AXL TYROSINE KINASE INHIBITORS AND METHODS OF MAKING AND USING THE SAME
Disclosed are novel inhibitors of the Axl 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.
This application is a divisional of U.S. patent application Ser. No. 13/439,176, filed on Apr. 4, 2012, which is a divisional of U.S. patent application Ser. No. 12/526,094 filed on Dec. 15, 2009, which is a national stage application under 35 U.S.C. 371 of PCT Application No. PCT/US2008/053337, having an international filing date of Feb. 7, 2008, which designated the United States, which PCT application claimed the benefit of U.S. Application Ser. No. 60/888,741, filed Feb. 7, 2007. The entire disclosure of each of these related applications is incorporated herein by reference.
REFERENCE TO SEQUENCE LISTINGThis application contains a Sequence Listing submitted as an electronic text file named “Sequence_Listing.txt”, having a size in bytes of 183 kb, and created on Sep. 30, 2010. The information contained in this electronic file is hereby incorporated by reference in its entirety pursuant to 37 CFR §1.52(e)(5).
FIELD OF THE INVENTIONThe present invention generally relates to novel inhibitors of the Axl receptor tyrosine kinase (RTK) and to the use of such inhibitors in a variety of compositions and therapeutic approaches in the areas of cancer therapy and anti-thrombosis (anti-clotting) therapy.
BACKGROUND OF THE INVENTIONDrug therapies for many cancers continue to be inadequate, having either limited efficacy, prohibitive toxicities, or in many cases both. As an example, effective therapies are sorely needed for non-small cell lung cancers (NSCLC), of which there are over 162,000 deaths per year according to the National Cancer Institute. Eighty percent of the over 200,000 new diagnoses of lung cancer each year are non-small cell carcinomas. While some patients are successful candidates for surgical resection or radiation therapy, most patients have disseminated disease at the time of diagnosis and are therefore not candidates for these approaches. Most patients diagnosed in the later stages will need to be treated with a variety of therapies including chemotherapies and biologically targeted therapies, neither of which work well for the majority of patients. Results of standard treatment are poor except for the most localized cancers, and currently, no single chemotherapy or biologic regimen can be recommended for routine use. Furthermore, according to the National Cancer Institute, there are nearly 12,000 new diagnoses of myeloid leukemia and over 9,000 deaths from this cancer each year. Thirty to 40% of patients will not attain complete remission of this disease following standard chemotherapy, and only 25% of those attaining complete remission are expected to live longer than 3 years. Thus as with most cancers, there continues to be a need for new therapies that can keep the cancer in remission and increase survival.
There are several new, biologically targeted agents under investigation for NSCLC and other cancers in the hopes that these new agents will expand the pool of patients who respond to and receive a survival benefit from these therapies. In recent years, inhibition of specific cancer-associated tyrosine kinases has emerged as an important approach for cancer therapy. 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 nearly thirty novel cancer targets extensively studied in the past ten years, one third of these are tyrosine or other kinases. Of the ten truly novel anti-cancer therapies approved in the past five years, five of these 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.
The Axl receptor tyrosine kinase (Axl), originally identified as a protein encoded by a transforming gene from primary human myeloid leukemia cells, is overexpressed in a number of different tumor cell types and transforms NIH3T3 fibroblasts (O′Bryan et al., Mol. Cell Bio. 11:5016-5031 (1991)). Axl 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. Axl is associated with the development and maintenance of various cancers including lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, gliomas, melanoma, prostate cancer, breast cancer, gastric cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer, among others. Furthermore, in some cancer types, particularly non-small cell lung cancer (NSCLC), myeloid leukemia, and gastric cancers, the over-expression of this cell signaling molecule indicates a poor prognosis for the patient. Researchers have found that siRNA knockdown of Axl in NSCLC cell lines reduced invasive capacity of the tumor cells (Holland et al., 2005, Cancer Res. 65:9294-9303). Vajkoczy et al. have shown that expression of a dominant-negative Axl construct decreased brain tumor proliferation and invasion (Vajkoczy et al., 2006, PNAS 15:5799-804; European Patent Publication No. EP 1 382 969 Al). Furthermore, in clinical patient samples of NSCLC, Axl protein over-expression has been statistically associated with lymph node involvement and advanced clinical stage of disease.
Axl signaling also plays important roles in spermatogenesis (Lu et al., 1999, Nature 398:723-728), immunity (Lu and Lemke, 2001, Science 293: 306-311; Scott et al, 2001, Nature 411: 207-211), platelet function (Angelillo-Scherrer et al, 2001, 2005) and even kidney pathology (Yanagita et al, 2002, J Clin Invest 110:239-246).
Axl is related to two other receptor tyrosine kinases, Mer 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)). 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. Vasc. Biol. 24:1118-1123 (2004)). The platelet dysfunction caused by Gas6 is thought to be mediated through the Axl, Mer, 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., 2005, J. Clin Invest. 115:237-246). Therefore, in addition to its association with neoplastic disease, Axl 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. Therefore, there is a continued need in the art 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 can continue to be developed and ultimately used in combination with other agents to affect the best outcome for patients.
SUMMARY OF THE INVENTIONOne embodiment of the invention relates to an Axl inhibitor, wherein the Axl inhibitor is preferably an Axl fusion protein. The Axl fusion protein comprises: (a) a first protein comprising, consisting essentially of, or consisting of, at least a portion of the extracellular domain of an Axl receptor tyrosine kinase (Axl RTK) that binds to an Axl ligand; and (b) a second protein that is a heterologous fusion protein, wherein the second protein is fused to the first protein.
In one aspect, the first protein comprises, consists essentially of, or consists of the Gas6 major binding site of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of the Gas6 major binding site and the Gas6 minor binding site of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of the Ig1 domain of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of the Ig1 domain and the Ig2 domain of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of a portion of the extracellular domain of Axl RTK in which at least one of the FBNIII motifs in the first protein is deleted or mutated of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of a portion of the extracellular domain of Axl RTK in which both of the FBNIII motifs is deleted or mutated of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of, the entire Axl RTK extracellular domain of Axl. In one aspect, the first protein comprises, consists essentially of, or consists of positions 1-445 of Axl RTK, with respect to SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of positions 1-324 or 1-325 of Axl RTK, with respect to SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of position 1 to position 222, 223, 224, or 225 of Axl RTK, with respect to SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of at least: position 10 to position 222, 223, 224, or 225 of Axl RTK, position 20 to position 222, 223, 224, or 225 of Axl RTK, position 30 to position 222, 223, 224, or 225 of Axl RTK, position 40 to position 222, 223, 224, or 225 of Axl RTK, position 50 to position 222, 223, 224, or 225 of Axl RTK, or position 60 to position 222, 223, 224, or 225 of Axl RTK, with respect to SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of: at least positions 63-225 of SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of at least: positions 1-137 of Axl RTK, positions 10-137 of Axl RTK, positions 20-137 of Axl RTK, positions 30-137 of Axl RTK, positions 40-137 of Axl RTK, positions 50-137 of Axl RTK, or positions 60-137 or Axl RTK, with respect to SEQ ID NO:2. In one aspect, the first protein comprises, consists essentially of, or consists of at least positions 63 to 218 of SEQ ID NO:2. In one aspect, the first protein comprises at least positions 63-99, 136, 138, and 211-218 of SEQ ID NO:2, arranged in a conformation that retains the tertiary structure of these positions with respect to the full-length extracellular domain of Axl RTK (positions 1-445 of SEQ ID NO:2).
In any of the above aspects of the invention, the invention the Axl RTK 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 or SEQ ID NO:4. In one aspect, the Axl RTK comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
In any of the above aspects of the invention, the fusion protein can be produced as a dimer of Axl proteins.
In any of the above aspects of the invention, the heterologous fusion protein (the second protein) is an immunoglobulin Fc domain. In one aspect, the immunoglobulin Fc domain consists essentially of or consists of a heavy chain hinge region, a CH2 domain and a CH3 domain. In one aspect, the immunoglobulin Fc domain is from an IgG immunoglobulin protein. In one aspect, the immunoglobulin Fc domain is from an IgG1 immunoglobulin protein. In one aspect, the immunoglobulin Fc domain is from a human immunoglobulin.
In another aspect of any of the above aspects of the invention, the fusion protein can further comprise a third protein, fused to the first or to the second protein. In one aspect, the third protein is a pro-apoptosis protein or an anti-clotting protein.
In any of the above aspects related to an Axl fusion protein of the invention, in one aspect, the Axl ligand is Gash.
In any of the above aspects related to an Axl fusion protein of the invention, in one aspect, the Axl fusion protein binds to the Axl ligand with an equal or greater affinity as compared to a naturally occurring Axl receptor tyrosine kinase. In one aspect, the Axl fusion protein inhibits binding of the Axl ligand to an endogenous Axl receptor tyrosine kinase by at least 50%. In another aspect, the Axl fusion protein inhibits binding of the
Axl ligand to an endogenous Axl receptor tyrosine kinase by at least 60%. In another aspect, the Axl fusion protein inhibits binding of the Axl ligand to an endogenous Axl receptor tyrosine kinase by at least 70%. In another aspect, the Axl fusion protein inhibits binding of the Axl ligand to an endogenous Axl receptor tyrosine kinase by at least 80%.
In any of the above aspects related to an Axl fusion protein of the invention, in one aspect, the Axl fusion protein does not activate Mer or Tyro-3.
Another embodiment of the invention relates to a composition comprising, consisting essentially of, or consisting of any of the Axl fusion proteins described herein. 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 treatment of cancer. In another aspect, the composition further comprises at least one therapeutic agent for treatment of a clotting disorder. In another aspect, the composition further comprises a Mer-Fc or a Tyro-3-Fc. In this latter aspect, preferably, the Mer-Fc does not activate Axl or Tyro-3.
Yet another embodiment of the present invention relates to a method of treating cancer in an individual, comprising administering to the individual any of the Axl fusion proteins or the compositions described herein. In one aspect, the cancer is an Axl-positive cancer. In another aspect, the cancer is a Mer-positive cancer. In another aspect, the cancer is a Tyro-3-positive cancer. In one aspect, the cancer is selected from: lung cancer, myeloid leukemia, uterine cancer, ovarian cancer, gliomas, melanoma, prostate cancer, breast cancer, gastric cancer, osteosarcoma, renal cell carcinoma, or thyroid cancer. In one aspect, the cancer is a leukemia or lymphoma. In another aspect, the cancer is myeloid leukemia. In another aspect, the cancer is non-small cell lung cancer (NSCLC).
Yet another embodiment of the invention relates to a method of treating or preventing a clotting disorder in an individual, comprising administrating to the individual any of the Axl 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 invention relates to the use of any of the Axl fusion proteins or compositions described herein in the preparation of a medicament for the treatment of cancer.
Yet another embodiment of the invention relates to the use of any of the Axl fusion proteins or compositions described herein in the preparation of a medicament for the prevention or treatment of a clotting disorder.
The present invention generally relates to novel inhibitors of the Axl 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. The present inventors describe herein a family of Axl RTK inhibitors and have demonstrated that such therapeutic agents can bind the ligand, Gas6, and inhibit activation of membrane-bound Axl in the A549 non small cell lung cancer (NSCLC) cell line. The inventors propose herein to use these agents as biologic therapeutics for the treatment of many Axl-overexpressing cancers, including NSCLC. Many other human cancers have been found to have over-expression of Axl, including myeloid leukemia, and the novel Axl inhibitors described herein are believed to be useful for the treatment of these cancers. In addition, the inhibitors of the present invention are useful for the treatment of clotting disorders (e.g., as an anti-clotting agent).
More particularly, the present inventors have developed inhibitors of Axl that are capable of preventing Axl activation by sequestration of Axl ligands. In one particular embodiment, the present inventors have developed Axl inhibitors that inhibit the activation of Axl, and do not activate Mer (in the presence of Gas6). Specifically, the inventors have demonstrated that this novel therapeutic can bind the ligand Gas6 and inhibit activation of membrane-bound Axl in the A549 non small cell lung cancer (NSCLC) cell line. It is proposed herein that this Axl ligand “sink” can be used as a biologic therapeutic agent for the sequestration of Axl ligands and accordingly, for the treatment of Axl 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. The inhibitors of the invention are useful for treating both Axl-positive and Mer-positive cancers. In addition, the novel therapeutic agents of the invention are useful in the treatment of clotting disorders (anti-thrombotic therapy).
Axl Inhibitors of the InventionThe invention includes, as one embodiment, an Axl inhibitor, and compositions comprising such inhibitor. The Axl inhibitors of the present invention generally comprise the extracellular domain of Axl or more preferably, a portion thereof (described below), fused (linked, joined) to a fusion partner, e.g., an Fc region of an immunoglobulin, to allow crosslinking. The extracellular domain of Axl or the portion thereof includes at least one domain that binds to and sequesters ligand (at least one ligand binding domain), and/or at least one domain that binds to a TAM receptor (at least one TAM receptor binding domain) directly to inhibit activation and signaling through the TAM receptor (e.g., by preventing/blocking ligand binding or by preventing receptor dimerization, trimerization or formation of any receptor-protein complex). TAM (Tyro-Axl-Mer) receptors include Tyro, Axl, and Mer receptor tyrosine kinases. The inhibitors can be further combined with other therapeutic reagents to enhance or supplement other therapeutic treatments for neoplastic and thrombotic disorders or conditions. Also included in the invention are peptides and mimetics thereof that bind to the ligand binding site of Axl and thereby inhibit the binding of Axl to Gas6 or another ligand. The inhibitors of the invention are described in detail below.
General reference to an “Axl inhibitor” refers to any of the Axl inhibitors described herein, and include the Axl proteins described herein fused to any suitable fusion partner encompassed by the invention. General reference to an Axl-Fc can refer to any Axl protein described herein fused to any Fc portion of an immunoglobulin as described herein. However, in some instances, “Axl-Fc” or “AxlFc” is used to particularly describe a full-length extracellular domain of Axl (described below) fused to an Fc domain. Truncated versions of an Axl extracellular domain as described herein can be denoted by more specific names reflecting the Axl fusion protein. For example, an “Axl Ig/Fc” protein can refer herein to a portion of Axl comprising only the Ig domains, which is fused to an Fc portion.
The Axl RTK is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the Axl protein represents a unique structure in its extracellular region that juxtaposes immunoglobulin (IgL) repeats and fibronectin type III (FNIII) repeats, a structure it shares with TAM (Tyro-Axl-Mer) family members, Mer and Tyro-3.
The Axl gene is evolutionarily conserved between vertebrate species. Indeed, the nucleic acid sequence (genomic and/or mRNA) and amino acid sequence for Axl RTK from several different species are known in the art. There are two transcript variants of Axl. In humans, transcript variant 1 encodes the full-length Axl isoform (isoform 1), and transcript variant 2 lacks exon 10, resulting in a protein (isoform 2) lacking an internal 9 amino acids, but which is otherwise the same as the full length protein encoded by transcript variant 1. The nucleic acid sequence of the transcript variant 1 of human Axl is represented herein by SEQ ID NO:1 (see also NCBI Accession No. NM—021913.2, GI:21536465). SEQ ID NO:1 encodes human Axl isoform 1, represented herein by SEQ ID NO:2 (see also NCBI Accession No. NP—068713.2, GI:21536466). The nucleic acid sequence of the transcript variant 2 of human Axl is represented herein by SEQ ID NO:3 (see also NCBI Accession No. NM—001699.3, GI:21536467). SEQ ID NO:3 encodes human Axl isoform 2, represented herein by SEQ ID NO:4 (see also NCBI Accession No. NP—001690.2, GI:21536468).
The nucleic acid sequence and encoded amino acid sequence of the Axl RTK is also known for mouse (Mus musculus), rat (Rattus norvegicus), dog (Canis familiaris), cow (Bos taurus), chicken (Gallus gallus), and zebrafish (Danio rerio), as well as other vertebrates. The nucleic acid sequence of mouse Axl and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:5 and SEQ ID NO:6, respectively (see also NCBI Accession No. BCO58230.1, GI:34849483). The nucleic acid sequence for rat Axl (transcript variant 1) and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:7 and SEQ ID NO:8, respectively (see also NCBI Accession No. NM—031794.1, GI:93204848). The nucleic acid sequence for chicken Axl and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:9 and SEQ ID NO:10, respectively (see also NCBI Accession No. U70045.1, GI:1572686). The nucleic acid sequence for cow Axl and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:11 and SEQ ID NO:12, respectively (see also NCBI Accession No. XM—594754.3, GI:119910556). The nucleic acid sequence for dog Axl (transcript variant 1) and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:13 and SEQ ID NO:14, respectively (see also NCBI Accession No. XM—541604.2, GI:73947521). The nucleic acid sequence for zebrafish Axl (transcript variant 1) and the amino acid sequence of the protein encoded thereby are represented by SEQ ID NO:15 and SEQ ID NO:16, respectively (see also NCBI Accession No. XM—695874.1, GI:68427805).
Sasaki et al. (Sasaki et al., 2006, EMBO Journal (2006) 25, 80-87) resolved at 3.3A resolution a minimal human Gas6/Axl complex, revealing substantial information regarding the ligand binding structure of Axl. The coordinates and structure factors of the Gash-LG/Axl-IG complex have been deposited in the Protein Data Bank (PDB Accession code 2c5d). With respect to the sequences described below, it is noted that the position numbering in Sasaki et al. starts with a methionine that is 7 amino acids downstream from the first methionine in SEQ ID NO:2 disclosed herein. Therefore, all numbering referenced with respect to Sasaki et al. is based on the Sasaki et al. positions (Sasaki et al., 2006, EMBO Journal (2006) 25, 80-87).
The extracellular domain of human Axl (SEQ ID NO:2 or SEQ ID NO:4) spans amino acid positions from about 1 to about 445, with respect to SEQ ID NO:2, and contains two Ig domains and two FNIII domains. The first Ig domain, denoted herein as Ig1, includes from about position 33 to about position 137 of SEQ ID NO:2). The second Ig domain, denoted herein as Ig2, includes from about position 139 to about position 222 of SEQ ID NO:2. The first FNIII domain, denoted herein as FNIII(a), includes from about position 225 to about position 328 of SEQ ID NO:2. The second FNIII domain, denoted herein as FNIII(b), includes from about position 337 to about position 418 of SEQ ID NO:2. The corresponding domain in other splice variants and species can be readily determined by aligning the sequences. However, the present invention includes Axl-Fc proteins in which the Axl portion of the protein consists of smaller fragments of the extracellular domains than this full-length extracellular domain.
For example, Axl proteins useful in the invention can include any smaller portions (fragments) of the extracellular domain of Axl that retain the ability to bind to an Axl ligand (e.g., Gas6), and/or that retain the ability to bind to a TAM receptor (at least one TAM receptor binding domain) to inhibit activation and signaling through the TAM receptor (e.g., by preventing/blocking ligand binding or by preventing receptor dimerization, trimerization or formation of any receptor-protein complex). Preferably, such portions do not activate Mer. Sasaki et al. (Sasaki et al., 2006, supra) teach that an Axl fragment spanning the two N-terminal Ig domains (denoted Ig1 and Ig2) and lacking carbohydrate modifications (Axl-IG) retains full Gas6-LG binding activity (Gas6-LG is the C-terminal portion of Gas6 required for Axl binding). As taught by Sasaki et al., supra, there are two distinct Gas6/Axl contacts of very different size, denoted therein as the major binding site and the minor binding site, both featuring interactions between edge β-strands. Structure-based mutagenesis, protein binding assays and receptor activation experiments performed by Sasaki et al. demonstrated that both the major and minor Gas6 binding sites are required for productive transmembrane signaling, although for the purposes of creating a ligand sink via an Fc-Axl according to the present invention, where signaling is not required, lesser portions can be used. Sasaki et al., supra, taught that Gas6-mediated Axl dimerization is likely to occur in two steps, with a high-affinity 1:1 Gas6/Axl complex forming first. Only the minor Gas6 binding site is highly conserved in the other Axl family receptors, Tyro3 (also known as Sky and Rse) and Mer. Specificity at the major contact is suggested to result from the segregation of charged and apolar residues to opposite faces of the newly formed β-sheet (Sasaki et al., supra).
According to the present invention, the major binding site lies from about Glu63 to about Val99 in the Ig1 domain (with reference to the numbering in SEQ ID NO:2). Using the numbering in Sasaki et al., supra, the major binding site is represented by Glu56 to Val92 in Sasaki et al., 2006, supra. The minor binding site includes strand G (spanning from about position Lys211 to Thr218 with respect to SEQ ID NO:2 or from Lys204-Thr211, with respect to Sasaki et al., 2006, supra) and also includes a few residues in the linker region (Leu138 and Glu136 with respect to SEQ ID NO:2 or Leu129 and Glu131 with respect to Sasaki et al., supra).
Accordingly, a suitable Axl protein for use in the present invention excludes at least the cytoplasmic domain of Axl, and preferably all or the majority of the transmembrane domain of Axl, and includes a portion of the extracellular domain of Axl, up to the entire extracellular domain. Preferably, the portion of the extracellular domain includes at least the major Gas6 binding surface of Axl, and in other embodiments, includes at least the major and the minor Gas6 binding surface of Axl, and in other embodiments, contains at least the Ig1 and Ig2 domains of Axl, or residues therein that form a conformational structure sufficient to bind to an Axl ligand (e.g., Gas6). Glycosylation of the three predicted glycosylation sites in Axl-Ig (Asn36, Asn150 and Asn191 with respect to Sasaki et al., or Asn43, Asn57, and Asn198, with respect to SEQ ID NO:2) is not required for Gas6 binding. In another embodiment, a suitable portion of the extracellular domain includes at least one Ig domain and two FNIII domains.
In another embodiment, a suitable portion of the extracellular domain of Axl for use in the present invention includes both FNIII domains or a sufficient portion thereof to directly bind to a TAM receptor in a manner that inhibits binding of a ligand to the receptor or prevents receptor dimerization, receptor trimerization, or formation of any receptor-protein complex), but does not include not the Ig domains (i.e., ligand binding domains are not included). Such an Axl protein is believed to be useful for binding to a TAM receptor and preventing ligand binding or complexing of TAM receptors (dimerization, trimerization, or formation of any receptor complex), but does not itself bind ligand.
In one embodiment, a suitable Axl protein for use in an Axl inhibitor of the invention, and particularly an Axl-Fc protein, includes positions 1-445, or a ligand-binding portion thereof, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 1-324 or 1-325, or a ligand-binding portion thereof and/or a TAM binding portion thereof (i.e., sufficient to bind to a TAM receptor inhibit the binding of the natural ligand to its receptor or to inhibit the complexing of the receptor), with respect to SEQ ID NO:2. In any of the above-embodiments, the portion can be shorter than position 324 or 325 (e.g., 323, 322, 321, etc.), or extend beyond position 324 or 325 to any higher position within the extracellular domain of Axl, in whole number increments (e.g., 326, 327, . . . 398 . . . 445).
In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 1 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 10 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 20 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 30 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 33 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 40 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 50 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 60 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 63 to about position 222, 223, 224 or 225, with respect to SEQ ID NO:2. In any of the above-embodiments, the portion can be shorter than 222, 223, 224 or 225 (e.g., 221, 220, etc.), or extend beyond position 222, 223, 224 or 225 to any higher position within the extracellular domain of Axl, in whole number increments (e.g., 226, 227, 228, . . . 230 . . . 445).
In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 1 to 137, positions 10 to 137, positions 20 to 137, positions 30 to 137, positions 40 to 137, or positions 50 to 137, with respect to SEQ ID NO:2.
In another embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises, consists essentially of, or consists of positions 63 to 218 of SEQ ID NO:2 or any additional 1-20 amino acids on either side of these positions. In one embodiment, a suitable Axl protein for use in the Axl inhibitor of the invention, and particularly an Axl-Fc protein, comprises positions 63-99, 136, 138, and 211-218 of SEQ ID NO:2, arranged in a conformation that retains the tertiary structure of the full Axl extracellular domain with respect to the major and minor binding sites.
Fragments within any of these specifically defined fragments are encompassed by the invention, provided that, in one embodiment, the fragments retain ligand binding ability of Axl, preferably with an affinity sufficient to compete with the binding of the ligand to its natural receptor (e.g., naturally occurring Axl) and provide inhibition of a biological activity of Axl 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 Gas6 is retained. While both full-length and truncated forms of the Axl extracellular domains are effective to sequester Gas6, truncated forms that do not activate Mer are preferred for use in the invention. Fragments within any of the above-defined fragments are also encompassed by the invention if they additionally (ligand binding also required), or alternatively (ligand binding not retained), retain the ability to bind to a TAM receptor (at least one TAM receptor binding domain) sufficient to inhibit activation and signaling through the TAM receptor (e.g., by preventing/blocking ligand binding or by preventing receptor dimerization, trimerization or formation of any receptor-protein complex).
Assays for measuring binding affinities are well-known in the art. In one embodiment, a BlAcore machine can be used to determine the binding constant of a complex between the target protein (e.g., an Axl-Fc) and a natural ligand (e.g., Gas6). For example, the Axl inhibitor can be immobilized on a substrate. A natural or synthetic ligand is contacted with 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 (O′Shannessy et al. Anal. Biochem. 212:457-468 (1993); Schuster et al., Nature 365:343-347 (1993)). Contacting a second compound (e.g., a different ligand or a different Axl protein) at various concentrations at the same time as the first ligand 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.
In one embodiment, all or a portion of one or both of the FNIII sites of Axl can be deleted or mutated, as well as any intervening linker regions in the extracellular domain of Axl. Again, any deletions or other mutations (substitutions, additions, etc.) are encompassed by the invention, provided that the ligand-binding ability of the Axl-containing protein is retained. Accordingly, the present invention includes the deletion of at least one amino acid from one or both of the FNIII sites, up to all of the amino acids within one or both of the FNIII sites, in whole integers (e.g., one, two, three, four, five, six, seven, eight, nine, ten . . . twenty . . . thirty, etc. deleted amino acids).
In another embodiment, one or both of the FNIII sites of Axl are retained, and may include intervening linker regions in the extracellular domain of Axl. In one aspect of this embodiment, the Axl inhibitor includes only one or both of the FNIII sites of Axl, and more particularly, does not include one or both Ig domains. Such an inhibitor does not bind ligand, but should retain the ability to bind to a TAM receptor (at least one TAM receptor binding domain) sufficient to inhibit activation and signaling through the TAM receptor (e.g., by preventing/blocking ligand binding or by preventing receptor dimerization, trimerization or formation of any receptor-protein complex).
As discussed above, an Axl inhibitor of the invention typically includes a soluble form of Axl that is linked to a fusion partner that permits the formation of a dimer of Axl proteins (e.g., an Fc region of an immunoglobulin protein, other fusion partners that cause dimerization). In one embodiment, an Axl inhibitor of the invention includes a soluble form of Axl that is linked to a fusion partner that allows binding of a ligand without dimerization of the Axl proteins. As used herein, the term “soluble form” of Axl, “sAxl” or “soluble Axl” refers to an Axl receptor tyrosine kinase that does not contain cytoplasmic domains, and preferably no or little of the transmembrane domains of the natural protein (e.g., SEQ ID NO:2), and that includes any portion of the extracellular domain of Axl (described above) that has the ability to bind to an Axl ligand, e.g., a ligand including, but not limited to, Gash. There are multiple soluble forms of Axl that are operable in the invention. Structural and functional features required of these forms are discussed above. The soluble form of Axl is preferably generated by recombinant means, whereby a construct encoding an entire Axl-Fc protein is produced, although a soluble form of Axl can be generated by post-translational proteolytic cleavage and then later joined with an Fc domain, if desired.
According to the present invention, an Fc protein or fragment (also referred to as Fc domain or Fc region of an immunoglobulin) is a portion of an immunoglobulin (also referred to herein as antibody) lacking the ability to bind to antigen. More particularly, the Fc region (from “Fragment, crystallizable”) of an immunoglobulin, is derived from the constant region domains of an immunoglobulin and is generally composed of two heavy (H) chains that each contribute between two and three constant domains (depending on the isotype class of the antibody), also referred to as CH domains. The Fc region, as used herein, preferably includes the “hinge” region of an immunoglobulin, which joins the two heavy (H) chains to each other via disulfide bonds. Alternatively, if the hinge region is not included, then the Fc region is designed with a region that otherwise links the two heavy chains together, since the Axl-Fc protein is produced as a dimer of Axl extracellular domains (e.g., see U.S. Patent No. 6,323,323 for a generic description of a method for producing dimerized polypeptides).
There are five major H chain classes referred to as isotypes, and accordingly, an Fc protein used in the present invention may be derived from any one of these five classes. The five classes include immunoglobulin M (IgM or μ), immunoglobulin D (IgD or δ), immunoglobulin G (IgG or γ), immunoglobulin A (IgA or α), and immunoglobulin E (IgE or ε). The distinctive characteristics between such isotypes are defined by the constant domain of the immunoglobulin. Human immunoglobulin molecules comprise nine isotypes, IgM, IgD, IgE, four subclasses of IgG including IgG1 (γ1), IgG2 (γ2), IgG3 (γ3) and IgG4 (γ4), and two subclasses of IgA including IgAl (α1) and IgA2 (α2). The nucleic acid and amino acid sequences of immunoglobulin proteins and domains, including from all isotypes, are well-known in the art in a variety of vertebrate species. Preferably, the Fc region used in the Axl-Fc protein is from the same animal species as the Axl portion of the protein and most preferably, is from the same animal species as the animal species in which the Axl-Fc protein is to be used in vivo. For example, for use in humans, it is preferred that a human Axl protein and a human Fc protein are fused.
However, to the extent that Axl from one species will bind Gash from a different species and may be tolerated for use in such species, such cross-use is encompassed by the invention.
Fc regions used in the Axl-Fc proteins of the present invention include any Fc region. Preferred Fc regions include the hinge region and the CH2 and CH3 domains of IgG, and preferably, IgG1, although Fc regions of other immunoglobulins can be used. Preferably, the Fc protein does not interfere with the ability of the Axl-Fc protein to remain soluble and circulate in vivo, and does not interfere with the ability of the Axl portion to bind to its ligand. As discussed above, a suitable Fc protein may or may not include the hinge region of the immunoglobulin, but if not, should be otherwise capable of being linked to another Fc protein so that the Axl portion of the fusion protein can be expressed as a dimer.
The Axl inhibitors useful in the present invention may also be produced using a different fusion partner than the Fc region of an immunoglobulin, and are referred to generally as Axl fusion proteins. Suitable candidates include any protein (any fusion partner) that, when fused to the Axl protein described above, allows the Axl fusion protein to be produced as a dimer, does not interfere with the binding of Axl to its ligand, and allows the Axl 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, an Axl protein of the invention can be produced as a dimer by expressing two copies of the Axl protein as single peptide chains 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.
In one embodiment, a suitable fusion partner candidate does not interfere with the binding of Axl to its ligand, and/or does not necessarily allow the Axl fusion protein to be produced as a dimer. In another embodiment, Axl inhibitors can include fusion partners that improve the stability of the fusion protein, including, but not limited to, e.g., human serum albumin or the C-terminal sequence of the chorionic gonadotropin beta subunit.
Other suitable fusion partners for stabilizing a protein will be known to those of skill in the art.
A fusion (or chimeric) protein comprising an Axl protein and an Fc protein (or other suitable fusion partner) as described above is typically and preferably produced or constructed using recombinant technology, although the proteins can also be produced separately and then joined after expression using chemical conjugation. Fusion proteins suitable for use in the invention comprise a suitable Axl protein of the invention (described above) operatively linked to a heterologous protein or polypeptide (i.e., having an amino acid sequence not substantially homologous to the Axl polypeptide), which is a fusion segment or fusion partner (e.g., an Fc protein). “Operatively linked” indicates that the Axl protein and the heterologous fusion partner are fused in-frame. The fusion partner can be fused to the N-terminus or C-terminus of the Axl protein. Fusion proteins can be produced by standard recombinant DNA techniques well known in the art. Preferred fusion partners according to the present invention include, but are not limited to, any proteins or peptides that can: enhance a protein's stability; allow the Axl protein to be produced as a dimer; and/or assist with the purification of a protein (e.g., by affinity chromatography), or in some embodiments, provide another protein function. A suitable heterologous fusion partner can be a domain of any size that has the desired function. Preferably, the fusion partner is an Fc protein.
Axl-Fc proteins that have been produced and accordingly exemplify the invention include: an Fc region consisting of a hinge region, Cm and CH2 domain, fused to: (1) an Axl protein selected from positions 1 to 445 of human Axl (SEQ ID NO:2) (also referred to herein as AxlFc); (2) to positions 1 to 325 of human Axl (also referred to herein as Axl IgNFl/Fc); or (3) to positions 1 to 225 of human Axl (also referred to herein as AxlIgFc or AxlIg/Fc).
The present inventors have shown that two Axl-Fc inhibitors of the invention directly bind Gas6 (
Furthermore, the present inventors have shown that Mer is activated (p-Mer) in cells by Axl-Fc inhibitors of the invention (fusions comprising the full-length extracellular domain of Axl) in the absence of added Gas6 ligand. However, AxlIg/Fc, which does not include the FNIII domains of Axl, does not activate Mer. Therefore, the inventors have discovered a preferred Axl inhibitor that sequesters Gas6 and thereby inhibits ligand-mediated activation of both Axl and Mer, but does not activate Mer itself. Accordingly, preferred Axl fusion proteins of the invention include less than the full-length extracellular domain of Axl, and specifically, do not activate Mer, while retaining the ability to sequester Gas6 ligand.
Accordingly, general embodiments of the present invention described in more detail below pertain to any isolated polypeptides described herein, including various portions of full-length Axl, and including those expressed by nucleic acids encoding Axl or a portion or variant thereof.
As used herein, reference to an isolated protein or polypeptide in the present invention, including an isolated Axl protein, includes full-length proteins, fusion proteins, or any fragment or other homologue (variant) of such a protein. The amino acid sequence for Axl from several vertebrate species, including human, are described herein as exemplary Axl proteins (see above). Reference to a Axl protein can include, but is not limited to, purified Axl protein, recombinantly produced Axl protein, membrane bound Axl protein, Axl protein complexed with lipids, soluble Axl protein, an Axl fusion protein, a biologically active homologue of an Axl protein, and an isolated Axl protein associated with other proteins. More specifically, an isolated protein, such as an Axl protein, 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 Axl protein” refers to a Axl protein (generally including a homologue of a naturally occurring Axl protein) from a human (Homo sapiens) or to a Axl protein that has been otherwise produced from the knowledge of the structure (e.g., sequence) and perhaps the function of a naturally occurring Axl protein from Homo sapiens. In other words, a human Axl protein includes any Axl protein that has substantially similar structure and function of a naturally occurring Axl protein from Homo sapiens or that is a biologically active (i.e., has biological activity) homologue of a naturally occurring Axl protein from Homo sapiens as described in detail herein. As such, a human Axl protein 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 Axl (or nucleic acid sequences) described herein. An isolated protein useful as an antagonist or agonist 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 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 can include an agonist of a protein or an antagonist of a protein. A homologue of a human Axl protein can include a non-human Axl protein (i.e., an Axl protein from a different species).
Variants or homologues include a substantially homologous polypeptide encoded by the same genetic locus in an organism, i.e., an allelic variant, as well as other splicing variants. A naturally occurring allelic variant of a nucleic acid encoding a protein is a gene that occurs at essentially the same locus (or loci) in the genome as the gene which encodes such protein, but which, due to natural variations caused by, for example, mutation or recombination, has a similar but not identical sequence. Allelic variants typically encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. One class of allelic variants can encode the same protein but have different nucleic acid sequences due to the degeneracy of the genetic code. Allelic variants can also comprise alterations in the 5′ or 3′ untranslated regions of the gene (e.g., in regulatory control regions). Allelic variants are well known to those skilled in the art.
The terms variant or homologue may also encompass polypeptides derived from other genetic loci in an organism, but having substantial homology to any of the previously defined soluble forms of the extracellular Axl receptor tyrosine kinase, or polymorphic variants thereof. Variants also include polypeptides substantially homologous or identical to these polypeptides but derived from another organism. Variants also include polypeptides that are substantially homologous or identical to these polypeptides that are produced by chemical synthesis.
In one embodiment, a Axl homologue comprises, consists essentially of, or consists of, an amino acid sequence that is at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95% identical, or at least about 95% identical, or at least about 96% identical, or at least about 97% identical, or at least about 98% identical, or at least about 99% identical (or any percent identity between 45% and 99%, in whole integer increments), to a naturally occurring Axl amino acid sequence or to any of the extracellular fragments of a naturally occurring Axl amino acid sequence as described herein. A homologue of Axl differs from a reference (e.g., wild-type) Axl protein and therefore is less than 100% identical to the reference Axl at the amino acid level.
As used herein, unless otherwise specified, reference to a percent (%) identity refers to an evaluation of homology which is performed using: (1) a BLAST 2.0 Basic BLAST homology search using blastp for amino acid searches and blastn for nucleic acid searches with standard default parameters, wherein the query sequence is filtered for low complexity regions by default (described in Altschul, S. F., Madden, T. L., Schääffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.” Nucleic Acids Res. 25:3389-3402, incorporated herein by reference in its entirety); (2) a BLAST 2 alignment (using the parameters described below); (3) and/or PSI-BLAST with the standard default parameters (Position-Specific Iterated BLAST. It is noted that due to some differences in the standard parameters between BLAST 2.0 Basic BLAST and BLAST 2, two specific sequences might be recognized as having significant homology using the BLAST 2 program, whereas a search performed in BLAST 2.0 Basic BLAST using one of the sequences as the query sequence may not identify the second sequence in the top matches. In addition, PSI-BLAST provides an automated, easy-to-use version of a “profile” search, which is a sensitive way to look for sequence homologues. The program first performs a gapped BLAST database search. The PSI-BLAST program uses the information from any significant alignments returned to construct a position-specific score matrix, which replaces the query sequence for the next round of database searching. Therefore, it is to be understood that percent identity can be determined by using any one of these programs.
Two specific sequences can be aligned to one another using BLAST 2 sequence as described in Tatusova and Madden, (1999), “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250, incorporated herein by reference in its entirety. BLAST 2 sequence alignment is performed in blastp or blastn using the BLAST 2.0 algorithm to perform a Gapped BLAST search (BLAST 2.0) between the two sequences allowing for the introduction of gaps (deletions and insertions) in the resulting alignment. For purposes of clarity herein, a BLAST 2 sequence alignment is performed using the standard default parameters as follows.
For blastn, using 0 BLOSUM62 matrix:
Reward for match=1
Penalty for mismatch 32 −2
Open gap (5) and extension gap (2) penalties
gap x_dropoff (50) expect (10) word size (11) filter (on)
For blastp, using 0 BLOSUM62 matrix:
Open gap (11) and extension gap (1) penalties
gap x_dropoff (50) expect (10) word size (3) filter (on).
In one embodiment of the present invention, any of the amino acid sequences described herein, including homologues of such sequences (e.g., Axl extracellular domains), can be produced with from at least one, and up to about 20, 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 fragments of full-length Axl proteins of the invention. The invention also encompasses fragments of the variants of the polypeptides described herein. As used herein, a fragment comprises at least 6 contiguous amino acids and includes any fragment of a full-length Axl protein described herein, and more preferably includes the entire extracellular domain of Axl or any portion thereof that retains the ability to bind to a Axl ligand (described in detail above). 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 (e.g., 7, 8, 9 . . . 67, 68, 69 . . . 278, 279, 280 . . . amino acids).
As used herein, the phrase “Axl agonist” refers to any compound that is characterized by the ability to agonize (e.g., stimulate, induce, increase, enhance, or mimic) the biological activity of a naturally occurring Axl as described herein, and includes any Axl homologue, binding protein (e.g., an antibody), agent that interacts with Axl or mimics Axl, or any suitable product of drug/compound/peptide design or selection which is characterized by its ability to agonize (e.g., stimulate, induce, increase, enhance) the biological activity of a naturally occurring Axl protein in a manner similar to the natural agonist, Axl.
Similarly, the phrase, “Axl antagonist” refers to any compound which inhibits (e.g., antagonizes, reduces, decreases, blocks, reverses, or alters) the effect of an Axl agonist as described above. More particularly, a Axl antagonist is capable of acting in a manner relative to Axl activity, such that the biological activity of the natural agonist Axl, is decreased in a manner that is antagonistic (e.g., against, a reversal of, contrary to) to the natural action of Axl. Such antagonists can include, but are not limited to, a protein (e.g., soluble Axl, including the Axl-Fc proteins of the invention), peptide, or nucleic acid (including ribozymes, RNAi, aptamers, and antisense), antibodies and antigen binding fragments thereof, or product of drug/compound/peptide design or selection that provides the antagonistic effect.
Homologues of Axl, including peptide and non-peptide agonists and antagonists of Axl (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., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., 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, 1997, Methods Enzymol. 289:3-13; Wade et al., 1993, Australas Biotechnol. 3(6):332-336; Wong et al., 1991, Experientia 47(11-12):1123-1129; Carey et al., 1991, Ciba Found Symp. 158:187-203; Plaue et al., 1990, Biologicals 18(3):147-157; Bodanszky, 1985, Int. J. Pept. Protein Res. 25(5):449-474; or H. Dugas and C. Penney, BIOORGANIC CHEMISTRY, (1981) at pages 54-92, 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 polypeptides (including 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 desired function 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 Axl protein, including a biologically active homologue or fragment thereof, has at least one characteristic of biological activity of activity a wild-type, or naturally occurring Axl protein. Biological activity of Axl and methods of determining the same have been described previously herein. A particularly preferred Axl protein for use in the present invention is an Axl protein variant that binds a ligand of Axl. Signaling function is not required for most of the embodiments of the invention and indeed, is not desired in the case of an Axl fusion protein that is an Axl inhibitor as described herein. In one aspect, the Axl protein binds to any ligand of naturally occurring Axl, including Gash. In one aspect, the Axl protein binds to Protein S. In another aspect, the Axl protein preferentially binds to one Axl ligand as compared to another Axl ligand. In one aspect, the Axl protein does not activate Mer. In one aspect, the Axl protein binds to a TAM receptor, preferably sufficiently to inhibit the activation of the TAM receptor (e.g., such as by blocking or inhibiting the binding of a natural ligand to the TAM receptor and/or inhibiting receptor dimerization, trimerization or formation of any receptor-protein complex). In this aspect, ligand binding by the Axl protein can be retained or not retained. Most preferably, an Axl protein of the invention includes any Axl protein and preferably any Axl fusion protein with improved stability and/or half-life in vivo that is a competitive inhibitor of Axl (e.g., that preferentially binds to an Axl ligand as compared to an endogenous Axl cellular receptor). Such fusion proteins have been described in detail above.
Preferably, an Axl inhibitor of the invention, including an Axl fusion protein (e.g., an Axl-Fc fusion protein), binds to an Axl ligand with an equal or greater affinity as compared to the binding of the ligand to a naturally occurring Axl receptor tyrosine kinase (e.g., an Axl RTK expressed endogenously by a cell). In one embodiment, the Axl fusion protein inhibits the binding of an Axl ligand to a naturally occurring Axl receptor tyrosine kinase (or to a Mer or Tyro-3 receptor tyrosine kinase) and subsequent activation of the Axl RTK. For example, one can measure the Axl RTK activation using a phospho-Axl analysis by Western blot. In one embodiment, binding of an Axl ligand to a naturally occurring Axl receptor tyrosine kinase is inhibited 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.
Axl 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, Axl fusion proteins, in addition to comprising the Axl protein and fusion partner as described above, 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 Axl fusion protein 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 Axl inhibitors described herein, and may additional comprise one or more pharmaceutical carriers or other therapeutic agents.
In one aspect, the Axl 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. Common suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
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 compound 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., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp. 353-365 (1989). To reduce its systemic side effects, this may be a preferred method for introducing the compound.
In yet another embodiment, the therapeutic compound can be delivered in a controlled release system. For example, a polypeptide 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 said 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, an Axl fusion protein inhibitor (e.g., Axl-Fc) can be provided in a composition with or administered with a Mer fusion protein (e.g., Mer-Fc) or a Tyro-3 fusion protein (e.g., Tyro-3-Fc). Mer-Fc proteins are described in detail in PCT Patent Publication No. WO 2006/058202, incorporated herein by reference in its entirety. A preferred Mer-Fc protein does not activate Axl. A preferred Axl-Fc protein does not activate Mer.
Nucleic Acid Molecules Encoding Axl Proteins and Other Proteins of the InventionAnother embodiment of the invention relates to an isolated nucleic acid molecule, or complement thereof, encoding any of the Axl 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 RNA, for example, mRNA, 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%, 95%). 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 on pages 2.10.1-2.10.16 and pages 6.3.1-6.3.6 in Current Protocols in Molecular Biology (Ausubel, F. M. et al., “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., ibid. to calculate the appropriate hybridization and wash conditions to achieve these particular levels of nucleotide mismatch. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10° C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 6×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:RNA hybrids include hybridization at an ionic strength of 6×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, Tm can be calculated empirically as set forth in Sambrook et al., supra, pages 9.31 to 9.62. 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 Tm 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 Tm of the particular hybrid. One example of hybridization conditions suitable for use with DNA:DNA hybrids includes a 2-24 hour hybridization in 6×SSC (50% formamide) at about 42° C., followed by washing steps that include one or more washes at room temperature in about 2×SSC, followed by additional washes at higher temperatures and lower ionic strength (e.g., at least one wash as about 37° C. in about 0.1×-0.5×SSC, followed by at least one wash at about 68° C. in about 0.1×-0.5×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. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols. A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, Calif., 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. Pat. 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 and Wallace, Genomics, 4:560 (1989), Landegren et al., Science, 241:1077 (1988)), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989)), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 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, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 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) which 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 which 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 which 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 InventionThe present invention also relates to methods of treatment (prophylactic and/or therapeutic) for Axl-positive cancers, for Mer-positive cancers, and/or for clotting disorders, using the Axl 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 Axl or of its related receptors, Mer 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) Axl 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 Axl receptor tyrosine kinase or its ligands. The Axl inhibitors of the present invention block the activation of the full length native Axl by binding to Axl ligands including, but necessarily limited to, Gas6. Therefore, an effective amount of an inhibitor of a Gas6 receptor which is provided in the form of the Axl inhibitors described herein may be used as a treatment for diseases and conditions associated with Axl expression, as well as with Tyro-3 expression and/or Mer expression.
Accordingly, the method of the present invention preferably modulates the activity of Axl receptor tyrosine kinases, and specifically those that are naturally expressed by the cells of an individual (including an individual that has an Axl-associated disease or condition). The method of the invention for example, involves contacting a cell, tissue or system of an individual with an Axl inhibitor that modulates one or more of the activities of Axl. The Axl inhibitors act as competitive inhibitors of Axl 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, modulation of Axl 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 Axl expressed by platelets. Inhibition is provided by the present invention through the administration of the Axl inhibitor(s) described herein (e.g., Axl-Fc), which bind directly to Axl ligands and competitively inhibit the binding of such ligands to Axl, Mer, or Tyro-3, and therefore inhibit the activity of such receptors. The Axl inhibitor can be administered alone or together with another therapeutic agent, such as another anti-clotting agent. In one embodiment, the Axl inhibitor is administered together with an agent that inhibits the expression or biological activity of Mer. One such agent is a Mer-Fc protein, wherein the Mer-Fc protein 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, comprising modulating the level of Gas6 ligand that is available for interaction with an endogenous Axl RTK in the blood.
An effective amount of an Axl inhibitor to administer to an individual is any amount that achieves any detectable inhibition of the natural Axl receptor in the patient, or any detectable reduction in at least one symptom of the clotting disorder.
As discussed above, Axl 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 Axl activity as part of a therapeutic strategy which 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 the
Axl inhibitor(s) described herein (e.g., Axl-Fc), which bind directly to Axl ligands and competitively inhibit the binding of such ligands to Axl, Mer, or Tyro-3, and therefore inhibit the activity of such receptors. The Axl inhibitor can be administered alone or together with another therapeutic agent, such as another anti-clotting agent. In one embodiment, the Axl inhibitor is administered together with an agent that inhibits the expression or biological activity of Mer. One such agent is a Mer-Fc protein, wherein the Mer-Fc protein does not activate Axl.
Cancers that can be treated by the method of the invention include, but are not limited to, lung cancer (including, but not limited, to non-small cell lung cancer), myeloid leukemia, uterine cancer, ovarian cancer, gliomas, melanoma, prostate cancer, breast cancer, gastric cancer, colon cancer, osteosarcoma, renal cell carcinoma, and thyroid cancer. Because Axl-Fc of the present invention acts as a ligand “sink” for Gas6 and other ligands of the TAM family, the composition and method of the invention are useful for the treatment of not only any cancer in which Axl is expressed, but also any cancer in which Mer and/or Tyro-3 are expressed.
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 an Axl inhibitor of the invention typically comprises between about 0.01 microgram×kilogram−1 and about 10 milligram×kilogram−1 body weight of an animal. A more preferred single dose of such an agent comprises between about 1 microgram×kilogram−1 and about 10 milligram×kilogram−1 body weight of an animal. An even more preferred single dose of an agent comprises between about 5 microgram×kilogram−1 and about 7 milligram×kilogram−1 body weight of an animal. An even more preferred single dose of an agent comprises between about 10 microgram×kilogram−1 and about 5 milligram×kilogram−1 body weight of an animal. Another particularly preferred single dose of an agent comprises between about 0.1 microgram×kilogram−1 and about 10 microgram×kilogram−1 body weight of an animal, if the agent is delivered parenterally.
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.
Each publication or patent cited herein is incorporated herein by reference in its entirety.
EXAMPLES Example 1The following example demonstrates that Axl-Fc inhibitors of the invention bind to Gas6 and compete with Axl RTK for Gas6 ligand, preventing the activation of the Axl oncogene in cells.
The inventors have produced two Axl-Fc inhibitors. The first, the amino acid sequence of which is represented herein by SEQ ID NO:17, includes the entire Axl extracellular domain (i.e., positions 1 to 445 of SEQ ID NO:2), fused to the human IgG1 Fc domain, including the hinge, CH2 and CH3 regions. This Axl inhibitor is also referred to herein as Axl-Fc or AxlFc. The second Axl-Fc protein encodes the two IgG-like domains (positions 1-225 of SEQ ID NO:2) fused in the same manner to the human IgG1 Fc domain described above. This Axl inhibitor is also referred to herein as Axl Ig/Fc or AxlIgFc. Stable cell lines expressing these chimeric proteins are made in CHO cells. The CHO cells are grown in suspension culture in serum-free, protein-free, medium and the secreted Axl-Fc is purified from the medium using Protein A Sepharose® chromatography.
Axl activation occurs following binding of the Axl receptor to the Gas6 ligand. This interaction causes Axl dimerization and auto-phosphorylation (see
The Gas6 ligand can also bind an Axl-Fc protein, as is demonstrated in pulldown assays (see
Furthermore, Axl-Fc can successfully compete with Axl receptor for the Gas6 ligand, and the sequestration of Gas6 by Axl-Fc prevents activation of the Axl oncogene on A549 NSCLC cells (see
Together, these data demonstrate that Axl-Fc is a successful inhibitor and Axl activation in NSCLC and is expected to be capable of blocking the oncogenic activity of Axl.
Example 2The following example demonstrates that an Axl-Fc inhibitor of the invention inhibits platelet aggregation and prolongs clotting time.
Referring to
Referring to
The following example describes the effect on proliferation, survival, and invasiveness in Axl-positive non small cell cancer lung cell lines following inhibition of Axl activity with Axl-Fc.
The Axl-overexpressing A549 non-small cell lung cancer cell is treated with varying concentrations of Axl-Fc (50 -150 nM). Inhibition of Axl activation is detected by western blots by probing for phospho-Axl as shown in
The results of these assays are expected to demonstrate that Axl-Fc inhibits proliferation, survival, and invasiveness of Axl-positive non small cell cancer lung cells.
Example 4The following example describes the determination of the effect of Axl-Fc treatment on cancer development and overall survival in a NSCLC xenograft mouse model.
Varying concentrations (104 to 107) of A549 NSCLC cells are injected into the flank or intratracheally into nude mice. Mice are treated with 2.5-10 mg/kg Axl-Fc (or control Ret-Fc) injected I.P. twice per week. Tumors on flank of mice treated with Axl-Fc are compared to controls. Following 21 days of treatment for mice instilled with A549 cells intratracheally for orthotopic tumor model, mice are euthanized and tumor size measured. The orthotopic tumor model will be replicated using a luciferase labeled A549 cell line (Xenogen) and serial imaging is performed in vivo using the IVIS Imaging System 200. The bioluminescence imaging allows analysis of Axl-Fc efficacy over a range of treatment times.
The results of these experiments are expected to demonstrate that Axl-Fc inhibits tumor growth or reduces tumor burden, and/or increases survival of mice with tumors.
Example 5The following example demonstrates that both AxlFc and AxlIgFc bind to Gas6 ligand.
In this experiment, AxlFc, composed of the entire extracellular domain of Axl fused to Fc domain of human immunoglobulin (IgG) (SEQ ID NO:17), was expressed in HEK293 cells and was detected as a protein of approximately 115 kD when analyzed by Western blot (
The following example demonstrates that Axl Ig/Fc Does Not Activate Mer.
In this experiment, the results of which are shown in
The following example demonstrates that Axl Ig/Fc blocks ligand-mediated activation of Axl and Mer.
Referring to
Referring to
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
1. An Axl fusion protein comprising:
- a) a first protein comprising, consisting essentially of, or consisting of, at least a portion of the extracellular domain of an Axl receptor tyrosine kinase (Axl RTK) that binds to an Axl ligand; and
- b) a second protein that is a heterologous fusion protein, wherein the second protein is fused to the first protein.
2. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of the Gas6 major binding site of Axl.
3. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of the Gas6 major binding site and the Gas6 minor binding site of Axl.
4. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of the Ig1 domain of Axl.
5. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of the Ig1 domain and the Ig2 domain of Axl.
6. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of a portion of the extracellular domain of Axl RTK in which at least one of the FBNIII motifs in the first protein is deleted or mutated of Axl.
7. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of a portion of the extracellular domain of Axl RTK in which both of the FBNIII motifs is deleted or mutated of Axl.
8. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of, the entire Axl RTK extracellular domain of Axl.
9. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of positions 1-445 of Axl RTK, with respect to SEQ ID NO:2.
10. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of positions 1-325 of Axl RTK, with respect to SEQ ID NO:2.
11. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of positions 1-225 of Axl RTK, with respect to SEQ ID NO:2.
12. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of at least: positions 10-222 of Axl RTK, positions 20-222 of Axl RTK, positions 30-222 of Axl RTK, positions 40-222 of Axl RTK, positions 50-222 of Axl RTK, or positions 60-222 of Axl RTK, with respect to SEQ ID NO:2.
13. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of at least: positions 10-225 of Axl RTK, positions 20-225 of Axl RTK, positions 30-225 of Axl RTK, positions 40-225 of Axl RTK, positions 50-225 of Axl RTK, or positions 60-225 of Axl RTK, with respect to SEQ ID NO:2.
14. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of: at least positions 63-225 of SEQ ID NO:2.
15. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of at least: positions 1-137 of Axl RTK, positions 10-137 of Axl RTK, positions 20-137 of Axl RTK, positions 30-137 of Axl RTK, positions 40-137 of Axl RTK, positions 50-137 of Axl RTK, or positions 60-137 or Axl RTK, with respect to SEQ ID NO:2.
16. The Axl fusion protein of claim 1, wherein the first protein comprises, consists essentially of, or consists of at least positions 63 to 218 of SEQ ID NO:2.
17. The Axl fusion protein of claim 1, wherein the first protein comprises at least positions 63-99, 136, 138, and 211-218 of SEQ ID NO:2, arranged in a conformation that retains the tertiary structure of these positions with respect to the full-length extracellular domain of Axl RTK (positions 1-445 of SEQ ID NO:2).
18. The Axl fusion protein of claim 1, wherein the Axl RTK comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:2 or SEQ ID NO:4.
19. The Axl fusion protein of claim 1, wherein the Axl RTK comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or SEQ ID NO:4.
20. The Axl fusion protein of claim 1, wherein the Axl RTK comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2 or SEQ ID NO:4.
21. The Axl fusion protein of claim 1, wherein the Axl RTK comprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
22. The Axl fusion protein of claim 1, wherein the fusion protein is produced as a dimer of Axl proteins.
23. The Axl fusion protein of claim 1, wherein the heterologous fusion protein is an immunoglobulin Fc domain.
24. The Axl fusion protein of claim 23, wherein the immunoglobulin Fc domain consists essentially of or consists of a heavy chain hinge region, a CH2 domain and a CH3 domain.
25. The Axl fusion protein of claim 23, wherein the immunoglobulin Fc domain is from an IgG immunoglobulin protein.
26. The Axl fusion protein of claim 23, wherein the immunoglobulin Fc domain is from an IgG1 immunoglobulin protein.
27. The Axl fusion protein of claim 23, wherein the immunoglobulin Fc domain is from a human immunoglobulin.
28-37. (canceled)
38. An Axl fusion protein comprising:
- a) a first protein comprising, consisting essentially of, or consisting of, at least a portion of the extracellular domain of an Axl receptor tyrosine kinase (Axl RTK) that binds to a receptor selected from the group consisting of Axl, Tyro and Mer and inhibits the binding of a ligand to said receptor or inhibits dimerization, trimerization or formation of any receptor-protein complex of said receptor; and
- b) a second protein that is a heterologous fusion protein, wherein the second protein is fused to the first protein.
39. The Axl fusion protein of claim 38, wherein the first protein consists of the FNIII domains of Axl.
40-62. (canceled)
Type: Application
Filed: Dec 29, 2014
Publication Date: Jul 2, 2015
Inventors: Douglas Kim GRAHAM (Aurora, CO), Susan Louise SATHER (Denver, CO)
Application Number: 14/584,555
