WO2012149018A1

WO2012149018A1 - Mer tyrosine kinase inhibitors and methods of making and using the same

Info

Publication number: WO2012149018A1
Application number: PCT/US2012/034994
Authority: WO
Inventors: Douglas K. GRAHAM; Susan Sather; Deborah Deryckere; Jorge DI PAOLA; Brian BRANCHFORD; Gary Brodsky
Original assignee: The Regents Of The University Of Colorado
Priority date: 2011-04-25
Filing date: 2012-04-25
Publication date: 2012-11-01

Abstract

Disclosed are inhibitors of the Mer receptor tyrosine kinase (RTK) and methods of using such inhibitors in a variety of therapeutic approaches in the areas of cancer therapy and anti-thrombosis (anti-clotting) therapy.

Description

MER TYROSINE KINASE INHIBITORS AND METHODS OF MAKING AND USING THE SAME

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 61/478,930 filed April 25, 2011, which is incorporated herein by reference.

TECHNICAL FIELD

The present invention generally relates to novel inhibitors of the Mer receptor tyrosine kinase (RTK) and to the use of such inhibitors in compositions and therapeutic approaches in the areas of anti-thrombosis (anti-clotting) therapy and cancer therapy.

BACKGROUND OF INVENTION

Mer is a transmembrane receptor tyrosine kinase that is likely the human homologue of the chicken retroviral gene, v-eyk, which causes many types of cancer in chickens. The human Mer gene and the mouse Mer gene and complementary DNA have been sequenced and characterized, and the expression of Mer has been profiled in cell lines and tissues. (Graham, et al., Cell Growth and Differentiation, 5:647-657 (1994); Graham, et al, Oncogene 10:2349-2359 (1995); and, U.S. Patent No. 5,585,269). The Mer receptor tyrosine kinase, initially cloned from a human B lymphoblastoid cell line, is expressed in a spectrum of hematopoietic, epithelial, and mesenchymal cell lines.

Interestingly, while the ribonucleic acid (RNA) transcript of Mer is detected in numerous T and B lymphoblastic cell lines, Mer RNA is not found in normal human thymocytes, lymphocytes or in phytohaemagglutinin/ phorbol myristate acetate (PMA/PHA) stimulated lymphocytes. Mer is composed of two immunoglobulin domains and two fibronectin III domains in the extracellular portion, and a tyrosine kinase domain in the intracellular portion. (Graham et al., (1994), supra and Graham et al., (1995), supra). Human Mer is known to be transforming and anti-apoptotic, and Mer overexpression has been linked to a number of different human cancers including subsets of B and T cell leukemia, lymphoma, pituitary adenoma, gastric cancer, and rhabdomyosarcoma.

Mer is related to two other receptor tyrosine kinases, Axl and Tyro-3. Axl, Mer, and Tyro-3 are all expressed in a spectrum of hematopoeitic, epithelial, and mesenchymal cell lines. Each protein has been shown to have the capability to transform cells in vitro. Axl, Mer, and Tyro-3 are all activated by the ligand Gas6. Gas6 is structurally similar to Protein S, a cofactor for anticoagulant Protein C, and shares 48% protein identity with Protein S, which has also been shown to be a binding ligand of at least Mer and Tyro-3. Gas6 plays a role in coagulation, (Angelillo-Scherrer, et al., Nature Medicine 7:215-21 (2002)), and Gas6 antibodies may be used to protect wild type mice against fatal thromboembolism. (Angelillo-Scherrer, et al., (2002), supra). Mice with an inactivated Gas6 gene {i.e., Gas6 knockout) have platelet dysfunction that prevents venous and arterial thrombosis. These knockout mice are protected against (have decreased mortality against) fatal collagen/epinephrine induced thromboembolism and inhibited ferric chloride-induced thrombosis in vivo. Gas6 amplifies platelet aggregation and secretion response of platelets to known agonists. (Chen, et al., Aterioscler. Thromb. Vase. Biol. 24: 1118-1123 (2004)). The platelet dysfunction caused by Gas6 is thought to be mediated through Axl, Mer, and/or Tyro-3. In addition, mice with an inactivated Mer gene, inactivated Axl gene, or an inactivated Tyro-3 gene, all have platelet dysfunction, as well as decreased mortality against thromboembolism (by both statis-induced thrombosis in the inferior vena cava and by collagen-epinephrine induced pulmonary embolism. (Angelillo-Scherrer, et al., J. Clin Invest. 115:237-246 (2005)). Therefore, in addition to its association with neoplastic disease, Mer is also involved in blood clotting.

Various types of thrombosis and the complications associated with thrombosis represent a major cause of morbidity and death in the world. Although there are a variety of agents to thin the blood, all have the potential for bleeding complications, and with the exception of heparin (which itself cannot be tolerated by many patients), are largely irreversible.

Malignant cellular growth or tumors (cancer) are also a leading cause of death worldwide. Accordingly, the development of effective therapy for cardiovascular and neoplastic disease is the subject of a large body of research. Although a variety of innovative approaches to treat and prevent such diseases have been proposed, these diseases continue to have a high rate of mortality and may be difficult to treat or relatively unresponsive to conventional therapies. Tyrosine kinases as mediators of cell signaling, play a role in many diverse physiological pathways including cell growth and

differentiation. Deregulation of tyrosine kinase activity can result in cellular

transformation leading to the development of human cancer. Of the cancer targets extensively studied in the past ten years, nearly one third are tyrosine or other kinases. Therefore, inhibition of specific cancer-associated tyrosine kinases has emerged as an important approach for cancer therapy. At least five of the anti-cancer therapies approved in the past five years have been directed against receptor tyrosine kinases (RTKs). In fact, many cancer treatment protocols now use a combination of traditional chemotherapy drugs and novel biologically targeted agents, several of which inhibit tyrosine kinase activity or downstream signaling pathways. For example, a small molecule drug that inhibits the abl tyrosine kinase has led to significant improvement in outcomes for patients with chronic myelogenous leukemia. Inhibitors of other tyrosine kinases, including the Flt-3, EGFR, and PDGF receptor tyrosine kinases are also in clinical trials.

Therefore, there is a continued need for new therapies that can effectively target and prevent or treat these diseases. Because it is generally the case in cancer therapy that no single agent can successfully treat a patient, new agents that are developed may be successfully used in combination with other new or known anti-cancer agents to affect the best outcome for patients.

SUMMARY OF INVENTION

One embodiment of the invention relates to a Mer inhibitor, wherein the Mer inhibitor comprises a Mer splice variant, a homologue or protein mimetic of the same or a fusion protein including the same. The Mer splice variant comprises at least a portion of the extracellular domain of a Mer receptor tyrosine kinase (Mer RTK). The Mer splice variant inhibits the activation of Mer by inhibiting or preventing Mer receptor dimerization, trimerization or formation of a receptor-protein complex. The Mer splice variant may act to inhibit Mer activation by mechanisms including direct binding of the Mer splice variant to a Mer receptor, inhibition of Axl and/or Tyro3 activity by heterodimerization with Axl and/or Tyro3, and/or binding the ligand Gas6 thereby acting as a ligand "sink" preventing Gas6 binding to Mer, Axl, and/or Tyro3 (TAM) receptors. The Mer splice variant can be used as a biologic therapeutic agent for the inhibition/sequestration of Mer, Axl, and/or Tyro3 ligands and accordingly, for the treatment of Mer, Axl and/or Tyro 3-overexpressing cancers, including, but not limited to, lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, gliomas, melanoma, prostate cancer, breast cancer, gastric cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer.

In one aspect, the Mer inhibitor is a Mer splice variant comprising, consisting essentially of, or consisting of a truncated isoform of Mer containing 3 of the 4 extracellular domains (1 of the 2 fibronectin type III domains and 2 Ig domains) that are found in the full-length Mer protein, which is transcribed from an alternatively-spliced

Mer gene transcript, which is formed when exon 7A of the Mer gene is inserted between exons 7 and 8. Exon 7 A contains an in- frame stop codon. In one aspect, the Mer inhibitor comprises, consists essentially of, or consists of SEQ ID NO:2 (the amino acid sequence of the Mer RTK splice variant). In a related aspect, the Mer inhibitor is encoded by the cDNA sequence comprising, consisting essentially of, or consisting of SEQ ID NO: 1.

In any of the above aspects of the invention, the Mer inhibitor can comprise an amino acid sequence that is at least 80% identical, at least 90% identical, or at least 95% identical, to SEQ ID NO:2. In one aspect, the Mer inhibitor comprises an amino acid sequence of SEQ ID NO:2.

In one aspect, the Mer inhibitor of the invention inhibits endogenous Mer receptor tyrosine kinase activity by at least 50%. In another aspect, the Mer inhibitor of the invention inhibits the activity of an endogenous Mer receptor tyrosine kinase by at least 60%. In another aspect, the Mer inhibitor of the invention inhibits the activity of an endogenous Mer receptor tyrosine kinase by at least 70%. In another aspect, the Mer inhibitor of the invention inhibits the activity of an endogenous Mer receptor tyrosine kinase by at least 80%.

Another embodiment of the invention relates to a composition comprising, consisting essentially of, or consisting of the Mer inhibitors of the invention. In one aspect of this embodiment, the composition further comprises a pharmaceutically acceptable carrier. In another aspect, the composition further comprises at least one therapeutic agent for the treatment of cancer. In another aspect, the composition further comprises at least one therapeutic agent for the treatment of a clotting disorder.

Another embodiment of the invention relates to a method of treating or preventing a clotting disorder in an individual, comprising administering to the individual any of the Mer fusion proteins or compositions described herein. In one aspect, the disorder is selected from the group consisting of: thrombophilia, thrombosis and thrombo-embolic disorder. In one aspect, the disorder is thrombophilia. In one aspect, the individual is taking a medication that increases the risk of clotting in the individual. In one aspect, the individual has a disease associated with thrombosis. In one aspect, the disease is selected from the group consisting of: cancer, myeloproliferative disorders, autoimmune disorders, cardiac disease, inflammatory disorders, atherosclerosis, hemolytic anemia, nephrosis, and hyperlipidemia. In one aspect, the individual is undergoing surgery, an interventional or cardiac procedure, is experiencing or has experienced trauma, or is pregnant.

Another embodiment of the present invention relates to a method of treating cancer in an individual, comprising administering to the individual any of the Mer inhibitors, or the compositions described herein. In one aspect, the cancer is a Mer-positive cancer. In another aspect, the cancer is an Axl-positive cancer. In another aspect, the cancer is a Tyro-3 -positive cancer. In one aspect, the cancer is selected from: glioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, colorectal cancer, endometrium cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, testicular tumor, Wilms' tumor, Ewing's tumor, bladder carcinoma, angiosarcoma,

endotheliosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland sarcoma, papillary sarcoma, papillary adenosarcoma, cystadenosarcoma, bronchogenic carcinoma, medullary carcinoma, mastocytoma, mesotheliorma, synovioma, melanoma,

leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, retinoblastoma, oligodentroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma,

craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, base cell carcinoma, fibrosarcoma, myxoma, myxosarcoma, liposarcorna, chondrosarcoma, osteogenic sarcoma, leukemia and metastatic lesions secondary to these primary tumors. In a related aspect, the cancer is a leukemia or lymphoma and in a preferred embodiment, the cancer is a myeloid leukemia or a lymphoid leukemia or lymphoma. In another preferred embodiment, the cancer is a non-small cell lung cancer (NSCLC). In another preferred embodiment, the cancer is an astrocytoma or glioblastoma.

Another embodiment of the invention relates to the use of any of the Mer inhibitors or compositions described herein in the preparation of a medicament for the prevention or treatment of a clotting disorder. Another embodiment of the invention relates to the use of any of the Mer inhibitors or compositions described herein for use in the prevention or treatment of a clotting disorder. Another embodiment of the invention relates to methods for treating an individual suffering from, or at risk of uncontrolled bleeding comprising administering an effective amount of the use of any of the Mer inhibitors or compositions described herein to the individual in need thereof.

Another embodiment of the invention relates to the use of any of the Mer inhibitors or compositions described herein in the preparation of a medicament for the treatment of cancer. Another embodiment of the invention relates to the use of any of the Mer inhibitors or compositions described herein for use in the treatment of a cancer. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 is a graphic representation of the MerTK gene showing exons 1-19, which encode the full length MerTK transcript. Exons 1-9 encode the extracellular domain of MerTK. Exon 10 contains the transmembrane domain and Exons 11-19 encode the intracellular domain. The Mer inhibitor transcript was derived by alternative splicing such that exon 7 A was inserted into the transcript between exons 7 and 8. Exon 7 A contains an in-frame stop codon.

Figure 2 shows alternatively spliced MerTK transcript encoding a truncated isoform consisting of 3 of the 4 extracellular domains (1 of the 2 fibronectin type III domains and 2 Ig domains) as found in the full-length protein.

Figure 3 shows the Mer isoform transcript as detected in a variety of human tissues using semi-quantitative RT-PCR. The top panel shows the Mer isoform, the middle panel shows the full-length Mer transcript, and the bottom panel shows the glyceraldehyde 3- phosphate dehydrogenase (G3PDH) serving as the loading control. The Mer isoform transcript was also detected in these tissues using the primers:

5'-TCTTCCTCCCCGCGCTCTG-3' (SEQ ID NO:7) and

5 ' -GGAGTC AACAGTAGAAGAGAG-3 ' (SEQ ID NO:6).

Figure 4 shows aggregation of human platelets treated with the Mer isoform of SEQ ID NO:2 (uppermost line) compared to control samples (second line from top) in response to fibrillar collagen (1 μg/ml) as an agonist. Decreased ATP release is also depicted in the samples treated with the Mer isoform of SEQ ID NO:2 (bottom line) compared to samples treated with vehicle only (second line from bottom).

Figure 5 is a graphical representation of mean values and standard errors derived from six individual patients showing platelet aggregation in response to fibrillar collagen from samples treated with the Mer isoform of SEQ ID NO:2 (white bar) compared to samples treated with vehicle only (black bar). */?=0.02 by paired t-test.

Figure 6 depicts platelet adhesion in a control sample when whole blood was run in a flow chamber at a flow rate of 650 sec^"1 perpendicularly across a strip of equine Type I fibrillar collagen versus a similar sample treated with 1.2 μΜ of the Mer isoform of SEQ ID NO:2.

Figure 7 is a graphical comparison of adhesion of untreated platelets to fibrillar collagen in a human whole blood microfluidic flow assay using a flow chamber at a wall shear rate of 650s^"1 (control), as compared to ex vivo samples treated with the Mer isoform of SEQ ID NO:2. Figure 8 depicts an analysis of platelet aggregate sizes in the flow assay (as described in Figure 7), comparing controls to samples treated with the Mer isoform of SEQ ID NO:2. Analysis provided by chi-square test.

Figure 9 depicts samples of washed platelets on 100 ug/mL fibrillar collagen visualized using Scanning Electron Microscopy at 1,000 and 5,000 magnification, including a PBS-treated control sample (negative control), a sample treated with the Mer isoform of SEQ ID NO:2 (iMer), and an abciximab-treated sample (positive control).

Figure 10 depicts a flow cytometric analysis, comparing the percentage of activated platelets identified by fluorescent anti-p-selectin antibodies in convulxin (CVX) and thrombin-stimulated control samples (Thromb) and in samples treated with the Mer isoform of SEQ ID NO:2 (iMer) and abciximab (ReoPro) treated samples. (n=4, analysis by paired t-test.)

Figure 11 depicts fluorescent-labeled PAC-1 binding to activated ¾¾β3 integrin in non-inhibited samples stimulated with CVX and Thromb (negative controls), in samples treated with the Mer isoform of SEQ ID NO:2, stimulated with CVX and Thromb, and in abciximab (ReoPro) samples stimulated with CVX and Thromb (positive controls).

Figure 12 shows phosphorylation of B-cell acute lymphoid leukemia cells (REH cell line) in response to Gas6 (a ligand for MerTK) in untreated and vehicle only samples, and in REH cells treated with 650 nM Mer inhibitor for 30 minutes prior to stimulation with Gas6.

Figure 13 shows phosphorylation of B-cell acute lymphoid leukemia cells (697 cell line) in response to Gas6 in untreated and vehicle only samples, and in 697 cells treated with 1.2 μηΜ Mer inhibitor for 10 and 20 minutes prior to stimulation with Gas6.

Figure 14 shows phosphorylation of β3 -integrin in human platelets in response to to Gas6 in untreated and vehicle only samples, and in human platelets treated with 1.2μΜ

Mer inhibitor for 30 minutes prior to stimulation with Gas6 for 0, 3, 5, and 10 minutes.

Figure 15 shows the aggregation of human platelets treated with the Mer isoform of SEQ ID NO:2 (uppermost line) compared to control samples (second line from top) in response to ADP (4 μΜ) as an agonist. Decreased ATP release is also depicted in the samples treated with the Mer isoform of SEQ ID NO:2 (bottom line) compared to samples treated with vehicle only.

DESCRIPTION OF EMBODIMENTS

The present invention provides inhibitors of the Mer receptor tyrosine kinase

(RTK) and methods of using such inhibitors in a variety of therapeutic approaches in the areas of anti-thrombosis (anti-clotting) therapy and cancer therapy. The present inventors describe herein Mer RTK inhibitors and have demonstrated that such therapeutic inhibitors can inhibit activation of cellular Mer. These inhibitors inhibit adhesion to collagen under physiologic flow rates, thereby decreasing both ADP- and collagen-induced platelet aggregation, and decreasing platelet spreading on collagen surfaces. As such, the inhibitors are useful as primary or secondary anti-platelet therapy for the treatment of pathologic thrombosis (i.e., as an anti-clotting agent). Previously-published studies have demonstrated that Mer receptor knock-out in mice affects platelet aggregation and clot stability but does not affect bleeding time. Thus, the Mer inhibitors of the present invention show reduced bleeding complications relative to current therapies. The Mer inhibitors of the invention also inhibit activation of Mer in cancer cells and therefore have additional therapeutic utility as anti-cancer therapies.

More particularly, the present inventors have developed inhibitors of Mer that are capable of preventing Mer activation. In one particular embodiment, the present inventors have developed Mer inhibitors that inhibit the activation of Mer in the presence of Gas6. Specifically, the inventors have demonstrated that the inhibitors of the invention can inhibit activation of membrane-bound Mer in B-cell acute lymphoid leukemia cells. Thus, the Mer inhibitors of the invention are useful in the treatment of Mer overexpressing cancers, including, but not limited to, glioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphangio sarcoma, lymphangioendotheliosarcoma, colorectal cancer, endometrium cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, testicular tumor, Wilms' tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endotheliosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland sarcoma, papillary sarcoma, papillary adenosarcoma, cystadenosarcoma, bronchogenic carcinoma, medullary carcinoma, mastocytoma, mesotheliorma, synovioma, melanoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, retinoblastoma, oligodentroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, base cell carcinoma, fibrosarcoma, myxoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, leukemia and metastatic lesions secondary to these primary tumors. In particular, these inhibitors are useful in the treatment of leukemia or lymphoma and particularly myeloid leukemia or lymphoid leukemia or lymphoma.

Mer Inhibitors of the Invention

The Mer inhibitors of the invention comprise a truncated isoform of the Mer receptor consisting of 3 of the 4 extracellular domains (1 of the 2 fibronectin type III domains and 2 Ig domains) that are found in the full-length Mer receptor tyrosine kinase protein, and homologues and mimetics thereof. These inhibitors function to inhibit activation and signaling through the Mer receptor {e.g., by preventing/blocking ligand binding or by preventing receptor dimerization, trimerization or formation of any receptor- protein complex or down-regulation of receptor expression).

The inhibitors can be further combined with other therapeutic reagents to enhance or supplement other therapeutic treatments for neoplastic and thrombotic disorders or conditions.

Reference to a "Mer inhibitor" refers to any of the Mer inhibitors described herein, and include the Mer proteins, protein variants and protein mimetics thereof described herein.

The Mer receptor is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the Mer protein represents a unique structure in its extracellular region that juxtaposes immunoglobulin (Ig) repeats and fibronectin type III (FNIII) repeats, a structure it shares with TAM (Tyro-Mer-Mer) family members, Axl and Tyro-3. Figure 1 provides a schematic illustrating the exons comprising the full length Mer receptor transcript. The extracellular Ig and FNIII motifs are believed to be important in cell adhesion and migration, and indicate a means through which the Mer oncogene contributes to tumor invasiveness and metastasis. Mer transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin Independent protein growth-arrest-specific gene 6 (Gas6). Mer activation occurs following binding of the Mer receptor to its ligand {e.g., Gas6). This interaction causes Mer dimerization and auto-phosphorylation.

Mer inhibitors useful in the invention may also include smaller portions (fragments) of the truncated isoform of the Mer extracellular domain that retain the ability to inhibit activation and signaling through the Mer receptor.

In one embodiment, a suitable Mer inhibitor of the invention includes SEQ ID NO:2. In another embodiment, a suitable inhibitor of the invention comprises, consists essentially of, or consists of fragments of SEQ ID NO:2. In any of the above- embodiments, the portion can be shorter or extend beyond position 393 to any higher position within the extracellular domain of the Mer isoform, in whole number increments.

In one embodiment, the Mer inhibitor of the invention comprises, consists essentially of, or consists of positions 1-393 of SEQ ID NO:2.

Fragments within the Mer isoform are encompassed by the invention, provided that, in one embodiment, the fragments retain Mer inhibitory activity, and provide inhibition of a biological activity of Mer or provide a therapeutic benefit to a patient. It will be apparent that, based on the knowledge of residues important for binding to Gas6 within these regions, various conservative or even non-conservative amino acid substitutions can be made, while the ability to bind to the Mer receptor and/or Gas6 is sufficient to inhibit activation and signaling through the Mer receptor.

Assays for measuring binding affinities are well-known in the art. In one embodiment, a BIAcore machine can be used to determine the binding constant of a complex between the target protein (e.g., a Mer isoform fragment) and a Mer receptor. For example, the Mer inhibitor can be immobilized on a substrate. A recombinant Mer receptor is contacted with the inhibitor bound to the substrate to form a complex. The dissociation constant for the complex can be determined by monitoring changes in the refractive index with respect to time as buffer is passed over the chip. Contacting a second compound (e.g., a Mer ligand or a different Mer protein) at various concentrations at the same time as the first Mer receptor and monitoring the response function (e.g. , the change in the refractive index with respect to time) allows the complex dissociation constant to be determined in the presence of the second compound and indicates whether the second compound is an inhibitor of the complex. Other suitable assays for measuring the binding of a receptor to a ligand include, but are not limited to, Western blot, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microarray, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry.

The Mer inhibitors useful in the present invention may also be produced as fusion proteins including any fusion partner that, when fused to the Mer isoform, or therapeutically-effective fragment thereof, does not interfere with the binding of the inhibitor to the Mer receptor or Gas6, and allows the Mer fusion protein to have a suitable half-life in vivo to be useful as a therapeutic agent in a method of the invention. In one embodiment, a Mer inhibitor of the invention can be produced as a dimer of the Mer isoform described above by expressing two copies of the Mer isoform transcript as single peptide chain connected by a linker region (e.g., a linker peptide). A variety of peptide linkers suitable for dimerizing two protein monomers are well known in the art.

Embodiments of the present invention described in more detail below pertain to isolated polypeptides, including various portions of full-length Mer isoform, and including those expressed by nucleic acids encoding Mer isoform, or a portion or therapeutically- effective variant thereof.

As used herein, reference to an isolated protein or polypeptide in the present invention, including an isolated Mer isoform, includes full-length proteins, fusion proteins, or any fragment or other homologue (variant) of such a protein. Reference to a Mer inhibitor can include, but is not limited to, the purified Mer isoform, recombinantly- produced Mer isoform, Mer isoform complexed with lipids, soluble Mer isoform, a Mer isoform fusion protein, a biologically active homologue of the Mer isoform, and an isolated Mer isoform associated with other proteins. More specifically, an isolated protein, such as the Mer isoform, according to the present invention, is a protein (including a polypeptide or peptide) that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include purified proteins, partially purified proteins, recombinantly produced proteins, and synthetically produced proteins, for example. As such, "isolated" does not reflect the extent to which the protein has been purified. The term "polypeptide" refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. As used herein, a polypeptide is said to be "purified" when it is substantially free of cellular material when it is isolated from recombinant and non-recombinant cells, or free of chemical precursors or other chemicals when it is chemically synthesized. A polypeptide, however, can be joined to another polypeptide with which it is not normally associated in a cell (e.g., in a "fusion protein") and still be "isolated" or "purified."

In addition, and by way of example, a "human Mer isoform" refers to a Mer protein (generally including a homologue of a naturally-occurring human Mer isoform) from a human (Homo sapiens) or to a Mer isoform that has been otherwise produced from the knowledge of the structure (e.g., sequence) and perhaps the function of a naturally occurring Mer isoform from Homo sapiens. In other words, a human Mer isoform includes any Mer isoform that has substantially similar structure and function of the naturally occurring Mer isoform from Homo sapiens or that is a biologically active (i.e., has biological activity) homologue of a naturally occurring Mer isoform from Homo sapiens as described in detail above. As such, a human Mer isoform can include purified, partially purified, recombinant, mutated/modified and synthetic proteins. According to the present invention, the terms "modification" and "mutation" can be used interchangeably, particularly with regard to the modifications/mutations to the amino acid sequence of Mer isoforms (or nucleic acid sequences) described herein. An isolated protein useful as an inhibitor according to the present invention can be isolated from its natural source, produced recombinantly or produced synthetically.

The polypeptides of the invention also encompass fragment and sequence variants, generally referred to herein as homologues. As used herein, the term "homologue" is used to refer to a protein or peptide which differs from a naturally occurring protein or peptide (i.e., the "prototype" or "wild-type" protein) by minor modifications to the naturally occurring protein or peptide, but which maintains the basic protein and side chain structure of the naturally occurring form. Such changes include, but are not limited to: changes in one or a few amino acid side chains; changes in one or a few amino acids, including deletions (e.g., a truncated version of the protein or peptide) insertions and/or substitutions; changes in stereochemistry of one or a few atoms; and/or minor derivatizations, including but not limited to: methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol. A homologue can have enhanced, decreased, or substantially similar properties as compared to the naturally occurring protein or peptide. A homologue of a human Mer isoform can include a non-human Mer isoform (i.e., a Mer isoform from a different species). Variants ("also referred to as "mimetics") also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis.

An "amino acid analog" is an amino acid, or a small molecule mimetic of an amino acid, that shares a common chemical, charge, steric, or other property of a given amino acid. For example, analogs of alanine include, e.g., β-alanine, ethylglycine, a- aminoisobutryic acid, and D-alanine; analogs of cysteine include, e.g., homocysteine, D- cysteine, and penicillamine; analogs of phenylalanine include, e.g., 3-fluorophenylalanine,

4-methylphenylalanine, phenylglycine, 1-naphthylalanine, and 3,3-diphenylalanine, 4- aminophenylalanine, pentafluorophenylalanine, 2-pyridylalanine, 3-pyridylalanine, 4- nitrophenylalanine, 2-pyrrolidinylalanine, 3-piperidylalanine, 4-piperidylalanine; and analogs of histidine include, e.g., 1-methylhistidine, 2,4-diaminobutyric acid, thiazolylalanine, 2,3-diaminopropionic acid, guanylalanine, 2-pyridylalanine, 3- pyridylalanine, 4-pyridylalanine, thienylalanine, ornithine, 4-guanylphenylalanine, and 4- aminopheny lalanine .

Such peptide analogs are commonly used in the pharmaceutical industry as non- peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compounds are termed "peptide mimetics" or "peptidomimetics." (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, TINS at 392 (1985); and, Evans, et al., J. Med. Chem. 30: 1229 (1987)). Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce a similar therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biochemical property or pharmacological activity), such as human antibody, but have one or more peptide linkages optionally replaced by a linkage selected from: -CH₂NH-, -CH₂S-, -CH₂-, -CH₂-, -CH=CH- (cis and trans), -COCH₂-, -CH(OH)CH₂-, and -CH₂SO-, by methods well known in the art. Systematic substitution of one or more amino acids of a consensus sequence with a D- amino acid of the same type (e.g., D-lysine in place of L-lysine) may be used in certain embodiments to generate more stable peptides. In addition, constrained peptides comprising a consensus sequence or a substantially identical consensus sequence variation may be generated by methods known in the art, such as, for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

Conservative amino acid substitutions that may be made to the Mer isoform of the invention may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.

For the purposes of this disclosure, the transitional term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open- ended and does not exclude additional, unrecited elements or method steps. The transitional phrase "consisting of excludes any element, step, or ingredient not specified in the claim, but does not exclude additional components or steps that are unrelated to the invention such as impurities ordinarily associated therewith. The transitional phrase "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

Any of the polypeptide Mer inhibitors of the invention, including homologues of such sequences (e.g., Mer isoforms), can be produced with additional heterologous amino acids flanking each of the C- and/or N-terminal end of the given amino acid sequence. The resulting protein or polypeptide can be referred to as "consisting essentially of a given amino acid sequence. According to the present invention, the heterologous amino acids are a sequence of amino acids that are not naturally found (i.e., not found in nature, in vivo) flanking the given amino acid sequence or which would not be encoded by the nucleotides that flank the naturally occurring nucleic acid sequence encoding the given amino acid sequence as it occurs in the gene, if such nucleotides in the naturally occurring sequence were translated using standard codon usage for the organism from which the given amino acid sequence is derived. Similarly, the phrase "consisting essentially of, when used with reference to a nucleic acid sequence herein, refers to a nucleic acid sequence encoding a given amino acid sequence that can be flanked by from at least one, and up to as many as about 60, additional heterologous nucleotides at each of the 5' and/or the 3' end of the nucleic acid sequence encoding the given amino acid sequence. The heterologous nucleotides are not naturally found (i.e., not found in nature, in vivo) flanking the nucleic acid sequence encoding the given amino acid sequence as it occurs in the natural gene.

The invention is primarily directed to the use of the Mer isoform proteins of the invention. The invention also encompasses fragments of the variants of these Mer isoform polypeptides. Fragments can be discrete (not fused to other amino acids or polypeptides) or can be within a larger polypeptide (as in a fusion protein of the present invention). Therefore, fragments can include any size fragment between about 6 amino acids and one amino acid less than the full length protein, including any fragment in between, in whole integer increments.

Similarly, the phrase, "Mer inhibitor" refers to any compound that acts in a similar manner relative to Mer activity, such that the biological activity of a natural Mer agonist, is decreased in a manner that is antagonistic (e.g., against, a reversal of, contrary to) to the natural action of Mer.

Homologues of the Mer isoform, including peptide and non-peptide agonists and antagonists of Mer (analogues), can be products of drug design or selection and can be produced using various methods known in the art. Such homologues can be referred to as mimetics. A mimetic refers to any peptide or non-peptide compound that is able to mimic the biological action of a naturally occurring peptide, often because the mimetic has a basic structure that mimics the basic structure of the naturally occurring peptide and/or has the salient biological properties of the naturally occurring peptide. Mimetics can include, but are not limited to: peptides that have substantial modifications from the prototype such as no side chain similarity with the naturally occurring peptide (such modifications, for example, may decrease its susceptibility to degradation); anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous portions of an isolated protein (e.g., carbohydrate structures); or synthetic or natural organic molecules, including nucleic acids and drugs identified through combinatorial chemistry, for example. Such mimetics can be designed, selected and/or otherwise identified using a variety of methods known in the art. Various methods of drug design, useful to design or select mimetics or other therapeutic compounds useful in the present invention are disclosed in Maulik, et al., Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc. (1997), which is incorporated herein by reference in its entirety.

Homologues can be produced using techniques known in the art for the production of proteins including, but not limited to, direct modifications to the isolated, naturally occurring protein, direct protein synthesis, or modifications to the nucleic acid sequence encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. For smaller peptides, chemical synthesis methods may be preferred. For example, such methods include well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Such methods are well known in the art and may be found in general texts and articles in the area such as: Merrifield, Methods Enzymol. 289:3-13 (1997); Wade, et al., Australas Biotechnol. 3(6):332-336 (1993); Wong, et al, Experientia 47(11-12): 1123-1129 (1991); Carey, et al, Ciba Found Symp. 158: 187-203 (1991); Plaue, et al., Biologicals 18(3): 147-157 (1990); Bodanszky, Int. J. Pept. Protein Res. 25(5):449-474 (1985); or, Dugas, et al, BIOORGANIC CHEMISTRY at 54-92 (1981), all of which are incorporated herein by reference in their entirety. For example, peptides may be synthesized by solid-phase methodology utilizing a commercially available peptide synthesizer and synthesis cycles supplied by the manufacturer. One skilled in the art recognizes that the solid phase synthesis could also be accomplished using the FMOC strategy and a TFA/scavenger cleavage mixture. The Mer inhibitor polypeptides (including Mer inhibitor fusion proteins) of the invention can be purified to homogeneity. It is understood, however, that preparations in which the polypeptide is not purified to homogeneity are useful. The critical feature is that the preparation allows for the Mer inhibitory activity of the polypeptide, even in the presence of considerable amounts of other components. Thus, the invention encompasses various degrees of purity. In one embodiment, the language "substantially free of cellular material" includes preparations of the polypeptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins.

According to the present invention, an isolated Mer inhibitor, including a biologically active homologue or fragment thereof, has at least one biological activity of the wild-type Mer isoform. A Mer protein of the invention includes any Mer protein and preferably any Mer fusion protein with improved stability and/or half-life in vivo that is an inhibitor of Mer.

Preferably, a Mer inhibitor of the invention, inhibits activation of the Tyro-3, Axl, and/or Mer RTKs (TAM family RTKs). For example, one can measure the Mer RTK activation using a phospho-Mer analysis by Western blot. In one embodiment, a Mer inhibitor of the invention inhibits a naturally occurring Mer receptor tyrosine kinase by at least 50%), at least 60%>, at least 70%>, at least 80%>, at least 90%>, or greater, using any suitable method of measurement of binding, as compared to an appropriate control.

Mer inhibitor fusion proteins of the invention can, in some embodiments, be produced as chimeric proteins with additional proteins or moieties (e.g., chemical moieties) that have a second biological activity. For example, Mer inhibitor fusion proteins, in addition to comprising the Mer isoform protein, or theraptutically-effective fragment thereof, and fusion partner, may comprise a protein that has a biological activity that is useful in a method of the invention, such as a pro-apoptotic protein, in the case of treating a neoplastic disease. Alternatively, the additional protein portion of the chimera may be a targeting moiety, in order to deliver the Mer inhibitor to a particular in vivo site (a cell, tissue, or organ). Such additional proteins or moieties may be produced recombinantly or post-translationally, by any suitable method of conjugation.

Some embodiments of the present invention include a composition or formulation

(e.g., for therapeutic purposes). Such compositions or formulations can include any one or more of the Mer inhibitors described herein, and may additionally comprise one or more pharmaceutical carriers or other therapeutic agents. In one aspect, the Mer inhibitors of the invention can be formulated with a pharmaceutically-acceptable carrier (including an excipient, diluent, adjuvant or delivery vehicle). The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

The compositions can be formulated for a particular type or route of delivery, if desired, including for parenteral, transmucosal, (e.g., orally, nasally or transdermally). Parental routes include intravenous, intra-arteriole, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration.

In another embodiment, the therapeutic Mer inhibitor or composition of the invention can be delivered in a vesicle, in particular a liposome. (See Langer, Science 249: 1527-1533 (1990); Treat, et al., Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, at 353-365 (1989). To reduce its systemic side effects, this may be a preferred method for introducing the inhibitor.

In yet another embodiment, the therapeutic inhibitor can be delivered in a controlled release system. For example, a polypeptide inhibitor may be administered using intravenous infusion with a continuous pump, in a polymer matrix such as poly- lactic/glutamic acid (PLGA), a pellet containing a mixture of cholesterol and the anti- amyloid peptide antibody compound (U.S. Pat. No. 5,554,601) implanted subcutaneously, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.

The pharmaceutical compositions of the invention may further comprise a therapeutically effective amount of another agent or therapeutic compound, preferably in respective proportions such as to provide a synergistic effect in the prevention or treatment. Alternatively, the pharmaceutical compositions of the invention can be administered concurrently with, or sequentially with, another pharmaceutical composition comprising such other therapeutic agent or compound. A therapeutically effective amount of a pharmaceutical composition of the invention relates generally to the amount needed to achieve a therapeutic objective. For example, inhibitors and compositions of the invention can be formulated with or administered with (concurrently or sequentially), other chemotherapeutic agents or anti-cancer methods, when it is desired to treat a neoplastic disease, or with other anti-thrombotic/anti-clotting agents, when it is desired to treat a cardiovascular or thrombotic disease or condition.

In one embodiment of the invention, a Mer inhibitor can be provided in a composition with, or administered with an Axl inhibitor or a Tyro-3 inhibitor.

Nucleic Acid Molecules Encoding Mer Iso forms of the Invention

Another embodiment of the invention relates to an isolated nucleic acid molecule, or complement thereof, encoding any of the Mer inhibitor proteins, including fragments and homologues thereof, fusion partners, fusion proteins, or other proteins described herein. Isolated nucleic acid molecules of the present invention can be R A, for example, mR A, or DNA, such as cDNA and genomic DNA. DNA molecules can be double- stranded or single-stranded; single stranded RNA or DNA can include the coding, or sense, strand or the non-coding, or antisense, strand. The nucleic acid molecule can include all or a portion of the coding sequence of a gene or nucleic acid sequence and can further comprise additional non-coding sequences such as introns and non-coding 3' and 5' sequences (including regulatory sequences, for example).

An "isolated" nucleic acid molecule, as used herein, is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences {e.g., as in an RNA library). For example, an isolated nucleic acid of the invention may be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix. In other circumstances, the material may be purified to essential homogeneity, for example as determined by PAGE or column chromatography such as HPLC.

The nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. Thus, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells, as well as partially or substantially purified DNA molecules in solution. "Isolated" nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention. An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means. Therefore, recombinant DNA contained in a vector is included in the definition of "isolated" as used herein. Also, isolated nucleotide sequences include partially or substantially purified DNA molecules in solution. In vivo and in vitro RNA transcripts of the DNA molecules of the present invention are also encompassed by "isolated" nucleotide sequences. Such isolated nucleotide sequences are useful in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping {e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by northern blot analysis.

Nucleic acid molecules useful in the invention include variant nucleic acid molecules that are not necessarily found in nature but which encode novel proteins of the invention. Such variants can be naturally occurring, such as in the case of allelic variation or single nucleotide polymorphisms, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides that can result in conservative or non-conservative amino acid changes, including additions and deletions. Other alterations of the nucleic acid molecules of the invention can include, for example, labeling, methylation, internucleotide modifications such as uncharged linkages {e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates), charged linkages {e.g., phosphorothioates, phosphorodithioates), pendent moieties {e.g., polypeptides), intercalators {e.g., acridine, psoralen), chelators, alkylators, and modified linkages {e.g., alpha anomeric nucleic acids). Also included are synthetic molecules that mimic nucleic acid molecules in the ability to bind to designated sequences via hydrogen bonding and other chemical interactions. Such molecules include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

The invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein {e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein, and, optionally, have an activity of the polypeptide). In one embodiment, the invention includes variants described herein which hybridize under high stringency hybridization conditions (e.g., for selective hybridization) to a nucleotide sequence encoding an Axl protein inhibitor of the invention, or the complements thereof.

"Stringency conditions" for hybridization is a term of art which refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly (i.e., 100%) complementary to the second, or the first and second may share some degree of complementarity which is less than perfect (e.g., 70%, 75%), 85%o, 95%o). For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity. "High stringency conditions", "moderate stringency conditions" and "low stringency conditions" for nucleic acid hybridizations are explained in Current Protocols in Molecular Biology. (Ausubel, et ah, Current Protocols in Molecular Biology, John Wiley & Sons, (1998), the entire teachings of which are incorporated by reference herein). Typically, conditions are used such that sequences at least about 60%>, at least about 70%>, at least about 80%, at least about 90% or at least about 95% or more identical to each other remain hybridized to one another. By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize {e.g., selectively) with the most similar sequences in the sample can be determined.

More particularly, moderate stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70%> nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 30% or less mismatch of nucleotides). High stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 80%> nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 20% or less mismatch of nucleotides). Very high stringency hybridization and washing conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 90%> nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction (i.e., conditions permitting about 10% or less mismatch of nucleotides). As discussed above, one of skill in the art can use the formulae in Meinkoth, et al., Anal. Biochem. 138: 267-284 (1984), to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:R A or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10°C less than for DNA:R A hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 20°C and about 35°C (lower stringency), more preferably, between about 28°C and about 40°C (more stringent), and even more preferably, between about 35°C and about 45°C (even more stringent), with appropriate wash conditions. In particular embodiments, stringent hybridization conditions for DNA:R A hybrids include hybridization at an ionic strength of 6X SSC (0.9 M Na⁺) at a temperature of between about 30°C and about 45°C, more preferably, between about 38°C and about 50°C, and even more preferably, between about 45°C and about 55°C, with similarly stringent wash conditions. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G + C content of about 40%. Alternatively, T_m can be calculated empirically as set forth in Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., CSHP, NY (1989). In general, the wash conditions should be as stringent as possible, and should be appropriate for the chosen hybridization conditions. For example, hybridization conditions can include a combination of salt and temperature conditions that are approximately 20- 25°C below the calculated T_m of a particular hybrid, and wash conditions typically include a combination of salt and temperature conditions that are approximately 12-20°C below the calculated T_m of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6X SSC (50% formamide) at about 42°C, followed by washing steps that include one or more washes at room temperature in about 2X SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37°C in about 0.1X- 0.5X SSC, followed by at least one wash at about 68°C in about 0.1X-0.5X SSC).

Reference herein to "probes" or "primers" is to oligonucleotides that hybridize in a base-specific manner to a complementary strand of nucleic acid molecules. By "base specific manner" is meant that the two sequences must have a degree of nucleotide complementarity sufficient for the primer or probe to hybridize. Accordingly, the primer or probe sequence is not required to be perfectly complementary to the sequence of the template. Non-complementary bases or modified bases can be interspersed into the primer or probe, provided that base substitutions do not substantially inhibit hybridization. The nucleic acid template may also include "non-specific priming sequences" or "nonspecific sequences" to which the primer or probe has varying degrees of complementarity. Such probes and primers include polypeptide nucleic acids, as described in Nielsen, et al, Science, 254, 1497-1500 (1991). Typically, a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and more typically about 40, 50, 75, 100, 150, 200, or more, consecutive nucleotides of a nucleic acid molecule.

The nucleic acid molecules of the invention such as those described above can be identified and isolated using standard molecular biology techniques and the sequence information provided herein. For example, nucleic acid molecules can be amplified and isolated by the polymerase chain reaction using synthetic oligonucleotide primers designed based on a nucleotide sequence encoding a soluble form of Axl receptor tyrosine kinase or the complements thereof. (See generally PCR Technology: Principles and Applications for DNA Amplification, ed. Erlich, Freeman Press, NY, NY (1992); PCR Protocols: A Guide to Methods and Applications, eds. Innis, et al., Academic Press, San Diego, CA (1990); Mattila, et al, Nucleic Acids Res., 19:4967 (1991); Eckert, et al, PCR Methods and Applications, 1 : 17 (1991); PCR, eds. McPherson, et al, IRL Press, Oxford; and, U.S. Patent No. 4,683,202. The nucleic acid molecules can be amplified using cDNA, mRNA or genomic DNA as a template, cloned into an appropriate vector and characterized by DNA sequence analysis.

Other suitable amplification methods include the ligase chain reaction (LCR), (see Wu, et al, Genomics, 4:560 (1989), Landegren, et al, Science, lAVAQll (1988)), transcription amplification, (Kwoh, et al, Proc. Natl. Acad. Sci., 86: 1173 (1989)), and self-sustained sequence replication (Guatelli, et al, Proc. Nat. Acad. Sci., 87: 1874 (1990)) and nucleic acid based sequence amplification (NASBA).

The amplified DNA can be labeled (e.g., with radiolabel or other reporter molecule) and used as a probe for screening a cDNA library derived from human cells, mRNA in zap express, ZIPLOX or other suitable vector. Corresponding clones can be isolated, DNA can obtained following in vivo excision, and the cloned insert can be sequenced in either or both orientations by art recognized methods to identify the correct reading frame encoding a polypeptide of the appropriate molecular weight. For example, the direct analysis of the nucleotide sequence of nucleic acid molecules of the present invention can be accomplished using well-known methods that are commercially available. {See, e.g., Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd ed., CSHP, NY (1989); Zyskind et al., Recombinant DNA Laboratory Manual, Acad. Press, (1988). Using these or similar methods, the polypeptide and the DNA encoding the polypeptide can be isolated, sequenced and further characterized.

Preferably, the nucleotide sequences of the invention can be used to identify and express recombinant polypeptides for analysis, for characterization or for therapeutic use. Such nucleic acid sequences can be incorporated into host cells and expression vectors that are well known in the art. According to the present invention, a recombinant nucleic acid molecule includes at least one isolated nucleic acid molecule of the present invention that is linked to a heterologous nucleic acid sequence. Such a heterologous nucleic acid sequence is typically a recombinant nucleic acid vector (e.g., a recombinant vector) which is suitable for cloning, sequencing, and/or otherwise manipulating the nucleic acid molecule, such as by expressing and/or delivering the nucleic acid molecule into a host cell to form a recombinant cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention, although the vector can also contain regulatory nucleic acid sequences (e.g., promoters, untranslated regions) which are naturally found adjacent to nucleic acid molecules of the present invention. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. The vector can be maintained as an extrachromosomal element (e.g., a plasmid) or it can be integrated into the chromosome. The entire vector can remain in place within a host cell, or under certain conditions, the plasmid DNA can be deleted, leaving behind the nucleic acid molecule of the present invention. The integrated nucleic acid molecule can be under chromosomal promoter control, under native or plasmid promoter control, or under a combination of several promoter controls. Single or multiple copies of the nucleic acid molecule can be integrated into the chromosome. As used herein, the phrase "recombinant nucleic acid molecule" is used primarily to refer to a recombinant vector into which has been ligated the nucleic acid sequence to be cloned, manipulated, transformed into the host cell (i.e., the insert).

The nucleic acid sequence encoding the protein to be produced is inserted into the vector in a manner that operatively links the nucleic acid sequence to regulatory sequences in the vector (e.g., expression control sequences) that enable the transcription and translation of the nucleic acid sequence when the recombinant molecule is introduced into a host cell. According to the present invention, the phrase "operatively linked" refers to linking a nucleic acid molecule to an expression control sequence (e.g., a transcription control sequence and/or a translation control sequence) in a manner such that the molecule can be expressed when transfected (i.e., transformed, transduced, transfected, conjugated or conduced) into a host cell. Transcription control sequences are sequences that control the initiation, elongation, or termination of transcription. Particularly important transcription control sequences are those that control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in a host cell into which the recombinant nucleic acid molecule is to be introduced.

Recombinant molecules of the present invention, which can be either DNA or RNA, can also contain additional regulatory sequences, such as translation regulatory sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell. In one embodiment, a recombinant molecule of the present invention, including those that are integrated into the host cell chromosome, also contains secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed protein to be secreted from the cell that produces the protein. Suitable signal segments include a signal segment that is naturally associated with a protein of the present invention or any heterologous signal segment capable of directing the secretion of a protein according to the present invention.

One or more recombinant molecules of the present invention can be used to produce an encoded product of the present invention. In one embodiment, an encoded product is produced by expressing a nucleic acid molecule as described herein under conditions effective to produce the protein. A preferred method to produce an encoded protein is by transfecting a host cell with one or more recombinant molecules to form a recombinant cell. Suitable host cells to transfect include, but are not limited to, any bacterial, fungal (e.g., yeast), insect, plant or animal cell that can be transfected. Host cells can be either untransfected cells or cells that are already transfected with at least one nucleic acid molecule.

According to the present invention, the term "transfection" is used to refer to any method by which an exogenous nucleic acid molecule (i.e., a recombinant nucleic acid molecule) can be inserted into the cell. The term "transformation" can be used interchangeably with the term "transfection" when such term is used to refer to the introduction of nucleic acid molecules into microbial cells, such as bacteria and yeast. In microbial systems, the term "transformation" is used to describe an inherited change due to the acquisition of exogenous nucleic acids by the microorganism and is essentially synonymous with the term "transfection." However, in animal cells, transformation has acquired a second meaning that can refer to changes in the growth properties of cells in culture after they become cancerous, for example. Therefore, to avoid confusion, the term "transfection" is preferably used with regard to the introduction of exogenous nucleic acids into animal cells, and the term "transfection" will be used herein to generally encompass both transfection of animal cells and transformation of microbial cells, to the extent that the terms pertain to the introduction of exogenous nucleic acids into a cell. Therefore, transfection techniques include, but are not limited to, transformation, electroporation, microinjection, lipofection, adsorption, infection and protoplast fusion.

Methods of the Invention

The present invention also relates to methods of treatment (prophylactic and/or therapeutic) for Mer-positive cancers, for Axl-positive cancers, for Tyro3 -positive cancers, and/or for clotting disorders, using the Mer inhibitors described herein.

The method of use of the inhibitors and therapeutic compositions of the present invention preferably provides a benefit to a patient or individual by inhibiting at least one biological activity of Mer or of its related receptors, Axl and/or Tyro-3.

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and may be performed either for prophylaxis and/or during the course of clinical pathology. Desirable effects include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, lowering the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Accordingly, a therapeutic benefit is not necessarily a cure for a particular disease or condition, but rather, preferably encompasses a result which most typically includes alleviation of the disease or condition, elimination of the disease or condition, reduction of a symptom associated with the disease or condition, prevention or alleviation of a secondary disease or condition resulting from the occurrence of a primary disease or condition (e.g., metastatic tumor growth resulting from a primary cancer), and/or prevention of the disease or condition.

In the case of cancer, the method of the invention preferably increases the death of tumor cells, decreases the invasive potential of tumor cells, increases the survival of an individual with cancer, and/or increases tumor regression, decreases tumor growth, and/or decreases tumor burden in the individual.

In the case of clotting disorders and/or cardiovascular disease, the method of the invention preferably prevents or reduces clotting, platelet aggregation, and/or secretion response of platelets to known agonists, or any other symptom of thrombosis or any clotting disorder, without causing bleeding side effects.

A beneficial effect can easily be assessed by one of ordinary skill in the art and/or by a trained clinician who is treating the patient. The term, "disease" refers to any deviation from the normal health of a mammal and includes a state when disease symptoms are present, as well as conditions in which a deviation (e.g., infection, gene mutation, genetic defect, etc.) has occurred, but symptoms are not yet manifested.

According to the present invention, the methods and assays disclosed herein are suitable for use in or with regard to an individual that is a member of the Vertebrate class, Mammalia, including, without limitation, primates, livestock and domestic pets (e.g., a companion animal). Most typically, a patient will be a human patient. According to the present invention, the terms "patient", "individual" and "subject" can be used interchangeably, and do not necessarily refer to an animal or person who is ill or sick (i.e., the terms can reference a healthy individual or an individual who is not experiencing any symptoms of a disease or condition).

Diseases and disorders that are characterized by altered (relative to a subject not suffering from the disease or disorder) Mer receptor tyrosine kinases, levels of this protein, and/or biological activity associated with this protein, are treated with therapeutics that antagonize (e.g., reduce or inhibit) the Mer receptor tyrosine kinase. The Mer inhibitors of the present invention block the activation of the full length native Mer. Therefore, an effective amount of an inhibitor of a Mer receptor which is provided in the form of the Mer inhibitors described herein may be used as a treatment for diseases and conditions associated with Mer expression, as well as with Tyro-3 expression and/or Axl expression.

The methods of the invention for example, involve contacting a cell, tissue or system of an individual with a Mer inhibitor that inhibits one or more of the activities of Mer. The Mer inhibitors act as competitive inhibitors of Mer expressed by cells. Such methods are preferably performed in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder, specifically a clotting disorder or a cancer. In one embodiment of the invention, inhibition of Mer is contemplated to prevent thrombosis or any clotting disorder, preferably without causing bleeding side effects. According to the present invention, "modulation" refers to any type of regulation, including upregulation, stimulation, or enhancement of expression or activity, or downregulation, inhibition, reduction or blocking of expression or activity. Preferably, the method of the present invention specifically inhibits the activity of Mer expressed by platelets. Inhibition is provided by the present invention through the administration of the Mer inhibitor(s) described herein, which inhibit the activity of Mer receptors. The Mer inhibitor can be administered alone or together with another therapeutic agent, such as another anti-clotting agent. In one embodiment, the Mer inhibitor is administered together with an agent that inhibits the expression or biological activity of Axl. One such agent is an Axl-Fc protein, wherein the Mer inhibitor does not activate Axl.

Clotting disorders that can be treated by the method of the invention include, but are not limited to, thrombophilia (including inherited traits predisposing an individual to have a higher risk of clotting), thrombosis or thrombo-embolic disorder. Specifically, this method of treatment could be applied to patients on medications (including, but not limited to, estrogens and chemotherapy) which increase the risk of clotting as well as diseases associated with thrombosis (including, but not limited to, cancer, myeloproliferative disorders, autoimmune disorders, cardiac disease, inflammatory disorders, atherosclerosis, hemolytic anemia, nephrosis, and hyperlipidemia). In addition, this method of treatment could be applied to predisposing factors to increased clotting including cardiovascular interventions, surgery, trauma, or pregnancy. Finally, this method of treatment may be appropriate for patients with adverse side effects from other anticoagulant or anti-platelet therapies, including heparin-induced thrombocytopenia (a severe immune-mediated drug reaction that occurs in 2-5% of patients exposed to heparin.)

Accordingly, the present invention provides for a method of treating an individual who has or is likely to develop a clotting disorder or disease, comprising inhibiting Mer receptors. An effective amount of a Mer inhibitor to administer to an individual is any amount that achieves any detectable inhibition of the natural Mer receptor in the patient, or any detectable reduction in at least one symptom of the clotting disorder. One specific embodiment is the use of a Mer inhibitor of the invention in the manufacture of a medicament for the treatment of a clotting disorder or disease. Another specific embodiment is a Mer inhibitor of the invention for use in the treatment of a clotting disorder or disease. Another specific embodiment is a method of treating an individual at risk of developing a clotting disorder or bleeding uncontrollably by administering an effective amount of a Mer inhibitor of the invention to the individual in need thereof.

As discussed above, TAM-family RTK signaling has been shown to favor tumor growth through activation of proliferative and anti-apoptotic signaling pathways, as well as through promotion of angiogenesis and tumor invasiveness. Accordingly, it is another embodiment of the present invention to inhibit Mer activity as part of a therapeutic strategy that selectively targets cancer cells. Any of the above-described methods and agents for treating a clotting disorder can be applied to the treatment of cancers. Inhibition is also provided by the present invention in this embodiment through the administration of Mer inhibitors described herein. The Mer inhibitor can be administered alone or together with another therapeutic agent, such as another anti-cancer agent. In one embodiment, the Mer inhibitor is administered together with an agent that inhibits the expression or biological activity of Mer. One such agent is an Axl-Fc protein, wherein the Axl-Fc protein does not activate Mer.

Cancers that can be treated by the method of the invention include, but are not limited to glioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, colorectal cancer, endometrium cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, testicular tumor, Wilms' tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endotheliosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland sarcoma, papillary sarcoma, papillary adenosarcoma, cystadenosarcoma, bronchogenic carcinoma, medullary carcinoma, mastocytoma, mesotheliorma, synovioma, melanoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, retinoblastoma, oligodentroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, base cell carcinoma, fibrosarcoma, myxoma, myxosarcoma, liposarcorna, chondrosarcoma, osteogenic sarcoma, leukemia and metastatic lesions secondary to these primary tumors. In a related aspect, the cancer is a leukemia or lymphoma and in a another related embodiment, the cancer is a myeloid leukemia or a lymphoid leukemia or lymphoma. In another embodiment, the cancer is a non-small cell lung cancer (NSCLC).

In another embodiment, the cancer is an astrocytoma or glioblastoma. Accordingly, the present invention provides for a method of treating an individual who has or is likely to develop a cancer, comprising inhibiting TAM-family receptors. An effective amount of a Mer inhibitor to administer to an individual is any amount that achieves any detectable inhibition of the natural Mer receptor in the patient, or any detectable reduction in at least one symptom of the cancer. One specific embodiment is the use of a Mer inhibitor of the invention in the manufacture of a medicament for the treatment of cancer. Another specific embodiment is a Mer inhibitor of the invention for use in the treatment of cancer. Another specific embodiment is a method of treating an individual at risk of developing a cancer by administering an effective amount of a Mer inhibitor of the invention to the individual in need thereof.

In the therapeutic methods of the invention, suitable methods of administering a composition of the present invention to a subject include any route of in vivo administration that is suitable for delivering the composition. The preferred routes of administration will be apparent to those of skill in the art, depending on the type of delivery vehicle used, the target cell population, and the disease or condition experienced by the patient.

A preferred single dose of a protein such as a Mer inhibitor of the invention typically comprises between about 0.01 microgram x kilogram^"1 and about 10 milligram x kilogram^"1 body weight of an animal. A more preferred single dose of such an agent comprises between about 1 microgram x kilogram^"1 and about 10 milligram x kilogram^"1 body weight of an animal. An even more preferred single dose of an agent comprises between about 5 microgram x kilogram^"1 and about 7 milligram x kilogram^"1 body weight of an animal. An even more preferred single dose of an agent comprises between about 10 microgram x kilogram^"1 and about 5 milligram x kilogram^"1 body weight of an animal. Another particularly preferred single dose of an agent comprises between about 0.1 microgram x kilogram^"1 and about 10 microgram x kilogram^"1 body weight of an animal, if the agent is delivered parenterally.

Each publication or patent cited herein is incorporated herein by reference in its entirety.

The invention now being generally described will be more readily understood by reference to the following examples, which are included merely for the purposes of illustration of certain aspects of the embodiments of the present invention. The examples are not intended to limit the invention, as one of skill in the art would recognize from the above teachings and the following examples that other techniques and methods can satisfy the claims and can be employed without departing from the scope of the claimed invention.

EXAMPLES

Example 1

This example illustrates that a naturally-occuring splice variant of MerTK is expressed in a variety of human tissues.

Figure 1 shows a graphic representation of the MerTK gene showing exons 1-19, which encode the full length MerTK transcript. Exons 1-9 encode the extracellular domain of MerTK. Exon 10 contains the transmembrane domain and Exons 11-19 encode the intracellular domain. The Mer inhibitor transcript is derived by alternative splicing such that exon 7 A is inserted into the transcript between exons 7 and 8. Exon 7 A contains an in- frame stop codon. Thus, the alternatively spliced MerTK transcript encodes a truncated isoform consisting of three of the four extracellular domains (1 of the two fibronectin III domains and two Ig domains) that are found in the full-length protein. See Figure 2.

To detect the expression of the Mer isoform, RT-PCR was performed using primers that amplify both forms of the human MerTK transcript:

5'-CACCTCTGCCTTACCACATCT-3' (SEQ ID NO: 3)

5 ' -ATCC AC AAAAGC AGCC AAAGA-3 ' (SEQ ID NO: 4)

or only the alternatively spliced Mer isoform transcript:

5 ' -GATGATGAAGTTAC AGC AAT-3 ' (SEQ ID NO: 5)

5 ' -GGAGTC AACAGTAGAAGAGAG-3 ' (SEQ ID NO: 6).

The locations of the primers are shown in Figure 1. The Mer isoform transcript was detected in a variety of human tissues, as shown in Figure 3. The middle panel of Figure 3 shows the regular transcript encoding full-length Mer, demonstrating that the Mer isoform has a similar expression profile to the transcript encoding full-length Mer. The Mer isoform transcript was also detected in these tissues using these primers, SEQ ID NOs: 5 and 6, demonstrating the existence of the full-length Mer isoform transcript (data not shown).

Example 2

This example shows that the Mer isoform of SEQ ID NO:2 decreases platelet aggregation in response to fibrillar collagen.

Aggregometry was performed on platelet-rich human plasma using fibrillar collagen (1 μg/ml) as an agonist. In Figure 4, a representative sample shows decreased aggregation in samples treated with the Mer isoform of SEQ ID NO:2 (uppermost line) compared to control samples (second line). Decreased ATP release is also observed in samples treated with the Mer isoform of SEQ ID NO:2 (bottom line) compared to samples treated with vehicle only (second line from bottom). Samples treated with the Mer isoform of SEQ ID NO:2 (white bar in Figure 5) exhibited a significantly lower mean maximum aggregation % compared to samples treated with vehicle only (black bar in Figure 5). Mean values and standard errors derived from six individual patients are shown. */?=0.02 by paired t-test.

Example 3

This example shows that the Mer isoform of SEQ ID NO:2 reduces stable platelet aggregate formation under physiologic flow conditions.

Whole blood was run in a flow chamber at a flow rate of 650 sec^"1 perpendicularly across a strip of equine Type I fibrillar collagen. Samples treated with 1.2 μΜ of the Mer isoform of SEQ ID NO:2 exhibit decreased stability of platelet binding under shear stress conditions compared to control samples treated with vehicle only, resulting in fewer large platelet aggregates bound at the end of each run. See Figure 6.

A human whole blood microfluidic flow assay demonstrated that adhesion of untreated platelets to fibrillar collagen in a flow chamber at a wall shear rate of 650s^"1 resulted in 8.7 +/- 2.3% mean surface area coverage, while ex vivo samples treated with the Mer isoform of SEQ ID NO:2 showed only 1.9 +/- 1% coverage (/?<0.001, n=7). Analysis provided by paired t-test. See Figure 7. Samples treated with the Mer isoform of SEQ ID NO:2 have a higher percentage of small platelet aggregates (1-2 or 3-10 platelets/aggregate) compared to controls which had a higher proportion of large aggregates (11-50 and 51-100 plateelts/aggregate). Analysis provided by chi-square test. See Figure 8.

Example 4

This example shows the Mer isoform of SEQ ID NO:2 decreases spreading of washed platelets on 100 μg/mL fibrillar collagen.

PBS-treated control samples (negative control) demonstrate clusters of activated platelets with visibly extended filopodia and lamellipodia. See Figure 9 (-Control). The samples treated with the Mer isoform of SEQ ID NO:2 shows many clusters of round, unactivated platelets, after 15 minutes of incubation. See Figure 9 (iMer). Scanning Electron Microscopy was used for imaging. Abciximab-treated sample (pos control) shows almost complete lack of platelet spreading. See Figure 9 (+Control).

Example 5 This example shows that the Mer isoform of SEQ ID NO:2 decreases platelet- surface expression of activation markers in flow cytometric analysis.

Samples treated with the Mer isoform of SEQ ID NO:2 show a decreased percentage of activated platelets identified by fluorescent anti-p-selectin antibodies compared to either convulxin (CVX) or thrombin (thromb)- stimulated control samples, which were normalized to 100% activation after background fluorescence was subtracted from all samples. (32 +/- 7%, p=0.04, decrease from baseline following 10 ng/mL CVX stimulation and 35 +/- 10% , p=0.007, reduction following 0.2 U/mL thromb stimulation) The abciximab (ReoPro) treated samples did not differ significantly from control, since this drug is an antibody to ¾¾β₃ integrin and therefore not expected to have a major effect on P-selectin expression. (n=4, analysis by paired t-test.) See Figure 10.

Samples treated with the Mer isoform of SEQ ID NO:2 also showed a reduction in fluorescent-labeled PAC-1 binding to activated α¾β₃ integrin after stimulation with either CVX or thromb (82 +/- 9%, /?=0.06, decrease from baseline following CVX stimulation and 80 +/- 10%>, /?=0.05, reduction following thromb stimulation) The samples treated with the Mer isoform of SEQ ID NO:2 demonstrated a decreased activation phenotype that was intermediate between that of non-inhibited samples (neg control) and those that were completely inhibited with abciximab (ReoPro) (pos control). (n=4, analysis by paired t- test.) See Figure 11.

Example 6

This example illustrates that Mer inhibitor inhibits activation of MerTK in B-cell acute lymphoid leukemia cells.

REH cells were treated with 650 nM Mer inhibitor for 30 minutes prior to stimulation with Gas6 (a ligand for MerTK) or vehicle only. Phosphorylation of MerTK in response to Gas6 was observed in the untreated and vehicle only samples. See Figure 12. In addition, 697 cells were treated with 1.2 μΜ Mer inhibitor for 10 and 20 minutes prior to stimulation with Gas6 or vehicle only. See Figure 13. In both instances, MerTK was not efficiently phosphorylated in the presence of Mer inhibitor. See Figures 12 and 13.

Example 7

This example illustrates that Mer inhibitor decreases activation of β3 -integrin in human platelets.

Human platelets were treated with 1.2 μΜ Mer inhibitor for 30 minutes prior to stimulation with Gas6 or vehicle only for 3, 5, and 10 minutes. Phosphorylation of β3- integrin decreased in the presence of the Mer inhibitor. See Figure 14. Example 8

This example demonstrates that iMer decreases platelet aggregation in response to ADP and collagen.

Aggregometry was performed on platelet-rich plasma from healthy volunteers using ADP (4 μΜ) or collagen (0.5 μg/ml) as agonists. Figure 15A shows that decreased aggregation is observed in samples treated with 940 nM of the Mer isoform of SEQ ID NO:2 (iMer; top line) compared to control samples (bottom line). Decreased ATP release is also observed in samples treated with the Mer isoform of SEQ ID NO:2 compared to samples treated with vehicle only. More pronounced decreases in aggregation and ATP release were observed when platelets were treated with a higher concentration of the Mer isoform of SEQ ID NO:2 (1.4 μΜ), indicating a dose-dependent effect. The data shown were derived using samples from the same patient. Figure 15 B shows platelets treated with 940 nM of the Mer isoform of SEQ ID NO:2 (iMer; right bar) exhibit statistically significant decreases in maximum percent aggregation in response to collagen and ADP compared to control samples treated with vehicle only (left bar). Mean values and standard errors derived from individual patients are shown.

While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims.

Claims

What is claimed is:

1. A Mer inhibitor comprising, consisting essentially of, or consisting of, at least a portion of an isolated isoform of a Mer receptor tyrosine kinase (Mer RTK) that binds to at least one naturally-occurring Tyro3, Axl, or Mer receptor.

2. A Mer inhibitor comprising, consisting essentially of, or consisting of, at least a portion of an isolated isoform of a Mer receptor tyrosine kinase (Mer RTK) that binds to a Mer receptor ligand.

3. The Mer inhibitor of Claim 1 or Claim 2, wherein the Mer inhibitor decreases activation of at least one of cellular Mer, cellular Axl, and cellular Tyro3.

4. The Mer inhibitor of Claim 1 or Claim 2, wherein the Mer inhibitor decreases adhesion to collagen under physiologic flow rates.

5. The Mer inhibitor of Claim 1 or Claim 2, wherein the Mer inhibitor decreases ADP- induced platelet aggregation.

6. The Mer inhibitor of Claim 1 or Claim 2, wherein the Mer inhibitor decreases collagen- induced platelet aggregation.

7. The Mer inhibitor of Claim 1 or Claim 2, wherein the Mer inhibitor decreases platelet spreading on collagen surfaces.

8. The Mer inhibitor of Claim 1 or Claim 2, wherein the isolated inhibitor comprises, consists essentially of, or consists of SEQ ID NO:2.

9. The Mer inhibitor of Claim 1 or Claim 2, wherein the isolated inhibitor comprises, consists essentially of, or consists of a therapeutically effective fragment of SEQ ID No:2.

10. The Mer inhibitor of Claim 1 or Claim 2, wherein the isolated inhibitor comprises, consists essentially of, or consists of a fusion protein, wherein the fusion protein is produced as a dimer of Mer isoform proteins.

11. The Mer inhibitor of Claim 1 or Claim 2, wherein the isolated inhibitor comprises, consists essentially of, or consists of a fusion protein, wherein the fusion protein is produced as a dimer of therapeutically-effective Mer isoform protein fragments.

12. The Mer inhibitor of Claims 10 or 11, wherein the isolated fusion protein comprises a pro-apoptosis protein fused to the Mer inhibitor protein.

13. The Mer inhibitor of Claims 10 or 11, wherein the isolated fusion protein comprises an anti-clotting protein fused to the Mer inhibitor protein.

14. The Mer inhibitor of any one of the preceding claims, wherein the Mer inhibitor binds to the Mer ligand with an equal or greater affinity as compared to a naturally occurring Mer receptor tyrosine kinase ligand.

15. The Mer inhibitor of any one of the preceding claims, wherein the Mer inhibitor binds to the Mer ligand with lower affinity as compared to a naturally occurring Mer receptor tyrosine kinase ligand.

16. A pharmaceutical composition comprising the Mer inhibitor of any one of Claims 1 to 15 and at least one pharmaceutical carrier.

17. The composition of Claim 16, wherein the composition further comprises a pharmaceutically-acceptable carrier.

18. The composition of Claim 16, wherein the composition further comprises at least one therapeutic agent for the treatment of cancer.

19. The composition of Claim 16, wherein the composition further comprises at least one therapeutic agent for treatment of a clotting disorder.

20. The composition of Claim 16, wherein the composition further comprises an Axl or a Tyro3 inhibitor.

21. A method of treating or preventing a clotting disorder or disease in an individual, comprising administering to an individual in need thereof, the Mer inhibitor or composition of any one of Claims 1 to 21.

22. The method of Claim 21, wherein the disorder or disease is selected from the group consisting of thrombophilia, thrombosis and thrombo-embolic disorder.

23. The method of Claim 21, wherein the disorder or disease is thrombophilia.

24. The method of Claim 21, wherein the individual is taking a medication that increases the risk of clotting in the individual.

25. The method of Claim 21, wherein the individual has a clotting disorder or disease associated with thrombosis.

26. The method of Claim 25, wherein the clotting disorder or disease is selected from the group consisting of cancer, myeloproliferative disorders, autoimmune disorders, cardiac disease, inflammatory disorders, atherosclerosis, hemolytic anemia, nephrosis, and hyperlipidemia.

27. The method of Claim 21, wherein the individual is undergoing surgery or an interventional procedure, is experiencing or has experienced a trauma, or is pregnant.

28. The use of the Mer inhibitor or composition of any one of Claims 1 to 20 in the preparation of a medicament for the treatment of a clotting disorder.

29. The use of the Mer inhibitor or composition of any one of Claims 1 to 20 in the preparation of a medicament for the treatment of a cancer.

30. A Mer inhibitor or composition of any one of Claims 1 to 20 for use in the treatment of a clotting disorder.

31. A method of treating an individual suffering from, or at risk of bleeding comprising administering an effective amount of a Mer inhibitor or composition of any one of Claims 1 to 20 to the individual in need thereof.

32. A Mer inhibitor or composition of any one of Claims 1 to 20 for use in the treatment of a cancer.

33. A method of treating cancer in an individual, comprising administering to an individual in need thereof, the Mer inhibitor or composition of any one of Claims 1 to 20.

34. The method of Claim 33, wherein the cancer is a Mer-positive cancer.

35. The method of Claim 33, wherein the cancer is selected from the group consisting of: glioma, gliosarcoma, anaplastic astrocytoma, medulloblastoma, lung cancer, small cell lung carcinoma, cervical carcinoma, colon cancer, rectal cancer, chordoma, throat cancer, Kaposi's sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, colorectal cancer, endometrium cancer, ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, hepatic carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, testicular tumor, Wilms' tumor, Ewing's tumor, bladder carcinoma, angiosarcoma, endotheliosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland sarcoma, papillary sarcoma, papillary adenosarcoma, cystadenosarcoma, bronchogenic carcinoma, medullary carcinoma, mastocytoma, mesotheliorma, synovioma, melanoma, leiomyosarcoma, rhabdomyosarcoma, neuroblastoma, retinoblastoma, oligodentroglioma, acoustic neuroma, hemangioblastoma, meningioma, pinealoma, ependymoma, craniopharyngioma, epithelial carcinoma, embryonal carcinoma, squamous cell carcinoma, base cell carcinoma, fibrosarcoma, myxoma, myxosarcoma, liposarcorna, chondrosarcoma, osteogenic sarcoma, leukemia and metastatic lesions secondary to these primary tumors.

36. The method of claim 33, wherein the cancer is a leukemia or lymphoma.

37. The method of claim 33, wherein the cancer is myeloid leukemia.

38. The method of claim 33, wherein the cancer is non-small cell lung cancer (NSCLC).