated in the description of FIG. 12 and migration was assessed in the continual presence of antibody. To examine the formation of tubules on MATRIGEL, cells (8E4 cells/well) were added to a 96-well plate coated with MATRIGEL (BD Bioscience), incubated overnight and photographed at 200-400× magnification.
As shown in FIG. 12A, CHO-K1 cells exhibited modest cell migration, whereas CHO-TEM1 cells showed >10-fold enhanced migration. M4 antibody treatment, but not control IgG, abolished CHO-TEM1 cell migration. Similar results were observed in migration experiments using transwell chambers coated with FN (FIG. 12B).
Example 14
Endosialin Increases MMP-9 Activity
Endosialin, MMP-2, and COL IV have been shown to colocalize in tissue areas characterized by finger-like protrusions of early angiogenic processes (Virgintino et al. (2007) Angiogenesis, 10:35-45). To assess MMP activity, cells were seeded in a 6-well plate and serum starved for 48 h. The culture supernatant was collected and clarified by centrifugation (13,000 rpm, 15 min.) at 4° C. to remove any debris. Equal amounts of protein were subjected to gelatin and casein zymography under nonreducing conditions (Invitrogen) according to the manufacturer's protocol. Positive controls human MMP-2 and 9 (Chemicon, International) were used to indicate the migration of MMP-2 and MMP-9 and used as a reference for our CHOK1 and CHO-TEM-1 supernatants. As shown in FIG. 13, MMP-9 activity was significantly enhanced in CHO-TEM1 cells compared to parental CHO-K1 cells. The enhanced MMP-9 activity correlated with increased secretion of MMP-9 protein in the supernatant of CHO-TEM 1 cells as measured by a MMP-specific ELISA (data not shown). These data indicate that induced MMP-9 secretion contributes to the enhanced migration capability of CHO-TEM1 cells through MATRIGEL and FN-coated transwells demonstrated herein.
Example 15
Endosialin Increases β-integrin Activity
Integrins (e.g., α4β1, α5β1) are well-characterized receptors that mediate FN-dependent cell adhesion (Wierzbicka-Patynowski & Schwarzbauer 2003; Magnusson & Mosher 1998). In addition, an unidentified cellular receptor has been functionally described that binds the N-terminal 70 kDa region of FN (McKeown-Longo & Mosher (1983) J. Cell Biol., 97:466-472) and is required to expose the cryptic integrin-binding site (RGD motif) of soluble FN involved with FN-integrin and FN-FN interactions (Tomasini-Johansson et al. (2006) Matrix Biol., 25:282-293; McKeown-Longo & Mosher (1985) J. Cell Biol., 100:364-374). Endosialin is identified herein as a novel receptor that interacts with the N-terminal 70 kDa FN region and enhances FN-dependent cell adhesion. The enhanced FN binding measured in these cellular systems in vitro could be the result of sequential interaction with endosialin and integrins.
Human embryonic kidney 293 (HEK293) cells were transfected with a vector expressing endosialin or mock cDNA. Cells were confirmed to express cell surface endosialin (293TEM1) while those transfected with mock (293T) did not. Cells were the tested for the ability to upregulate integrin expression and activity in the presence of antibody M4. FIG. 14B shows that over-expression of endosialin results in increased integrin β1 activity relative to control cells. FIG. 14A shows that cell surface integrin β1 expression is not changed. Treatment of cells with the endosialin inhibitor M4 resulted in suppressed integrin activity while no effect on cell surface levels were observed (FIG. 14B). While not wishing to be bound to any one theory, the interaction between endosialin and the N-terminal 70 kDa fragment of soluble FN may be responsible for initiating the assembly of FN into a multimeric high affinity form able to bind integrins.
Example 16
M4.1 Recognizes Unreduced Human TEM-1 but not Murine TEM-1
CHO-TEM-1 cells, mouse 2H11 cells, parental CHO-K1 cells, mouse NS0 cells, and mouse MS1 cells were cultured in complete RPMI1640 (RPMI1640; sodium pyruvate; nonessential amino acids; L-glutamine, and FBS; Invitrogen Corp.). Human primary pericytes were cultured in Perciyte Medium (500 ml of basal medium (Cat# 1201), 10 ml (2%) of fetal bovine serum (FBS, Cat. No. 0025), 5 ml of pericyte growth supplement (PGS, Cat. No. 1252) and 5 ml of penicillin/streptomycin solution (P/S, Cat. No. 0503); Invitogen Corp.). Cells were grown at 37° C. and 5% CO2 in a humidified incubator. Cells were deadhered with TrypLETM Select (Invitrogen Corp., catalog no. 12563-011), washed, and counted. Cells were lysed at 2×107 cells in RIPA Lysis buffer containing protease inhibitors and incubated on ice for 10 minutes. Insoluble material was pelleted at 10,000×G for 10 minutes at 4° C., and supernatants were transferred to fresh tubes. Aliquots were mixed with an equal volume of 2× protein loading buffer with or without 10% 2-mercaptoethanol (reducing agent). 15 μL (1.5×105 cells) of lysate were loaded onto a 15 well 4-12% Bis/Tris SDS-PAGE gel and electrophoresed for 30 minutes at 200V in MES running buffer. The gel was electroblotted onto PVDF, then blocked for 1 hour at room temperature with rocking in 5% milk-TBST (5% M-TBST). M4.1 blots were probed with M4.1 in 5% M-TBST at 3.3 ug/mL overnight at 4° C.
Rabbit anti-tem-1 polyclonal antibody blots were probed with a 1:300 dilution (4.5 mg/ml stock Lot #: NB487-76) of antibody in 5% M-TBST overnight at 4° C. Antibody M4.1, like antibody M4, is a humanized antibody to human endosialin. Membranes were washed 5 times for 5 minutes each with 30 mL TBST at room temperature. HRP-conjugated goat anti-human IgG (H+L) (Jackson Immuno, 1 mg/mL stock) was diluted 1:20,000 in 5% M-TBST as a secondary antibody for probing the M4.1 blots for 30 minutes at room temperature. HRP-conjugated goat anti-rabbit (H+L) secondary antibody was used to blot the polyclonal anti-TEM-1 blots for 30 minutes at room temperature. Membranes were washed 5 times for 5 minutes each with 30 mL TBST at room temperature. Signal was detected by chemiluminescence using the Femto Western Blot Detection System (Pierce) as per manual.
As illustrated in FIG. 15, M4.1 recognizes unreduced human TEM-1 in CHO-TEM-1 cells and human primary pericytes but not murine TEM-1 (SEQ ID NO:2) in mouse 2H11 cells (FIG. 15). Rabbit polyclonal against human TEM-1 (rabPAb TEM-1) recognizes human TEM-1 in CHO-TEM-1 cells and human pericytes, but also murine TEM-1 in mouse 2H11 cells. Neither M4.1 nor rabPAb TEM-1 reacted against lysates from parental CHO-K1 cells or mouse NS0 and MS1 cells due to lack of TEM-1 expression in these cells. Only rabPAb TEM-1 reacted with reduced human TEM-1, albeit to a lesser extent when compared to unreduced TEM-1.
The present invention is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.

## Claims

1. A method for identifying agonists or antagonists of the interaction of endosialin with a ligand for endosialin, said ligand for endosialin comprising collagen I, collagen IV, fibronectin, the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, or the 30 kDa heparin binding fragment of fibronectin, said method comprising contacting endosialin with said ligand for endosialin in the presence and absence of a test compound and quantifiably measuring the interaction of endosialin with the ligand, wherein an increase or a decrease in the level of interaction of endosialin with said ligand in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of the interaction of endosialin with said ligand.

2. The method of claim 1, wherein the endosialin is bound to a cell membrane, a cell membrane fragment, or an artificial lipid bilayer.

3. The method of claim 2, wherein the wherein the cell membrane or cell membrane fragment is from a mammalian cell.

4. The method of claim 3, wherein the mammalian cell is a stable cell or stable cell line expressing endosialin.

5. The method of claim 1, wherein the ligand is collagen I, collagen IV or fibronectin.

6. The method of claim 1, wherein the endosialin is bound to a solid support.

7. The method of claim 1, wherein the ligand is bound to a solid support.

8. A method for identifying an agonist or antagonist that modulates cell adhesion comprising
(a) contacting a mammalian cell expressing endosialin with a ligand for endosialin in the presence and absence of a test compound, said ligand for endosialin comprising collagen I, collagen IV, fibronectin, the 70 kDa N-terminal fragment of fibronectin, the 45 kDa gelatin binding fragment of fibronectin, or the 30 kDa heparin binding fragment of fibronectin; and
(b) quantifiably measuring cell adhesion,
wherein an increase or decrease in the level of adhesion in the presence of the test compound indicates that the test compound is an agonist or antagonist, respectively, of cell adhesion.