Axl signalling inhibition in antimetastatic therapy
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology, more specifically to AXL signalling pathway inhibitors, and can be used in medicine. What is produced is a soluble AXL polypeptide version free from AXL transmembrane domain, which contains at least one amino acid modification in position No. n, wherein n is specified in 32, 72, 87, 92 or 127 or a combination thereof, wherein n+7 is described by numbering SEQ ID NO: 1 that are wild-type AXL sequences, wherein the above modification increases a AXL polypeptide binding affinity to protein 6 specifically inhibiting the growth (GAS6), which is twice as strong as the wild-type AXL polypeptide affinity. The polypeptide can be fused with Fc fragment and used in a method of treating, reducing or preventing tumour dissemination and invasion in a mammalian patient.
EFFECT: invention enables inhibiting the AXL/GAS6 signalling pathways effectively.
8 cl, 15 dwg, 6 tbl, 3 ex
The invention claims priority of Provisional application U.S. No. 61/336478, filed on January 22, 2010, the contents of which are incorporated herein by reference.
Area of technology
The present invention relates to tumor invasion and metastasis, for example, treatment or diagnosis of tumor invasion and metastasis through the path associated with AXL and/or GAS6.
The level of technology
Invasion and metastasis is the most insidious and life-threatening aspects of cancer. While tumors with minimally invasive or non-invasive can be successfully removed, once the tumor becomes invasive, it can spread through the lymphatic and/or vascular channels in several places, and complete removal becomes very difficult. Invasion and metastasis destroy the host through two processes: local invasion and colonization of remote bodies with their damage. Local invasion can lead to dysfunction of the tissue from local compression, local destruction or obstruction of normal organ function. The most important turning point in cancer, however, is the development of distant metastases. The patient cannot be cured by local therapy at this stage.
The process of metastasis is a cascade of linked sequential steps involving multiple interactions� host-tumor. This complex process requires the input cells in the blood or lymphatic system, delays in a remote section of vascular or lymphatic stream, the active penetration of vessels into the interstitial tissue and the parenchyma of the body, as well as growth as a secondary colony. Metastatic potential depends on the local microenvironment, angiogenesis, interactions stroma - tumor, the production of local cytokines tissues, and molecular phenotypes of tumor and host cells.
Local initial stage of infiltrating tumor growth may occur early, despite the fact that the remote distribution not obviously or perhaps has not yet started. The tumor cells penetrate the epithelial basement membrane and enter the main interstitial stroma during the transition from in situ to invasive carcinoma. Once the tumor cells infiltrating the primary stroma, they have access to lymphatic and blood vessels to remotely distribute in the release of fragments of matrix and growth factors. During the transition from benign tumor to invasive carcinoma occurs common and widespread changes in the organization, distribution and number of epithelial basement membranes.
Therapeutic efforts for the prevention and treatment of cancer currently concen�Governor will be at the level of signaling pathways or selective modulating proteins. The activity of protein kinases, homeostasis and activation of the oncoprotein are control signals and, therefore, can play a key role regulatory sites for therapeutic intervention. Kinases in signaling pathways that regulate invasion and angiogenesis may be an important regulator of metastasis. One of the major classes of biochemical molecular targets is the family of tyrosine kinase receptors (RTKs). The most common molecular targets related to the tyrosine kinase receptors are the receptors for EGF and vascular endothelial growth factor (VEGF). Newest kinase molecular targets include the type III RTK family of c-kit and abl. Inhibitors of these molecules were prescribed in combination with classical chemotherapy.
Metastases ultimately responsible for much of the suffering and mortality from cancer. There is a need to identify molecular and functional markers that define metastatic cancer cells and to target them, and create reagents for their specific inhibition.
Publications in this area include, in particular, Li et al. Oncogene. (2009) 28(39):3442-55; Application for U.S. patent, 20050186571 Ullrich et al.; The patent application U.S. 20080293733 Bearss et al.; Sun et al. Oncology. 2004; 66(6):450-7; Gustafsson et al. Clin Cancer Res. (2009) 15(14):4742-9; Wimmeletal. Eur J Cancer. 2001 37(7):2264-74; Koorstra et al. Cancer Biol Ther. 2009 8(7):618-26; Tai et al. Oncogene. (2008) 27(29):4044-55.
The receptor tyrosine kinase AXL (also known as Ufo and Tyro7) belongs to the family of receptor tyrosine which includes Tyro3 (Sky) and Mer (Tyrol 2). A common ligand for AXL family is GAS6 (Specifically retarding growth protein 6). AXL person contains an open reading frame in p.about. able to direct the synthesis 894-amino acid polypeptide. Two mRNA variants have been described, with the variant 1 transcript can be found in Genbank, the number of access NM_021913.3, and option 2 of the transcript can be found at number NM_001699.4. Polypeptide sequence of the native protein is defined as SEQ ID NO:1, and specific reference may be made to the sequence based the amino acid modifications. An important function of the cell GAS6/AXL include cell adhesion, migration, phagocytosis, and inhibition of apoptosis. Family of receptors, GAS6 and AXL are strictly regulated in tissues and in a certain way because of the disease.
AXL is characterized by a unique molecular structure, which consists in the fact that the intracellular region has the typical structure of a receptor tyrosine kinase, and the extracellular domain contains motifs of fibronectin III and Ig-like adhesion molecules katherinovka type. During development, AXL is expressed in various organs, including the brain, h�about suggests that this RTK is involved in the development of the mesenchyme and the nervous system. In adults, the expression of AXL low, but in a variety of tumors, the expression is returned to high levels. GAS6 is still only an activating ligand for AXL.
The receptor tyrosine kinase (RTK), typically activated by ligands that contribute to the dimerization of the receptor and, in turn, autophosphorylating tyrosine residues in the cytosolic domain. The binding of signaling proteins to these phosphorylated tyrosine residues leads to the subsequent signal transmission in the signal path. Family AXL RTKs is unique in that its members are activated by GAS6, a member of the family of vitamin K-dependent proteins, which resemble the coagulation factors, rather than the typical growth factors.
Summary of the invention
The present invention is based in part on the discovery that the path associated with AXL and/or GAS6 is associated with tumor invasion and/or metastasis. Accordingly, the present invention relates to compositions and methods that are used for the treatment of tumor invasion and/or metastasis, for example, by inhibiting pathways associated with AXL and/or GAS6. In addition, the present invention provides reagents and methods that are useful for determining the sensitivity or the likelihood that the tumor will become invasive and/or metastatic, �of primer, using the definition of the level of activity of AXL and/or GAS6.
In one of the embodiments of the present invention proposed a soluble variant of the AXL polypeptide, wherein the polypeptide does not have the transmembrane domain of AXL and, optionally, an intracellular domain, and contains at least one amino acid modification compared to the sequence of wild-type AXL, and where these changes increase the affinity of binding of the polypeptide to AXL GAS6. In some embodiments, the instant embodiment variant of the AXL polypeptide contains at least one amino acid modification in a phase selected from the group consisting of 1) between 15-50, 2) between 60-120 and 3) between 125-135 in the sequence of wild-type AXL (SEQ ID NO:1). In some other embodiments embodiment, the soluble variant of the AXL polypeptide contains at least one amino acid modification in position 19, 23, 26, 27, 32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109, 112, 113, 116, 118, 127 or 129 of the sequence of wild-type AXL (SEQ ID NO:1), or a combination. In some other embodiments of the instant embodiment variant of the AXL polypeptide contains at least one amino acid modification selected from the group consisting of 1) AT, 2) T23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6) N33S, 7) T38I, 8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15) Q86R, 16) D87G, 17) D88N, 18) I90M or I90V, 19) V92A, V92G or V92D, 20) I97R, 21) T98A or TR, 22) T105M 23) 0109R, 24) V112A, 25) F113L, 26) H116R, 27) T118A, 28) G127R or G127E and 29) E129K and their combinations and conservative equivalents.
In still some other embodiments of the instant embodiment variant of the AXL polypeptide includes substitutions of amino acids compared with the sequence of wild-type AXL (SEQ ID NO:1) for the following positions: (a) glycine 32; (b) aspartic acid 87; (C) valine 92; and (d) glycine 127. In still some other embodiments of the instant embodiment variant of the AXL polypeptide contains the following changes: the glycine residue 32 is replaced by a serine residue, an aspartic acid residue 87 is replaced by a glycine residue, a valine residue 92 is replaced by an alanine residue, or a glycine residue 127 is replaced by an arginine residue or a combination thereof, or conservative equivalent. In several other embodiments of the instant embodiment variant of the AXL polypeptide contains amino acid substitutions compared to the sequence of wild-type AXL (SEQ ID NO:1) for the following positions: (a) glutamic acid 26; (b) valine 79; (C) valine 92; and (d) glycine 127. In several other embodiments of the instant embodiment variant of the AXL polypeptide contains the following changes: the glutamic acid residue 26 is replaced by a glycine residue, a valine residue 79 is replaced by a methionine residue, a valine residue 92 is replaced by an alanine residue or a glycine residue 127 is replaced by a glutamic acid residue, or a combination or conservative EQ�the entry level.
In some other embodiments of the instant embodiment variant of the AXL polypeptide contains at least amino acids 1-437, 19-437, 130-437, 19-132, 1-132 polypeptide of wild-type AXL (SEQ ID NO:1). In several other embodiments of the instant embodiment variant of the AXL polypeptide is a hybrid protein containing the Fc region.
In one of the embodiments of the soluble variant of the AXL polypeptide has an affinity of at least 1×10-5M to GAS6. In another embodiment, a soluble variant of the AXL polypeptide has an affinity of at least 1×10-6M, to GAS6. In yet another embodiment, a soluble variant of the AXL polypeptide has an affinity of at least 1×10-7M to GAS6. In yet another embodiment, the soluble variant of the AXL polypeptide has an affinity of at least 1×10-8M to GAS6. In yet another embodiment, a soluble variant of the AXL polypeptide has an affinity of at least about 1×10-9M, 1×10-10M, 1×10-11M, or 1×10-12M to GAS6. In various embodiments, the embodiments described herein, a soluble variant of the AXL polypeptide shows an affinity to GAS6, at least about 2 times stronger than the affinity of the polypeptide wild-type AXL. In some embodiments, the instant embodiment variant of the AXL polypeptide shows an affinity to GAS6 that is at least 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, � 25 times or 30 times stronger than the affinity of the polypeptide wild-type AXL.
In another embodiment, the present invention provides the selected antibodies or fragments thereof that specifically bind to GAS6 protein (SEQ ID NO:2). In some embodiments embodiment the selected antibody or a fragment thereof is a monoclonal antibody, a humanized antibody, chimeric antibody, single-chain antibody (ScFv), or combinations thereof. In some other embodiments, the embodiment selected antibody or a fragment thereof binds an epitope which consists of one or several amino acid sites GAS6, are selected from the group consisting of R299-T317, V364-P372, R389-N396, D398-A406, E-N, and W450-M468. In several other embodiments, the embodiment selected antibody or a fragment thereof binds an epitope that includes amino acid site selected from the group consisting of RMFSGTPVIRLRFKRLQPT (SEQ ID NO:3), VGRVTSSGP (SEQ ID NO:4), RNLVIKVN (SEQ ID NO:5), DAVMKIAVA (SEQ ID NO:6), ERGLYHLNLTVGGIPFH (SEQ ID NO:7) and WLNGEDTTIQETVKVNTRM (SEQ ID NO:8).
In yet another embodiment, the present invention relates to methods of treating, reducing or preventing the metastasis or tumor invasion into the patient's body related to mammals. In one embodiment, the method includes the introduction of a specified patient an effective dose of a soluble version of the AXL polypeptide or dedicated �STI-GAS6 antibodies, or its fragment.
In yet another embodiment, the present invention relates to methods of treating, reducing or preventing the metastasis or tumor invasion into the patient's body related to mammals. In one embodiment, the method comprises administering one or more inhibitors selected from the group consisting of (a) inhibitor of AXL activity, (b) an inhibitor of activity of GAS6; and (C) an inhibitor of the interaction of AXL-GAS6. In various embodiments, the embodiments described herein, the inhibitor is a polypeptide, polynucleotide, small molecule, antibody, antibody fragment, or drug conjugate to the antibody.
In another embodiment, the present invention provides methods for determining the ability of the tumor by invasion or metastasis in the subject. In one of the embodiments the method includes determining the level of activity of AXL and/or GAS6 activity in a biological sample from a subject with a tumor; and comparing the level of activity of AXL and/or GAS6 in the biological sample with a predetermined level, in which the increase compared with a predetermined level indicates a predisposition of tumors to invade or metastasize.
Brief description of figures
Figure 1. The AXL expression correlates with tumor progression and metastasis �aka breast and ovarian cancer person. A. Representative images of immunohistochemical staining of AXL in normal breast tissue (normal), primary ductal carcinoma infiltrates (stage 1, 2, and 3) and metastatic lymph nodes (lymph nodes). It should be noted that the high level of membrane AXL staining was observed at 2 stages (arrows) of the 3rd stage, and lymph node metastases. The absence of AXL staining was observed in normal or tumor stroma (*). B. Representative images of immunohistochemical staining of AXL in normal ovarian epithelium (arrow), stage II, stage III, and metastatic omentum obtained from patients with serous adenocarcinoma. Please note that normal and tumor stroma were negative for staining AXL (*).
Figure 2. Genetic inactivation of AXL enough to block metastasis of breast and ovarian cancer. A. Immunohistochemical staining of H & E and AXL in the lungs of mice through the tail vein, the mice were injected cell line MDA-231 shscramble (small hairpin RNA of random sequence shSCRM) and shAXL (small hairpin RNA to a sequence of AXL-shAXL). The photo shows 5 mice from the group. Graphs show the results of real-time PCR the expression of human GASPDH and AXL in healthy lungs of mice, which were injected with the cell line MDA-231 shSCRM or shAXL (n=5). �. Photographs of mice taken at 28 days after injection of SKOV3ip.1 shscramble cells (shSCRM) and shAXL (shAXL). Please note that the introduction shSCRM mice led to the development of multiple metastases in the abdominal cavity (marked by circles). For shAXL group shows the mouse with the highest tumor load. The graphs on the right show the average number of peritoneal metastasis mouse >5 mm in size and average weight of large tumors. The photo shows five mice from group. C. Photographs of mice taken through 34 days after injection OVCAR-8 cells shSCRM and shAXL. Please note that the introduction shSCRM mice led to the development of multiple metastases in the abdominal cavity (marked by circles). The graphs on the right show the average total number of peritoneal metastases in a mouse and an average total tumor mass. The photo shows eight mice from the group.
Figure 3. Genetic inactivation of AXL does not affect the proliferation of tumor cells of breast or ovarian cancer in vitro and growth in vivo. A. cell growth Curves for MDA-231, SKOV3ip.1 and OVCAR-8 cells stably expressing the sequence shPHK scramble control (shSCRM) or AXL (shAXL). The measurements were carried out three times, and the measurement error presents S. E. M. of V. the Average tumor volumes orthotopic MDA-231 (n=8 mice per group) and subcutaneous SKOV3ip.l tumors (n=4 mice per group) increased during the 48-day course. �okresnosti of the measurements are presented S. E. M.
Figure 4. AXL regulates cell invasion tumor ovarian and breast cancer in vitro. A. Analysis of invasion in the collagen under control (shSCRM) and AXL (shAXL) cells MDA-231, SKOV3ip.1 and OVCAR-8. The pictures were taken 7 days after introduction of the cells into the collagen and are represented by three samples from the group. Note the invasive phenotype observed in the AXL wild-type cells (branching) compared to AXL deficient cells (rounded). Graphs show quantitative analysis of invasion in collagen. V. Analysis method real-time PCR expression of MMP-2 in shAXL and shSCRM SKOV3ip.1 cells. Value expression normalized to 18S; n=3. Error definitions are S. E. M.. Asterisks indicate significant increase or decrease in expression compared to shSCRM, as determined by t-student test (**, p<0,001). C. MMP-2 values from the analysis shSCRM or shAXL SKOV3ip.1 cells (n=6). D. Gelatin zymography analysis of the activity of Pro - and active - MMP-2 in conditioned medium collected from serum depleted SKOV3ip.1 cells. E. Western blot analysis of phospho-AKT at Ser473 (P-AKT), total AKT (AKT), and expression of AXL in SKOV3ip.1 cells expressing Sh target sequence to scramble control (shSCRM) or AXL (shAXL) and depleted SKOV3ip.1 cells (strve), treated with GAS6 or PI3K inhibitor Ly294002 (Ly) with GAS6. MMP-2 values from the analysis of depleted SKOV3ip.1 cells (strve), about�botannic with GAS6 or GAS6 with the PI3K inhibitor Ly294002 (Ly+GAS6).
Figure 5. Ectodomain therapy soluble inhibits AXL AXL signaling and invasion in vitro. A. Schematic representation of the mechanism of therapy soluble AXL. Soluble AXL (sAXL) function as receptor-traps for inhibiting endogenous signal transmission AXL. B. Western blot analysis of phospho-AKT at Ser473 (P-AKT), total AKT (AKT) and AXL expression in MDA231 cells, SKOV3ip.1 and OVCAR-8, expressing the sequence ShPHK scramble control (shSCRM) or AXL (shAXL), and depleted of SKOV3ip cells.1 (strve) treated with GAS6 or PI3K inhibitor Ly294002 (Ly) with GAS6. C. Western blot analysis of the expression of phospho-AKT Ser473a cells treated in one of conditioned media containing soluble AXL receptor (sAXL) or control medium (-). All cells were cultured in serum-free medium for 48 hours and treated with GAS6 (+) or control medium (-). D. Analysis of invasion in collagen MDA-231 cells treated with conditioned medium containing control vector or sAXL.
Figure 6. Treatment with soluble AXL receptors inhibits metastatic tumor burden in mice with established metastases. A. Schematic representation of research on the treatment of soluble AXL receptor. Naked mice I. p. (intraperitoneally) was injected with 1×106SKOV3ip.1 cells. Five days after implantation was verified the presence of macroscopic lesions in mice (photography� shows the mouse with peritoneal metastasis at day 5 after injection, metastatic lesion (marked by circles). On day 7 the mice were injected adenovirus expressing IgG2a-Fc control (Ad-Fc) or soluble AXL receptor (Ad-sAXL). Serum levels of expression of sAXL was evaluated according to Western blotting every 3-4 days after adenovirus injection. In the subsequent 28 days after implantation of tumor cells, was evaluated by tumor burden in all mice. V. Presents photographs of mice treated by expression of adenovirus Ad-sAXL or Ad-Fc at 28 days after injection of tumor cells. Metastatic lesions marked by circles. Graphs show the average total number and weight of tumor for 7 mice per group. Measurement error presents S. E. M.. Note that the statistical difference in the number of tumors and weight (p=0.01, t-student test) was observed between mice treated with Ad-Fc or shPHK Ad-sAXL (*). C. Analysis of real-time PCR expression of MMP-2 in tumors of mice with Ad-Fc or Ad-AXL.
Figure 7. Therapy soluble ectodomain AXL does not cause toxicity in normal tissues. A. Full CBC and biochemical analysis of the serum of mice treated with control (Fc) or soluble AXL (sAXL). V. N & E staining of liver tissue and kidney taken from mice treated Fc or sAXL.
Figure 8. A schematic diagram illustrating the molecular mechanisms associated with the inhibition of metastasis of soluble receptor� AXL. Therapy soluble receptor AXL (sAXL) acts as a receptor trap, which binds to the AXL ligand GAS6. sAXL inhibits the path of endogenous GAS6-AXL signalling, which stimulates cell invasion and metastasis.
Figure 9. The creation of AXL-deficient lines of cancer cells of breast and ovarian cancer. A. Western blot analysis of AXL expression in the panel of human cancer cell lines of breast and ovarian cancer. Heat shock protein 70 (Hsp70) was used as load control protein. B. Western blot analysis of AXL expression in cancer cell lines of metastatic breast cancer (MDA-231), ovarian (SKOVSip.1 and OVCAR-8), stably transfected MSRC sequence control (scramble) (shSCRM) or AXL (shAXL). Please note that shAXL cell lines have a significant decrease in the expression of AXL.
Figure 10. AXL does not affect cell adhesion or survival of tumor cells of breast and ovarian cancer. A-B. the Percentage of cell migration of MDA-231 (A) and SKOVSip.1 (B) migration of cells to the serum, which acts as a chemoattractant, the analysis in the chamber of Boyden. C-D. Analyses of MDA-231 (A) SKOV3ip.1 (B) cell adhesion to extracellular matrix proteins. Abbreviations: bovine serum albumin (BSA), fibronectin (FN), collagen type I (Col I), collagen type IV (Col IV), laminin (LN), fibrinogen (FBN). Measurement errors represent the standard error� average. E-F. Analysis of the survival of wild-type AXL and AXL deficient MDA-231 (E) and SKOV3ip.1 (F) tumor cells after removal of the serum, as determined by analysis of HTT.
Figure 11. Treatment with soluble AXL receptors inhibits metastatic tumor burden in mice with established OVCAR-8 metastases. A. Schematic representation of research on the treatment of soluble AXL receptor. Nude mice were intraperitoneally introduced 5×106OVCAR-8 cells. After fourteen days after implantation was verified the presence of macroscopic lesions in mice (the photo shows a mouse with peritoneal metastasis on the 14th day after injection, metastatic lesion (marked by circles). On day 14 the mice were injected adenovirus expressing IgG2a-Fc control (Ad-Fc) or soluble AXL receptor (Ad-sAXL). Serum levels of expression of sAXL was evaluated according to Western blot analysis. In the next 34 days after implantation of tumor cells, tumor burden was evaluated in all mice. V. Presents photographs of mice treated with adenovirus expressing Ad-sAXL or Ad-Fc at 28 days after injection of tumor cells. Metastatic lesions marked by circles. C. Graphs show the average total number and weight of tumor for 8 mice in the group. Measurement error presents S. E. M.. Note that the statistical difference in the number and weight� tumors (p< 0,01, t-student test) were observed between mice that were treated with Ad-Fc and Ad-sAXL (*).
Figure 12. Analysis of binding of AXL library has identified 5 products for GAS6. In a scatter graph of flow cytometry of yeast cells expressing wild-type AXL (A) or pooled AXL, there are 5 products obtained by directed evolution (In). Analysis data of binding with subsequent dissociation described in example 2. The level of binding of 2 nm GAS6 shown in the left column, the level of binding to GAS6 after 4 hours is shown in the middle column, and the levels of binding to GAS6 after 6 hours are shown in the right column. For cells that are positive for the expression of a specific protein on the cell surface (upper right quadrant of each dot plot of flow cytometry, the levels of binding to GAS6 (y-axis) quantitatively presented in the chart below. Selected five products show significantly better binding to GAS6 compared to wild-type AXL.
Figure 13. Linking improved variants AXL with GAS6. The left panel shows equilibrium binding of GAS6 to AXL mutants S6-1 (red square) and S6-2 (blue diamonds) compared with wild-type AXL (green circles). Mutant S6-1 and S6-2 show significantly higher levels of binding of low concentrations of GAS6, demonstrating a stronger binding affinity for these mutant� compared to wild-type AXL. The right panel shows the kinetics of dissociation of the Gas6-AXL interaction for AXL wild-type or engineered AXL. When the interaction of Gas6 with wild-type AXL is formed Gas6-AXL "wild type", which decays quickly as a function of time, while constructed in the interaction of S6-1 ("S6-1") or S6-2 ("S6-2") with Gas6 shown a significant increase in the integrity of the binding.
Figure 14. Intraperitoneal delivery of cleared AXL S6-1-Fc shows the enhancement of therapeutic effect of wild-type AXL-Fc and AXL E59R/T77R-Fc. Shows two images of the autopsy of mice from the three treatment groups, AXL E59R/T77R-Fc, AXL-Fc wild-type, and AXL S6-1-Fc. Black circles in the pictures indicate the visible metastatic lesions, but not necessarily show all metastases. AXL-Fc wild-type shows a moderate inhibition of metastasis, but greater than negative control, AXL E59R/T77R, a AXL S6-1 shows an almost complete suppression of metastasis.
Figure 15. Inhibition of metastasis in xenographic model SKOV3ip.1. On the first two graphs the same data set presented in two different ways, to show how the average number of metastases counted in each treatment group. In addition, the lower two plots show how the same set of data that describes the total weight of all metastases, which are cut from the mice in each group. AXL-Fc di�wow type prevents the spread of metastases compared with negative E59R/T77R-Fc control as evidenced by the decrease in the number of injuries (upper panel) and total weight (bottom panel). AXL S6-1-Fc shows a significant reduction in tumor mass compared with wild-type AXL-Fc and AXL E59R/T77R-Fc, as can be gauged by the number of lesions (upper panel) and weight (lower panel). These data show that increased affinity AXL S6-1 provides enhanced therapeutic efficacy for the wild type, and that AXL S6-1-Fc is an effective method of treatment to combat metastasis.
In the description below, widely used a number of terms commonly used in the field of cell culture. In order to ensure a clear and consistent understanding of the specification and claims, as well as the volume defined by these terms, provided the following definitions.
"Inhibitors," "activators," and "modulators" AXL on metastatic cells or its ligand GAS6 are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays of binding the receptors or ligands, and signaling, e.g., ligands, receptors, agonists, antagonists, and their homologs and mimetics.
The term "polypeptide", "peptide" and "protein" are used herein interchangeably to designate�I polymer, consisting of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic corresponding to the natural amino acid and a natural amino acid polymers, and naturally occurring amino acid polymers.
The term "amino acid" refers to natural and synthetic amino acids, and analogs of amino acids and mimetics of amino acids that function like natural amino acids. Natural amino acids encoded by the genetic code, as well as amino acids, which are then modified, e.g., hydroxyproline, gamma-carboxyglutamate and O-phosphoserine. Analogs of amino acids are compounds that have the same basic chemical structure of natural amino acids, i.e. alfaguara, which is associated with hydrogen, carboxyl group, amino group and R group, for example, homoserine, norleucine, methanesulfonic, S-methylmethane. Such analogs have modified R groups (e.g., norleucine) or modified side of the peptide bond, but they saved the basic chemical structure of natural amino acids. The mimetics of amino acids are chemical compounds that have a structure different from the General chemical structure of amino acids, but functions � & similar natural amino acids. The present invention uses single letters to denote amino acids, which are generally accepted symbols of amino acids, widely used in this field, for example, alanine, C denotes cysteine, etc. When the amino acid is represented by one letter before and after the corresponding position, this means replacing the original amino acid in a given position on a modified amino acid (after). For example, IT means that the amino acid alanine at position 19 is changed to threonine.
The term "subject", "individual" and "patient" are used herein interchangeably to refer to mammals, which are analysed in the treatment process and/or who are receiving treatment. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" thus include persons with cancer, including, without limitation, ovarian adenocarcinoma, or prostate cancer, breast cancer, glioblastoma, etc., including those who have undergone or are candidates for removal of malignant tissue (surgery). Actors can be human, but also include other mammals, especially mammals, are useful as laboratory models for human diseases, for example, mouse, rat, etc.
The term "tumor", as used here, refers to all neoplastic� cellular entities regardless of malignant or benign character of cell proliferation, and all precancerous and cancerous cells and tissues.
The terms "cancer", "neoplasm" and "tumor" are used herein interchangeably to refer to cells that are characterized by Autonomous, unregulated growth so that they have abnormal growth phenotype, and characterized by significant loss of control over cell proliferation. In General, cells of interest for detection, analysis, classification or treatment in the present application includes premalignant (i.e., benign), malignant, premeditations, metastatic and non-metastatic cells. Examples of cancer include, but are not limited to, ovarian cancer, glioblastoma, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, kidney cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.
"Pathology" of cancer includes all phenomena that compromise the well-being of the patient. This includes, but is not limited to, violation or uncontrollable cell growth, metastasis, interference�tvo in the normal functioning of neighboring cells, secretion of cytokines or other secretory products at abnormal quantities, suppression or aggravation of inflammatory or immunological response, neoplasia, a precancerous condition, a malignant tumor, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
The terms "cancer recurrence" and "tumor recurrence", as used here, and their grammatical variants indicate further growth of tumor or cancer cells after the diagnosis of cancer. In particular, relapse is likely to occur when further cancer cell growth occurs in cancerous tissue. "The spread of the tumor also occurs when tumor cells spread to local or distant tissues and organs, therefore, spread of the tumor comprises tumor metastasis. "Tumor invasion" occurs when the growth of the tumor has spread to the local level, disrupting the function of involved tissues by compression, destruction or prevent the normal function of the body.
The term "metastasis", as used here, refers to the growth of cancerous tumors in the body or body parts which are not directly linked to the original body of the cancer. Metastasis also includes micrometastasis - the presence of some detectable amount �ekovich cells in an organ or body part, are not directly related to the initial body of the cancer. Metastasis can be defined as several process steps such as the release of cancer cells from the original tumor, migration and/or invasion of cancer cells in other parts of the body. Thus, the present invention provides a method of determining the risk for further growth of one or more cancerous tumors in the body or body parts which are not directly linked to the original body of the cancer, and/or any steps in preparation for this growth.
Depending on the nature of the cancer is obtained the corresponding patient sample. Used here, the expression "tumor tissue sample" refers to any cell derived from a cancer. In the case of solid tumors that do not metastasize, the tissue sample is obtained from a remote tumor surgically, and prepared for testing by conventional techniques.
To determine the used blood and other liquid samples of biological origin, solid tissue samples such as a biopsy or tissue cultures, or cells derived from it and their offspring. When determining also used samples that were modified after receiving them in any way, for example, by treatment with reagents; washing; or enrichment� certain cell populations, such as cancer cells. The sample can also be enriched by certain types of molecules, such as nucleic acids, polypeptides, etc., the Term "biological sample" encompasses a clinical sample, and the tissue obtained during surgical resection, the tissue obtained at biopsy, cells in culture, the supernatants of the cells, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. "Biological sample" includes a sample obtained from cancer cells of the patient, for example, a sample comprising polynucleotides and/or polypeptides that are derived from cancer cells of the patient (e.g., a lysate of cells or cellular extracts comprising the polynucleotides and/or polypeptides); and a sample comprising cancer cells of the patient. A biological sample containing cancer cells from the patient, may also include non-cancerous cells.
The term "diagnosis" is used here to refer to the definition of a molecular or pathological condition or disease, such as the identification of molecular subtypes of breast cancer, prostate cancer or other cancers.
The term "forecast" is used here to denote the prediction of the probability of cancer-related death or progression, including recurrence, Mets�of aerovane and drug resistance in neoplastic disease, such as ovarian cancer. The term "prediction" is used here to refer to the act of predictions or estimates based on observations, experience, and scientific reasoning. In one example, the physician can predict the likelihood that a patient will survive after surgical removal of the primary tumor and/or chemotherapy for a certain period of time without cancer recurrence.
As used herein, the terms "treating", "treat" and the like, refer to the introduction of an agent or procedure (e.g., radiation, surgery, etc.) in order to obtain the effect. The effect may be prophylactic from the point of view of complete or partial prevention of the disease or its symptoms, and/or may be therapeutic, from the point of view of the implementation of a partial or complete cure from a disease and/or symptoms of the disease. "Treatment" used herein includes any treatment of any metastatic tumors in mammals, particularly in humans, and includes: (a) preventing the disease or symptom of disease occurring in a subject which may be predisposed to the disease, but until now he had not been diagnosed (for example, if the subject of the diseases that may be associated with or caused by the underlying disease); (b) inhibiting the disease, i.e. the delay of its development and (C) facilitation of the disease, i.e. regression of the disease. In the treatment of a tumor (e.g., cancer), a therapeutic agent may directly decrease the metastasis of tumor cells.
Treatment can apply to any signs of success in the treatment or improvement, or prevention of cancer, including objective or subjective parameters, such as termination, remission; reduction of symptoms or making the disease condition more tolerable to the patient; slowing the rate of degeneration or deterioration; or making the final point of degeneration less debilitating. Treatment or relief of symptoms can be based on objective or subjective parameters, including the results of researches of the doctor. Thus, the term "treatment" includes introducing the compound or agent of the present invention to prevent or suspend, to alleviate, or to arrest or inhibit development of the symptoms or condition associated with neoplasia, such as tumor or cancer. The term "therapeutic effect" refers to the reduction, elimination or prevention of disease, symptoms of disease or side effects of the disease in the subject.
The terms "in combination with", "combination therapy" and "combination products" refer, in some embodiments, embodiments, to the concurrent administration to a patient a first therapeutic and�yente and connections which are used here. When administered in combination each component may be administered simultaneously or sequentially in any order at different points in time. Thus, each component may be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
In accordance with the present invention the first therapeutic agent may be any suitable therapeutic agent, for example, cytotoxic agents. One typical class of cytotoxic agents are chemotherapeutic agents, for example, which can be combined with treatment for the inhibition of AXL or GAS6 signaling. Typical chemotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, duocarmycin, epoetin Alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (�of Axel™), pilocarpine, prochlorperazine, rituximab, sprain, tamoxifen, Taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate. For the treatment of ovarian cancer preferred chemotherapy agent, which can be combined inhibitors of signaling AXL or GAS6 is paclitaxel (Taxol™).
Other combination therapies include radiation, surgery, and hormone deprivation (Kwon et al., Proc. Natl. Acad. Sci U. S. A., 96:15074-9, 1999). Angiogenesis inhibitors may be combined with the methods of the invention.
"Simultaneous administration" known anti-cancer therapeutic drug with a pharmaceutical composition of the present invention means administration of the drug and an inhibitor of AXL, in a time when the known drug and the composition of the present invention will have a therapeutic effect. Such co-administration may include concomitant (at the same time), prior, or subsequent administration of the drug in relation to the use of compounds of the present invention. The person skilled in the art will have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.
The phrase "survival without evidence of disease", as used here, refers otsutstvie of tumor recurrence and/or spread, and the fate of the patient after diagnosis, the impact of cancer on life expectancy of the patient. The phrase "overall survival" refers to the fate of patients after diagnosis, despite the possibility that the cause of death of the patient not directly related to the effects of cancer. The phrase "the probability of survival without evidence of disease", "risk of relapse" and their variants are converted to the probability of tumor recurrence or metastasis in a patient after a diagnosis of "cancer" in which the probability is determined according to the method of the invention.
The term "correlates" or "correlated with", and similar terms, as they are used here, refers to the statistical correlation between two events, where events include figures, datasets, and the like. For example, when events are associated with numbers, positive correlation (also referred to herein as "direct relationship" means that when one increases, the other also increases. A negative correlation (also referred to herein as "inverse correlation means that when one increases, the other decreases.
A "unit dosage" refers to physically discrete units corresponding to standard doses for the treatment of a specific individual. Each unit may contain a predetermined quantity of active substances(in) calculated for �of auchenia the desired therapeutic effect(s) in combination with the required pharmaceutical carrier. The particular shape of the dosing unit may be dictated by (a) the unique characteristics of the active compound(s) and, in particular, therapeutic effect(s) that must be achieved, and (b) the restrictions arising from the mixing of such active compound(s).
"Pharmaceutically acceptable excipient" means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and desirable, and includes excipients that are acceptable for veterinary use and for use as a pharmaceutical for humans. Such fillers may be solid, liquid, semisolid or, in the case of an aerosol composition, gaseous.
"Pharmaceutically acceptable salts and esters" means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed by an acidic proton present in the compound and is able to react with inorganic or organic bases. Suitable inorganic salts include salts formed with alkali metals, e.g. sodium and potassium, magnesium, calcium and aluminum. Suitable organic salts include salts formed with organic bases, �akimi as primary amines, for example, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. Such salts also include salts of joining acids, formed with inorganic acids (e.g. hydrochloric and Hydrobromic acids) and organic acids (e.g. acetic acid, citric acid, maleic acid, and the alkane - and arenesulfonates, such as methansulfonate and benzolsulfonat). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy and phosphonooxy groups present in compounds, for example, C1-6alkyl esters. When there are two acidic groups pharmaceutically acceptable salt or ester may be monocellate-monosol or ether or disol or ether; and where there are more than two acid groups, some or all of these groups participate in saleabration or esterification. Compounds described in the present invention, can be present in "mesolevel" or nonesterified form or in salt form, and/or esterified form, and at the mention of such compounds is meant as a source (naselenie and nonesterified) compounds and their pharmaceutically acceptable salts and esters. Furthermore, some compounds described in the present invention, may be represented at a few� the stereoisomeric forms and the mention of such compounds should include all individual stereoisomers and all mixtures (racemic or otherwise organized) of such stereoisomers.
The terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variants refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are applicable for use in, or for the person, without the existence of undesirable physiological effects to such an extent as to prohibit the use of the composition.
"Therapeutically effective amount" means an amount that, when administered to object to the treatment of diseases, is sufficient to effect treatment of these diseases.
In accordance with this invention, offers the soluble AXL options, for example, a soluble variant of the AXL polypeptide, which has the activity of binding to GAS6 essentially equal to or better than the activity of binding of the polypeptide of wild-type AXL. In some embodiments, embodiments of the invention, a soluble variant of the AXL polypeptide is used as therapeutic agents.
Protein AXL, with reference to the native sequence of SEQ ID NO:1 consists of immunoglobulin(Ig)-like domain formed by residues 27-128, the second Ig-like domain formed by residues 139-222, domains, fibronectin type 3 formed by residues 225-332 and 333-427, intracellular domain formed by residues 473-894, which includes domain �insincerity. Residues of tyrosine in 779, 866 821 and the provisions become autophosphorylated in the dimerization of the receptor and serve as sites of accession of intracellular signaling molecules. Native cleavage site for release of the soluble form of the polypeptide is located on the remains 437-451.
For the purposes of the invention, the soluble form of AXL is a portion of polypeptide that is sufficient for the binding of GAS6 with appreciable affinity, e.g., with high affinity, which is usually located between the signal sequence and transmembrane domain, that is, usually ranges from approximately residue 19 to residue 437 in SEQ ID NO:1 but which may include or consist mainly of a truncated version from about residue 19, 25, 30, 35, 40, 45, 50 to balance 132, 450, 440, 430, 420, 410, 400, 375, 350 and 321, for example, residues 19-132. In some embodiments embodiment the soluble form of AXL lacks a transmembrane domain, and possibly intracellular domain.
Soluble variant Polypeptides AXL (sAXL) in the present invention includes one or more modifications of amino acids within a soluble form of wild-type AXL, for example, one or more modifications of amino acids that increase its affinity for GAS6. In accordance with the present invention the modification of amino acids include natural or artificial modification of amino acids to�e known or later will be discovered in this area. In some embodiments, embodiments, modifications of amino acids include naturally occurring mutations, such as substitution, deletion, addition, insertion, etc. In some other embodiments, embodiments, modifications of amino acids include replacing the existing amino acids with another amino acid, for example, conservative equivalent. In some other embodiments, embodiments, modification of amino acids include replacing one or more existing amino acids, unnatural amino acids, or inserting one or more unnatural amino acids. In several other embodiments, embodiments, modifications of the amino acids comprise at least, 1, 2, 3, 4, 5 or 6, or 10 amino acid mutations or modifications.
In some typical embodiments, embodiments, one or more amino acid modifications can be used to modify the properties of soluble forms AXL, for example, affect the stability, binding activity and/or specificity, etc. Methods in vitro mutagenesis of cloned genes are known. Examples of protocols for scanning mutations may be found in Gustin et al., Biotechniques 14:22 (1993); Barany, Gene 37:111-23 (1985); Colicelli et al., Mol Gen Genet 199:537-9 (1985); and Prentki et al., Gene 29:303-13 (1984). Ways sitespecific mutagenesis can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.15.3-15.108; Weiner et al., Gene 126:35-41 (1993); Sayers et al., Biotechniques 13:592-6 (199); Jones and Winistorfer, Biotechniques 12:528-30(1992); Barton et al., Nucl Acids Res 18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70 (1989); and Zhu Anal Biochem 177:120-4 (1989).
In some embodiments of the incarnation sAXL variants of the present invention comprise one or more modifications of amino acids in one or more areas of 18 residue to 130, from 10 to balance 135, 15 residue up to 45, 60 residue 65, 70 residue 80, 85 balance to 90, 91 to 99 balance, residue from 104 to 110, 111 balance to 120, from residue 125 to 130, from 19 to balance 437, from 130 to balance 437, balance of 19 to 132, 21 balance to 132, from 21 to balance 121, balance of 26 to 132 or balance of 26 to 121 in the sequence of wild-type AXL (SEQ ID NO:1). In some other embodiments, the embodiment sAXL variants of the present invention comprise one or more modifications of amino acids in one or more sections of the residue from 20 to 130, residue from 37 to 124 or from residue 141 to 212 in wild-type AXL (SEQ ID NO:1). In some other embodiments, the embodiment sAXL variants of the present invention comprise one or more modifications of amino acids corresponding to position 19, 23, 26, 27, 32, 33, 38, 44, 61, 65, 72, 74, 78, 79, 86, 87, 88, 90, 92, 97, 98, 105, 109, 112, 113, 116, 118, 127 or 129 in the sequence of wild-type AXL (SEQ ID NO:1).
In some other embodiments, the embodiment sAXL variants of the present invention comprise one or more modifications of amino acids, including without any limitation 1) AT, 2) �23M, 3) E26G, 4) E27G or E27K, 5) G32S, 6) N33S, 7) T38I, 8) TA, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15) Q86R, 16) D87G, 17) D88N, 18) I90M or 190 V, 19) V92A, V92G or V92D, 20) I97R, 21) TO or TR, 22) CM, 23) Q109R, 24) V112A, 25) F113L, 26) H116R, 27) T118A, 28) G127R or G127E and 29) E129K, and combinations thereof.
In some other embodiments, the embodiment sAXL variants of the present invention include one or more modifications of amino acids at positions 32, 87, 92 or 127 wild-type AXL (SEQ ID NO:1), or a combination thereof, for example, G32S; D87G; V92A and/or G127R. In some other embodiments, the embodiment sAXL variants of the present invention comprise one or more modifications of amino acids at position 26, 79, 92, 127 wild-type AXL (SEQ ID NO:1) or a combination thereof, for example, E26G, V79M; V92A and/or G127E.
In accordance with the present invention sAXL variants of the present invention can be further modified, for example, joining a wide variety of other oligopeptides or proteins for a variety of purposes. For example, various posttranslational or postexplosion modifications can be made in respect of sAXL variants of the present invention. For example, when using the respective coding sequences, can provide farnesiana (the transfer process groups the enzyme farnesyltransferase) or prenisolone (posttranslational protein modification, which consists in joining farnetella and geranylgeranyl gr�PP to C-terminal cysteine residues). In some embodiments of the incarnation sAXL variants of the present invention can be Paglierani where polietilene groups provide increased life time in the bloodstream. Options sAXL of the present invention can be combined with other proteins, e.g., Fc of the IgG isotype, which can be complementarily with the toxin, such as ricin, abrin, diphtheria toxin, etc., or with specific binding agents that allow you to target specific fragments on the target cells.
In some embodiments, embodiments sAXL variants of the present invention are hybrid protein, for example, fused with a second polypeptide. In some embodiments, embodiments of the second polypeptide is able to increase the size of the hybrid protein, for example, so that a hybrid protein is not quickly removed from circulation. In some other embodiments, embodiments, the second polypeptide is a part of or the entire Fc region. In some other embodiments, embodiments, the second polypeptide is of any suitable polypeptide which is essentially similar to Fc, for example, providing an increased size and/or additional binding or interaction with the molecules of the Ig. In some other embodiments, embodiments, the second polypeptide is partially or completely protein albumin, e.g., human�Kim serum albumin.
In some other embodiments, embodiments of the second polypeptide useful for improving sAXL variants, for example, cleaning sAXL variants or to improve their stability in vitro or in vivo. For example, sAXL variants of the present invention can be combined with parts of the constant domains of immunoglobulin (IgG), resulting in chimeric or hybrid polypeptides. These hybrid proteins for easy cleaning and have a higher half-life in vivo. One stated example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of heavy or light chains of mammalian immunoglobulins. EP AND 394827; Trauneckeretal., Nature, 331:84-86, 1988. Hybrid proteins with disulfide-linked dimeric structures (due to the IgG) can also be more efficient in binding and neutralizing other molecules, than the Monomeric secreted protein or protein fragment. Fountoulakis et al., J. Biochem. 270:3958-3964, 1995.
In some other embodiments, embodiments, the second polypeptide is a marker sequence, such as a peptide which facilitates purification of the hybrid polypeptide. For example, the marker amino acid sequence may be a peptide hexastylis, such as is represented in the vector pQE (QIAGEN, Inc, 9259 Eton Avenue, Chatsworth, Calif., 91311), � particular, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989, hexastylis, for example, provides for convenient purification of the hybrid protein. Another peptide marker, useful for purification, "" the token corresponds to an epitope derived from the hemagglutinin protein of influenza. Wilson et al., Cell 37:767, 1984.
In some other embodiments, embodiments of the second polypeptide useful for improving characteristics of sAXL variants of the present invention. For example, in the region of additional amino acids, particularly charged amino acids, may be added to the N-Terminus of the polypeptide to improve stability and stability during purification from the host cell or subsequent handling and storage. In addition, peptide groups can be added to sAXL variants of the invention to facilitate cleaning and subsequently removed prior to final preparation of the polypeptide. In addition, peptide fragments to facilitate handling of polypeptides are known in conventional methods of research.
In still some embodiments, embodiments sAXL variants of the present invention have the activity of binding to GAS6 that is at least equal to or better than wild-type AXL. In some other embodiments, embodiments sAXL variants of the present invention have the activity of binding or affinity to GAS6, which, at least, 1 time, 2 times, 3 times�, 4 times, 5 times or 6 times more than that of the wild-type AXL. In some other embodiments, embodiments sAXL variants of the present invention have the activity of binding or affinity to GAS6, at least about 1×10-6, 1×10-7, 1×10-8or 1×10-9M. In certain other embodiments, embodiments sAXL variants of the invention are able to inhibit, suppress or compete with wild-type AXL for the binding of GAS6 in vivo, in vitro, or both. In some other embodiments, embodiments sAXL variants of the present invention inhibit or compete with the binding of AXL S6-1, AXL S6-2, and/or AXL S6-5, as shown in example 2 of the present description. In some other embodiments, embodiments sAXL variants of the present invention inhibit or compete with the binding to any option sAXL, as shown in example 2 of the present description.
The ability of a molecule to contact GAS6 may be determined, for example, by assessing the ability of the putative ligand to contact the plate, covered with GAS6. In one variation of the embodiments of the binding activity sAXL variants of the present invention with GAS6 can be analyzed by immobilization of ligands, e.g., GAS6 or sAXL variants. For example, the analysis may include the immobilization of GAS6, mixed with His marker on Ni-activated NTA polymer (resin) beads. Agents can batobalani in the appropriate buffer and beads were incubated for a certain period of time at a given temperature. After washing, intended to remove unbound material, the bound protein can be liberated, for example, SDS (sodium dodecyl sulphate), a buffer with a high pH and similar agents, and analyzed.
In other embodiments, embodiments sAXL variants of the present invention have higher thermal stability, thermal stability than wild-type AXL. In some embodiments, embodiments the melting point of sAXL variants of the present invention is not less than 5°C, 10°C, 15°C or 20°C above the melting temperature of wild-type AXL.
In accordance with the present invention sAXL variants of the present invention may also include one or more modifications that do not alter primary sequence sAXL variants of the present invention. For example, such modifications may include chemical conversion of polypeptides, e.g., acetylation, amidation, carboxylation, etc. Such modifications may also include modifications of glycosylation, e.g., made by modifying the glycosylation pattern of the polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to the action of enzymes that affect glycosylation, such as glycosylase or deglycosylated mammalian enzymes. In some�'s versions embodiments sAXL variants of the present invention include variant sAXL with phosphorylated amino acid residues, for example, phosphotyrosine, phosphoserine or posttraining.
In other embodiments, embodiments sAXL variants of the present invention include variants sAXL further modified to improve their resistance to proteolytic degradation or to optimize the properties of solubility, or to make them more suitable as a therapeutic agent. For example, sAXL variants of the present invention include analogs sAXL variants containing residues that differ from the natural L-amino acids, e.g., D-amino acids or naturally occurring synthetic amino acids. D-amino acids can be substituted for some or all of amino acid residues.
In some other embodiments, the embodiment sAXL variants of the present invention include at least two identical or different sAXL variants associated covalently or ecovalence. For example, in some embodiments of the incarnation sAXL variants of the present invention include two, three, four, five or six of the same or different sAXL variants associated covalently, for example, so that they will have the proper dimensions to avoid unwanted aggregation.
In accordance with the present invention sAXL variants of the present invention can be produced by any suitable method known or later of obnarugen�x in this field for example, is produced from eukaryotic or prokaryotic cells, synthesized in vitro, etc. If a protein is produced from prokaryotic cells, it can be further processed for deployment, e.g. heat denaturation, DTT and recovery etc., using methods known in this field, can be re-rolled.
The polypeptides can be obtained in the synthesis in vitro, using conventional methods known in this field. There are various commercial vehicles for the synthesis of, for example, an automated synthesizer from Applied Biosystems, Inc., Foster City, CA, Beckman, etc. With the help of synths natural amino acids can be replaced by unnatural amino acids. Determining the sequence and order of training will depend on convenience, Economics, and the required purity and the like.
Polypeptides can be isolated and purified in accordance with conventional methods of recombinant synthesis. The lysate can be obtained from the expression host, and purified using HPLC (high performance liquid chromatography), size exclusion chromatography (gel filtration), gel electrophoresis, affinity chromatography, or other purification technique. For the most part compositions used contain at least 20% by weight of the target product, more often at least about 75% by �asse, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, depending on the pollution associated with the method of preparation of the product and its purification. Generally, the percentage will be based on total protein.
Methods which are well known to specialists in this field can be used to construct expression vectors containing coding sequences and appropriate transcriptional/translational control elements. These methods include, for example, the technique of recombinant DNA in vitro synthetic methods and recombination/genetic recombination in vivo. Alternative RNA capable of encoding the polypeptides can be chemically synthesized. Any of the experts in this field can easily use the well-known table of codons and methods of synthesis for creating appropriate coding sequence for any of the polypeptides of the invention. Direct methods of chemical synthesis include, for example, phosphocreatine method of Narang et al. (1979) Meth. Enzymol. 68:90-99; fosfodiesterzy method Brown et al. (1979) Meth. Enzymol. 68:109-151; diethylphosphinic method of Beaucage et al. (1981) Tetra. Lett, 22:1859-1862; and the synthesis on a solid substrate U.S. patent No. 4458066. Chemical synthesis provides single-stranded oligonucleotide. It can be converted into duhamic�know DNA by hybridization with a complementary sequence, or polymerization DNA polymerase using the single strand as template. While chemical synthesis of DNA is often limited to a sequence of about 100 bases, large sequences can be obtained by the ligation of shorter sequences. Alternatively, subsequences may be cloned and the appropriate subsequences to split with the appropriate restriction enzymes.
Nucleic acids can be isolated and obtained in substantial purity. Typically, nucleic acids such as DNA or RNA, can be obtained in a form that is substantially free from impurities other natural nucleic acid sequences, and usually has at least 50%, at least about 90% purity. As a rule, they are also "recombinant", e.g., flanked by one or more nucleotides that are not typically associated with existing chromosomes. Nucleic acids of the invention can be represented as a linear molecule or a circular molecule, and can be represented in the Autonomous replicating molecules (vectors), or composed of molecules without the sequence for replication. Expression of nucleic acids can be adjusted independently, or other regulatory sequences�, known in this field. Nucleic acids of the invention can be introduced into suitable cells-owners, using a variety of available in this area, such as transferrin polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated DNA transfer, intracellular transportation of DNA coated latex beads, hybridization of protoplasts, viral infection, electroporation, gene gun mediated by calcium phosphate transfection, and the like.
In some embodiments, embodiments of the present invention offers expression vectors for in vitro or in vivo expression of one or more sAXL variants of the present invention, either permanently or under the control of one or more regulatory elements. In some embodiments, embodiments of the present invention provides a cell population containing one or more expression vectors for expression of sAXL variants of the invention, either permanently or under the control of one or more regulatory elements.
According to another aspect of the invention, it provides selected antibodies or fragments thereof that specifically bind to the protein GAS6. GAS6 (growth, delaying specific 6) structurally belongs to the family of vitamin �-dependent plasma proteins. GAS6 has a high structural homology with the natural anticoagulant protein S, sharing the same modular composition, and has 40% identity between sequences. GAS6 has properties similar to the growth factor through its interaction with the receptor tyrosine kinase family; Tyro3, AXL and MerTK. Protein Gas6 contains 678 amino acids, and consists of gamma carboxyglutamate (GLA) - rich domain that mediates the binding of the phospholipids of the membranes of the four domains that are similar to epidermal growth factor and two laminin G-like (LG) domains. With the sequence of transcriptional variants of the human GAS6 can be found in Genbank under the access number NM_001143946.1; NM_001143945.1; and NM_000820.2, respectively.
GAS6 uses a unique mechanism of action, interacting via a vitamin K-dependent GLA (gamma-carboxyglutamic acid) module with phosphatidylserine-containing membranes, and through the carboxy-terminal LamG domains with the receptor membrane THERE.
In accordance with the present invention the selected antibodies of the present invention include any of the selected antibody with a recognizable specificity of binding to GAS6. In some embodiments selected embodiments of the antibody partially or fully human antibodies. In some other embodiments, embodiments of the selected antibody - monoclonal reportsonline antibodies. In some other embodiments selected embodiments the antibodies are chimeric antibodies, for example, constant regions, variable regions and/or CDR3, or a combination of different sources. In some other embodiments, embodiments of the selected antibodies contain a combination of various features described herein.
In accordance with the present invention, the selected fragments of the antibodies of the present invention include a polypeptide containing a site of an antibody (or within the frame of antibodies within a different frame (not antibodies)), which is sufficient and necessary for the recognition and specific binding of the polypeptide with GAS6. In some embodiments, embodiments, the selected fragments of the antibodies of the present invention include a variable light chain, the variable heavy chain, one or more CDR of the heavy chains or light chains, or combinations thereof, for example. Fab, Fv, etc. In some embodiments, embodiments, the selected fragments of the antibodies of the present invention include a polypeptide containing a single-chain antibody, e.g., ScFv. In some embodiments, embodiments, the selected fragments of the antibodies of the present invention include only the variable regions or variable regions in combination with the portion of the Fc region, for example, CH1 plot. In some Varian�Ah embodiments, the selected fragments of the antibodies of the present invention include Manantial, for example, VL-VH-CH3 or banditella.
In some embodiments selected embodiments the antibodies of the present invention bind with the epitope comprising or represented by one or more amino acid sites that interact with AXL. In some other embodiments selected embodiments the antibodies of the present invention bind with the epitope consists of, or represented by one or more amino acid sites GAS6, for example, L295-T317, E-R, R389-N396, D398-A406, E-N and W450, M of GAS6.
In some other embodiments selected embodiments the antibodies of the present invention bind with the epitope comprising or represented by one or more amino acid sites, for example, LRMFSGTPVIRLRFKRLQPT (SEQ ID NO:3), EIVGRVTSSGP (SEQ ID NO:4), RNLVIKVN (SEQ ID NO:5), DAVMKIAVA (SEQ ID NO:6), ERGLYHLNLTVGIPFH (SEQ ID NO:7) and WLNGEDTTIQETVVNRM (SEQ ID NO:8).
In still some other embodiments selected embodiments the antibodies of the present invention bind with the epitope that contains, or presents at least one, two, three, four, five or six amino acids in the field L295-T317, E-R, R389-N396, D398-A406, E-N and W450-M468 of GAS6. In some other embodiments selected embodiments the antibodies of the present invention bind with the epitope that contains or presents at least one, two, three, four, five or six amino acids in about�Asti LRMFSGTPVIRLRFKRLQPT (SEQ ID NO:3), EIVGRVTSSGP (SEQ ID NO:4), RNLVIKVN (SEQ ID NO:5), DAVMKIAVA (SEQ ID NO:6), ERGLYHLNLTVGIPFH (SEQ ID NO:7) and WLNGEDTTIQETVVNRM (SEQ ID NO:8).
In several other embodiments selected embodiments the antibodies of the present invention are capable of inhibiting, suppressing or with opportunity of participating with the binding between GAS6 and wild-type AXL or sAXL variants of the present invention.
In accordance with the present invention and sAXL variants and selected antibodies of the present invention can be in pharmaceutical compositions suitable for therapeutic use, for example, for treatment of a person. In some embodiments, embodiments of the pharmaceutical compositions of the present invention include one or more therapeutic entities of the present invention, for example, sAXL variants and/or selected antibodies against GAS6 or pharmaceutically acceptable salts, esters or solvates, or prodrugs. In some other embodiments, embodiments of the pharmaceutical compositions of the present invention include one or more therapeutic entities of the invention in combination with another cytotoxic agent, e.g., another anticancer agent. In still some other embodiments, embodiments of the pharmaceutical compositions of the present invention include one or more therapeutic entities of the invention in combination with other farm�citiesi acceptable excipient.
In some other embodiments, embodiments of therapeutic agents in the invention are often administered as a pharmaceutical composition containing, for example, the active therapeutic agent and a variety of other pharmaceutically acceptable components. (Cm. Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company, Easton, Pa., 1980.) The preferred form depends on the intended route of administration and therapeutic application. The compositions can also include, depending on desired dosage form, a pharmaceutically acceptable non-toxic carriers or diluents, which are defined as vehicles commonly used in the formulation of pharmaceutical compositions for the treatment of animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffer solution, ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers and the like.
In some other embodiments, embodiments of the pharmaceutical compositions of the present invention can also include large, slowly metabolized macromolecules, such as, Bel�and, polysaccharides, such as chitosan, polylactic acid, polyglycol acid, and copolymers (such as latex functionalized Sepharose™, agarose, cellulose and the like), polymeric amino acids, copolymers of amino acids, and lipid aggregates (as oil droplets or liposomes). In addition, these carriers may function as immunostimulating agents (e.g., adjuvants).
According to one another aspect of the invention, it provides methods of treating, reducing or preventing metastasis of a tumor or tumor invasion by inhibiting the way AXL signaling and/or GAS6 signaling pathways. In some embodiments, embodiments of the methods of the present invention include the inhibition of the activity of AXL, GAS6 activity or interaction between AXL and GAS6. For example, the activity of AXL or GAS6 can be inhibited at the level of gene expression at the level of mRNA processing, translation, post-translational level, at the level of activation of the protein, etc. In some other examples, the activity of AXL or GAS6 can be inhibited by small molecules, biological molecules, such as polypeptides, polynucleotides, antibodies, conjugates of antibodies and drugs, etc. In some other examples, the activity of AXL or GAS6 can be inhibited by one or more sAXL variants, antibodies or dedicated� of the present invention.
In other embodiments, embodiments of the methods of the present invention include administration to a subject in need of treatment a therapeutically effective amount or effective dose of therapeutic object of the present invention, for example, the inhibitor activity of AXL or GAS6 activity, or an inhibitor of the interaction between AXL and GAS6. In some embodiments, embodiments of therapeutic effective dose of the objects of the present invention, for example, for the treatment of metastatic cancer, described herein vary depending on various factors including the route of administration, target site, physiological state of the patient, whether the patient is human or an animal, other drugs, and whether treatment is prophylactic or therapeutic. Typically, the patient is a person, but also can be treated mammalian, non-human, including transgenic animals. Therapeutic dose should be titrated to optimize performance and security.
In some embodiments, embodiments, the dosage may vary from about 0.0001-100 mg/kg, and more usually from about 0.01 to 5 mg/kg of body weight of the host. For example, the dose may be 1 mg/kg of body weight or 10 mg/kg of body weight, or within 1-10 mg/kg. Exemplary treatment regime entails the appointment once every two weeks or once a month, or once every 3 to 6 months Therapeutic entities of the present invention is generally administered several times. Intervals between single dosages can be weekly, monthly or yearly. The intervals may be irregular, as shown by measuring levels in the blood therapeutic entity. In addition, therapeutic entities of the present invention can be injected with a slow release, in this case, the frequency of introduction below. Dosage and frequency vary depending on the half-life of the polypeptide in the patient.
In prophylactic applications, a relatively low dosage is administered at relatively rare intervals over a long period of time. Some patients continue to receive treatment until the end of his life. For therapeutic purposes it is sometimes necessary to introduce a relatively high dosage at relatively short intervals to reduce or stop the progression of the disease, and preferably until the patient shows partial or complete improvement of symptoms. After this, the patient may be transferred to a preventive mode.
In other embodiments, embodiments of the methods of the present invention include the treatment, reduction or prevention of metastasis of a tumor, or tumor invasion of the ovaries, breast cancer, lung cancer, liver cancer, colon cancer, gallbladder cancer, pancreatic cancer, prostate cancer and/or g�of gioblastoma.
In some other embodiments, the embodiment for the prophylactic use of pharmaceutical compositions or medicaments are introduced to a patient susceptible to or at risk of developing the disease or condition in an amount sufficient to eliminate or reduce the risk, reduce the severity, or delay onset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes arising in the course of developing the disease.
In some other embodiments, the embodiment for therapeutic use therapeutic objects of the present invention is administered to a patient with suspected or already suffering from such a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and the development of intermediate pathological phenotypes of the disease. The amount that is adequate to achieve a therapeutic or prophylactic treatment is defined as a therapeutically or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually introduced in several dosages until a sufficient response will be achieved.Typically, the response control�varies and is given repeated doses, if there is a recurrence of cancer.
In accordance with the present invention compositions for the treatment of metastatic cancer can be administered parenterally, topically, intravenously, nutripure, orally, subcutaneously, intraarterially, nutrician, intraperitoneally, intranasally or intramuscularly. The most typical route of administration is intravenous or nutripure, although other routes can be equally effective.
For parenteral administration the compositions of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid, such as water, oil, saline, glycerol or ethanol. Additionally, auxiliary substances, such as moisturizing or emulsifying agents, surfactants, pH buffering substances, etc. may be present in the composition. Other components of pharmaceutical compositions are substances of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil and petroleum products. In General, glycols such as propylene glycol or polyethylene glycol, are preferred liquid carriers, particularly for solutions for injection. The antibodies can be administered in the form of an injection depot pre�Arata or implant, which can be formed in such a way as to provide sustained release of the active substance. A typical composition comprises monoclonal antibody at a dose of 5 mg/ml, placed in an aqueous buffer consisting of 50 mm L-histidine, 150 mm NaCl, brought to pH 6.0 with HCl.
Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; can also be obtained solid forms suitable for dissolution or suspension in liquid vehicles prior to injection. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for improving adjuvant effect, as described above. Longer, Science 249:1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28:97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant, which can be formed in such a way that it is possible to obtain a constant or pulsatile release of the active ingredient.
Additional forms suitable for other applications, include oral, intranasal and pulmonary forms, suppositories, and transdermal applications.
For candles binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing active ingredient in dia�the azone from 0.5 to 10%, preferably 1-2%. Oral forms include fillers such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose and magnesium carbonate. These songs used in the form of solutions, suspensions, tablets, pills, capsules, forms with prolonged release or powders and contain 10% -95% active ingredient, preferably 25-70%.
Local application may result in transdermal or intradermal delivery. Local administration may contribute to the simultaneous appointment of the agent recently isolated toxin or detoxified derivatives or subunits or other similar bacterial toxins. Glenn et al., Nature 391:851, 1998. Co-administration can be achieved using the components in a mixture or in the form of related molecules obtained by chemical crosslinking or expressed as a hybrid protein.
In addition, transdermal delivery can be achieved using dermal patches or by using transferases Paul et al., Eur. J. Immunol. 25:3521-24, 1995; Cevcetal., Biochem. Biophys. Acta 1368: 201-15, 1998.
Pharmaceutical compositions typically are prepared as sterile, essentially isotonic and in full compliance with all rules of production and quality control (GMP) of the administration of U.S. food and medicine.
Preferably therapeuti�Eski effective doses of the compositions of antibodies described herein will provide therapeutic effect without causing substantial toxicity.
Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50(the dose lethal to 50% of the population) or LD100(the dose lethal to 100% of the population). The ratio of doses between toxic and therapeutic effects is therapeutic index. The data obtained in the research on these cell cultures and animals, can be used in the development of a range of doses that are not toxic for use in humans. The dosage of the proteins described herein, is preferably in the range of circulating concentrations that include the effective dose and little or no toxic. The dosage may vary within this range depending upon the dosage form and the used routes of administration. The exact recipe, method of administration and dosage can be chosen by the attending physician considering the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1).
In addition, the scope of this invention includes kits containing a composition (e.g., soluble AXL options and their agents) of the invention and instructions for use. Sets can also�to keep at least one additional reagent. The kits typically include a label indicating the intended use of the contents of the set. The term includes any labels in written form, or accompanying written materials, or set in, or who are otherwise accompany the set.
According to another aspect of the invention provides methods for determining the ability of a tumor to tumor invasion and/or metastasis by detecting and/or determining the activity level of AXL or GAS6 activity in biological images of interest. In some embodiments, the activity level of AXL or GAS6 activity is measured by the level of mRNA expression, protein expression level, the level of activation of protein or any suitable indicator of the corresponding activity of AXL or GAS6 directly or indirectly. In some embodiments, embodiments, the level of AXL activity or GAS6 activity in a biological sample further, compared with a certain level, for example, a standard level obtained by establishing a normal level or range of activity of AXL or GAS6 activity, based on population samples of tumors where does not develop tumor invasion or metastasis of tumor or normal tissues. For example, increasing the activity of AXL or GAS6 activity more than a predefined level or standard level SVID�have confirmed the susceptibility of a tumor to tumor invasion or metastasis of the tumor.
All publications and patents cited in this description are incorporated herein by reference as if each publication or patent were specifically and individually indicated, shall be incorporated by reference and are included here by reference to the identification and description of the methods and/or materials are published. The citation of any publication disclosed before the filing date of the application should not be construed as an admission that the present invention does not take precedence retroactively to such publication prior to the entry into force of the invention. In addition, the publication dates of the conditional, and may differ from the actual publication dates, which may need independent confirmation.
As will be clear to experts in this field when reading this description, each embodiment described and illustrated here, has discrete components and features which may be readily separated from or combined with the features of any other one or more embodiments, without departing from the scope or spirit of the present invention. Any cited method can be carried out in the order cited events or in any other order that is logically possible. Subsequently will be described examples to illustrate the parts of the invention.
Terapeuticas�I blockade of AXL signaling inhibits metastatic progression of tumors.
Previously AXL as a therapeutic target for the treatment of metastatic disease has been little studied, and most importantly in vivo has not been demonstrated relationship with targeting AXL. We showed that AXL is a marker of metastasis in patients with breast cancer and patients with ovarian cancer, and the severity of the disease in these patients correlates with the amount of AXL protein in the primary tumor. Most importantly, we have shown that metastasis of the tumor can be successfully treated in mice that already have metastasis, through the introduction of soluble ectodomains AXL. Mechanistically, inhibition of AXL signaling in animals with metastatic disease results in reduction of infestation and activity of MMP. Our results show that inhibition of AXL signaling cascade in tumor cells by introduction of soluble ectodomains AXL sufficient for inhibiting metastatic progression of tumors.
In this study, we studied whether AXL is a critical factor in the metastasis of cancer in humans, and whether therapeutic blockade of AXL signaling can be an effective tool for the treatment of metastatic disease. We used both genetic and therapeutic approaches to directly assess the role of AXL in the initiation and progression of metastatic cancer m�dairy cancer and ovarian cancer.
AXL is a marker of tumor progression and metastasis for human cancer. We first compared the expression of AXL in normal tissues, primary tumors and metastases in patients with breast cancer or ovarian cancer. 100% normal epithelial cells adjacent to breast cancer cells, were present diffuse cytoplasmic and nuclear staining for AXL, that was made for the background color given the fact that AXL is a membrane-bound receptors (n=27, figure 1). However, in primary breast tumors membrane AXL staining in the tumor epithelium is present in 25% (1/4) of the 1st stage, 76% (10/13) stage 2, and 100% (18/18) of the 3rd stage samples (figure 1A and table 1). In addition, AXL was expressed in 88% (8/9) of lymph node metastases.
In serous ovarian cancer samples AXL expression was first examined in the normal surface epithelium of ovary (OSE), as the majority of ovarian tumors are thought to arise from these cells. In samples of patients with ovarian cancer, who have maintained normal OSE, AXL was expressed in 0% (0/5) of the samples (figure 1B). In contrast, staining of membrane AXL in the primary tumor epithelium was present in 66% (6/9) stage II and 83% (53/64) stage III patient samples (figure 1B and table I). In addition, tumor samples from common metastatic sites, the�them as the omentum and the abdominal cavity, showed high expression of AXL in 75% (24/32) and 90% (27/30) of samples, respectively (figure 1B and table I). These data show that AXL expression in primary tumors correlates with metastasis, as shown in the later stages of the disease and metastatic tumors. In addition, these data show that metastases derived from human breast and ovarian cancer Express high level of AXL.
AXL is a critical factor for tumor metastasis. To study the functional role of AXL in metastasis, we used a genetic approach to suppress AXL in mouse models of metastatic breast cancer and ovarian cancer. For this purpose, we analyzed a panel of human cancer cell lines of breast and ovarian cancer on the expression of AXL protein to identify metastatic cell lines with high expression of AXL. As in our clinical data, AXL is highly expressed in most metastatic cell lines of breast cancer (NCI-ADR-RES, MDA-231, HS 578T, BT-549) and ovarian (SKOV3, OVCAR-8, ES-2, MESOV, HEYA8), while cell lines with low metastatic potential (MCF7, MDA-MB435, T47D, IGROV1, OVCAR-3; figure 9) AXL expression was undetectable or low level. AXL-deficient cell line metastatic breast cancer (MDA-231) and ovarian (SKOV3ip.1 and OVCAR-8) were�researchers have described using msrdc, aimed at AXL. Western blot analysis confirmed that cells expressing the sequence shAXL, Express less than 5% AXL protein compared to cells expressing scramble control sequence MSRC (shSCRM, figure 9C).
To directly evaluate the role of AXL in the later stages of metastasis of breast cancer tumors, we injected AXL-wild-type (shSCRM) and AXL-deficient (shAXL) MDA-231 cells into the tail vein of naked mice and evaluated the tumor load in the lungs at the twenty-eighth day. Microscopic evaluation of the lungs showed that 5/5 mice, which were injected with scramble MSRC (shSCRM) MDA-231 cells developed metastatic foci stained positive for AXL (figure 2A). On the other hand, 0/5 mice, which were injected MSRC AXL (shAXL), developed lung metastases histological evaluation (figure 2A). In order to quantify tumor burden in the lungs of these animals, we conducted an analysis of real-time PCR for human GAPDH. Figure 2A shows that in the lungs of mice that were administered shSCRM MDA-231 cells expressed high levels of human GAPDH, indicating the presence of metastases derived from MDA-231 cells. In addition, shSCRM injected mice Express human AXL in the lungs, indicating the presence of AXL-positive tumor cells (figure 2A). In contrast, mice which�first introduced shAXL tumor cells, not Express human GAPDH or AXL in the lungs. These results show that genetic inactivation of AXL enough to completely inhibit the formation of lung metastases in this model.
To determine the influence of genetic inactivation of AXL on the ability of ovarian cancer cells to metastasize in vivo, we compared the ability shSCRM and shAXL SKOV3ip.1 cells to the formation of metastases using a peritoneal model of xenotransplanted ovarian cancer. This model repeats peritoneal dissemination of human ovarian metastases in a mouse, which develops rapidly progressive disease, consisting of ascites, and over 100 small metastatic lesions, which are attached to the mesentery, diaphragm, liver and other peritoneal surfaces after peritoneal injection of SKOV3ip.1 cells (figure 3B). Immunohistochemical analysis of AXL expression in SKOV3ip.1 peritoneal metastases showed that they are similar to the metastasis of ovarian cancer man, AXL is highly expressed in SKOV3ip.1 metastatic lesions, indicating that this is a relevant model system for studying the role of AXL in ovarian metastasis (data not shown). Twenty-eight days after peritoneal injection shSCRM and shAXL cells, shSCRM mice show signs of severe ascites and morbidity due �it was necessary to scarify mice and to investigate changes in tumor load between shSCRM and shAXL injected mice. When mice were injected shSCRM cells developed ascites and >100 peritoneal metastases, if the mouse was injected shAXL cells developed only a few metastases (figure 2B). The average number of peritoneal metastases greater than 5 mm in size was significantly reduced from 13,4 +/- 4.3 I shSCRM injected to mice 0,8 +/- 0,5 I shAXL injected mice (figure 2B). In addition, the average weight of these tumors was significantly reduced with 236 +/- 74 mg in shSCRM injected mice to 39.2 +/-18 mg in shAXL injected mice (figure 2B). In support of these findings, knockdown of AXL expression in OVCAR-8 cells significantly inhibited the total tumor mass and the number of peritoneal ovarian tumors (figure 2C). Collectively, these results indicate that AXL is a critical factor for metastasis of breast cancer and ovarian cancer.
Taking into account the important role of AXL in the formation of metastases in vivo, we next sought to determine whether AXL specifically regulates metastasis or AXL plays a General role in the regulation of proliferation of tumor cells and growth. To answer these questions, we conducted in vitro analyses of cell proliferation, in which the total number of cells between wild-type AXL (shSCRM) and AXL-deficient (shAXL) cells were counted for 10-14 days. We found no significant differences in cell growth curves between hSCRM and shAXL MDA-231, SKOV3ip.1 or OVCAR-8 cells (figure 3). In addition, no significant difference is observed between the orthotopic MDA-231 or subcutaneous SKOV3ip.1 tumor growth between shSCRM and shAXL cells (figure 3). These results show that AXL is not required for the proliferation of tumor cells or subcutaneous growth in vivo. Overall, our results show that AXL is specifically regulates metastasis of breast tumors and ovarian tumors.
AXL regulates tumor cell invasion. To determine possible mechanisms of AXL-mediated metastasis, we chose an unbiased approach and directly compared the role of AXL in critical cellular functions associated with metastatic cascade, including proliferation, invasion, migration, adhesion and survival. We found that the shAXL MDA-231, SKOV3ip.1 and OVCAR-8 cells were significantly weakened in the possibility of invasion through collagen type I (figure 4A). We also observed a slight decrease of cell migration in shAXL cells, but we were unable to find differences in adhesion to ECM proteins or in survival after the introduction of serum, indicating that AXL mostly affects invasion in the metastatic cascade.
Recently, it was determined that at the molecular level of MMP-9 is an effector of AXL-mediated invasion in breast cancer cells. Thus, we investigated was limR-9 expression or activity is altered in AXL-deficient cell tumors of the ovary. Although SKOV3ip.1 cells do not expressively MMP-9, we found that MMP-2 was highly expressed in these cells, and the cells shAXL mRNA of MMP-2 decreased significantly (figure 4B). Analyses of MMP-2 reporter gene luciferase showed that detected the promoter activity of MMP-2 decreased significantly in shAXL cells compared with cells shSCRM, which suggests that AXL regulates MMP-2 at the transcriptional level (figure 4C). Gelatin demograficheski analysis showed that the levels of secreted proteins of MMP-2 were significantly reduced in shAXL cells compared to shSCRM SKOV3ip.1 cells (figure 4D). Collectively, these data suggest a role of AXL as a step-up regulator of the expression of MMP-2 and its activity in cells of human ovarian cancer.
Next, we tried to determine the signalling pathways involved in AXL-mediated MMP-2 expression. Activation of AXL from GAS6, it was reported that directly induces a number of intracellular signaling pathways, including PI3K, PAS, MARK, SRC and PLC. Among these pathways, the PI3K signaling pathway has been shown as a way of regulating the expression of MMP-2 and invasion in ovarian cancer cells. To determine how PI3K signaling depends on the loss of AXL in SKOV3ip.1 cells, we conducted Western blot analyses of phospho-AKT at Ser473 (P-AKT) in AXL-wild-type and AXL-deficient SKOV3ip.1 cells. We saw strong inhibition of P-AKT expression in shAXL cell�x compared to shSCRM SKOV3ip.1 cells (figure 4E). In addition, GAS6 stimulation depleted SKOV3ip.1 cells as a result of PI3K-dependent induction of P-AKT, such as treatment with PI3K inhibitor Ly294002, completely overrides GAS6-induced P-AKT expression (figure 4E). To determine whether the PI3K pathway is involved in AXL-mediated MMP-2 expression, we conducted analyses of MMP-2 reporter gene luciferase in the presence of GAS6 and Ly294002. Induction of MMP-2 promoter activity after stimulation with GAS6 was completely blocked by Ly294002, suggesting that GAS6/AXL signaling regulates MMP-2 expression via PI3K signaling events (figure 4F).
Therapeutic inhibition of AXL significantly inhibits metastatic tumor progression in mice. Our results show that AXL is a critical factor for metastasis and support the hypothesis that therapeutic blockade can be an effective tool for the treatment of metastatic disease. To test this hypothesis, we used a soluble ectodomain AXL in therapeutic strategy for inhibition of AXL signaling. Soluble ectodomain AXL functionally acts as a receptor trap and it has been previously shown that GAS6 binds with mu affinity in vitro and in vivo (figure 5A). First, we investigated whether treatment with soluble ectodomain AXL sufficient to suppress AXL alarm� and invasion of metastatic tumor cells. PI3K/AKT signaling is regulated by AXL in different types of cells. We found that the PI3K/AKT signaling is regulated by GAS6/AXL signaling in SKOV3ip.1 cells, and treatment with soluble ectodomain AXL (sAXL) managed to reduce PI3K/AKT activation in treated GAS6 SKOV3ip cells.1 (figures 5B and C). In addition, the introduction of MDA-231 cells in collagen with sAXL was enough to drastically reduce cell invasion, demonstrating that the use of sAXL effect on AXL signaling and invasion in vitro (figure 5D).
Next, we examined how the introduction of sAXL will affect the metastatic tumor progression in vysokoplastichnymi model of ovarian cancer. First, we induced SKOV3ip metastases.1 in Nude mice (day 1) and started treatment sAXL on day 7 after checking macroscopic lesions. sAXL therapy was delivered using an adenoviral system in which the liver carries out a systematic release of sAXL protein in the serum of mice within 28 days after injection (figure 6A). Macroscopic analyses of tumor mass on day 28 showed that mice treated with therapy sAXL was statistically significant (p<0.01) reduction in tumor mass compared with mice treated with Fc control therapy. In the model of tumor SKOV3ip.1, the total mass of tumors and number of tumors decreased by 63% in mice treated sAXL compared to Fc mice (figure 6C). Similarly, in OVCAR-8 mod�Lee, the total weight of tumors and number of tumors decreased significantly by 47% and 42% respectively (figure 11). We examined the expression levels Mmrv tumors SKOV3ip.l using PCR analysis in real time and found that the levels MMR significantly decreased in tumors sAXL mice compared with Fc control mice (figure 6C). These results suggest that monotherapy AXL quite significantly reduced metastatic tumor burden in mice with identified disease. In addition, our results show that therapeutic effect of AXL in tumor growth, metastases may lead to inhibition of invasion, at least in part through the regulation of MMP activity.
Considering that previous antimetastatic inhibitors targeting MMP, it has been shown that they have a significant impact on normal tissue in terms of toxicity, we conducted a comprehensive analysis of the toxicity to normal tissues in mice treated with therapy sAXL within 21 days. We did not observe behavioral, macroscopic or microscopic abnormalities in Nude mice treated sAXL or Fc treatment (figure 7).
Invasion and migration are important intracellular properties that contribute to the pathogenesis of tumor metastasis. It has been suggested that therapeutic agents that target these processes can be� a useful strategy to suppress metastasis and may provide clinical benefit in patients with metastatic disease. We have shown that the receptor tyrosine kinase AXL is a critical determinant of the ability of cells to tumor invasion and metastasis. Most importantly, we showed that therapeutic blockade of AXL signaling with soluble AXL receptor significantly inhibited metastatic tumor progression in mice that already have metastatic disease. Mechanistically, our studies show that therapy with soluble AXL inhibits tumor metastasis, at least in part, through inhibition of MMP activity and invasion. Finally, we showed that AXL is highly expressed in metastases in advanced stage of the primary tumor of patients with ovarian cancer and breast cancer, underscoring the clinical significance of our findings.
Here it is shown that AXL is a critical factor for the metastasis of human cancer, and that therapeutic blockade of AXL signaling is effective for the treatment of metastatic disease. Also, we show that AXL is highly expressed in metastatic and advanced samples of primary breast tumors and ovarian cancer. We show that genetically AXL is crucial for early metastatic breast cancer and ovarian cancer using data obtained�tions on model organisms - naked mice. Most importantly, we have developed a very specific and non-toxic soluble AXL receptor, as an anti-AXL therapy, and demonstrated that treatment with soluble AXL receptor quite significantly inhibits metastatic progression of tumors in mice with existing metastatic disease. Our results show that inhibition of AXL signaling cascade in tumor cells can inhibit both the initiation and progression of metastatic disease. Our data imply AXL as a novel therapeutic target for advanced and metastatic stages of breast cancer and ovarian cancer, and suggest that anti-AXL therapy can control and the initiation and progression of metastatic disease.
MMP play an important role in the regulation of invasion of tumor cells and metastasis. However, the mechanisms by which tumor cells induce MMP activity, remain unclear. MMP expression is increased in cancer in humans and correlates with tumour progression and poor patient survival. Gene amplification and activating mutations in MMP rarely found in human cancer, suggesting that other factors are responsible for the increased expression of MMP in cancer. Our data indicate that expression of MMP 2 R�pulirula AXL at the transcriptional level in cells of human ovarian cancer. Although the exact mechanism by which AXL regulates the expression of MMP-2, yet to be determined, it is shown that pharmacological inhibition of PI3K path reduces the promoter activity of MMP-2 in GAS6-stimulated cells, indicating the role of the PI3K pathway (figure 8). It is important to note that our results suggest that therapeutic blockade of AXL may be an effective and non-toxic strategy to inhibit the activity of MMP in tumours. Broad spectrum MMP inhibitors have failed when tested in the treatment of cancer, in particular in connection with high levels of toxicity to normal tissue. Our results show that the predicted side effects of the anti-AXL therapy are minimal. We did not observe any toxicity to normal tissues associated with adenovirus-mediated delivery of soluble AXL of ectodomain in therapy in mice. In addition, germ line cells AXL and GAS6 mice are viable and phenotypically normal, as adults, suggesting that AXL or GAS6 is not required for the development and normal functioning of tissues.
We have shown that monotherapy AXL sufficient for inhibiting metastatic progression of tumors in viscoplasticity models of metastatic ovarian cancer. These findings have important clinical implications for the treatment of ovarian cancer. PR�approximately 14 600 people die from ovarian cancer annually in the United States. There are currently no FDA-approved biological methods for the treatment of ovarian cancer, although Avastin (mAb targeted VEGF) and Tarceva, and Masha (a small molecule inhibitor of EGFR kinase) are used in clinical trials for the treatment of advanced and recurrent ovarian cancer. Standard therapy for ovarian cancer includes surgery with optimal removal of the lesion and subsequent cytotoxic combination therapy platinum-Texan. Despite these efforts, eighty percent of patients diagnosed with ovarian cancer develop recurrent disease and only 30% of these patients survive 5 years after diagnosis.
Our data show that AXL therapy is an effective adjuvant therapy for the treatment of advanced and recurrent ovarian cancer. Model metastatic progression of ovarian tumors used in our research, like the development of disease recurrence in patients after surgical removal. We found that AXL therapy could reduce metastatic tumor burden in mice with identified disease by 63%. The creation of new metastatic lesions during the progression of the disease has decreased significantly. This observation is consistent with our findings, showing that AXL mainly affects cell invasion of tumors�and, and not cell proliferation or growth. Taken together, our results show that therapy AXL functions mainly as anti-metastatic agent and can be most effective in combination therapy to current cytotoxic agents.
Thus, AXL is a critical factor for metastasis, and the blockade of AXL signaling has a positive therapeutic effect in metastasis. These studies are important preclinical data on anti-AXL therapy for metastatic disease.
The cell lines. Cells SKOV3 ovarian, SKOV3ip.1 and HEYA8 were received as a gift from Dr. Gordon Mills (MD Anderson Cancer Center). Cells ovarian ES-2 and MESOV - a gift from Dr. Branimir Sikic (Stanford University). MDA-231, OVCAR-3 and MCF-7 cells were obtained from ATS. IGROV-1 and OVCAR-8 cells were purchased from NCI-Frederick DCTD repository cell line tumors. Cells are cultured in appropriate media with the addition of 10% thermoinactivation fetal calf serum and 1% penicillin and streptomycin at 37°C in 5% CO2incubator. Cell mass from the NCI60 panel of cancer cell lines of breast and ovarian cancer were provided by Dr. Giovani Melillo (NCI-Frederick).
Patients and tissue microarray. The microarrays of human breast tissue were acquired in US Biomax (BR1002). A tissue microarray of human ovaries were obtained�s from the Department of pathology at Stanford University. A total of 73 waxed sample tumors were previously obtained from patients with ovarian cancer at Stanford hospital from 1995 to 2001 at the time is not yet treated. These primary samples of ovarian tumors were collected in a tissue microarray containing two samples from each patient. Another 30 samples of tumors from the abdominal cavity were evaluated in this microchip. All patients had serous ovarian cancer, the information was obtained in accordance with the standards of the International Federation of gynecology and obstetrics. All samples and relevant clinical data were collected in accordance with protocols approved by the institutional review Board at Stanford University. Additional commercially available tumor microarrays were used to study 32 metastatic lesions of the omentum (US Biomax).
AXL immunohistochemistry. With paraffined tissue sections were made, which were deparaffinization in xylene, rehydrations and zamaskirovalis following standard immunohistochemical methods. Primary antibody AXL (RandD Systems) was used at a dilution of 1:500. Negative control for all samples was performed using only the secondary antibodies. Complexes of antigen-antibody were visualized using the Vectastain ABC system (Vector Laboratories) and DAB Substrate Kit for pen�of sidaz (Vector Laboratories) according to manufacturer's protocols. Sections were contrast stained with hematoxylin. AXL staining on the membrane of tumor cells was evaluated microscopically in accordance with the percentage of positive cells for AXL expression (0 if no, 1 for low quality samples, 2 for 5-60% and 3 for 61-100%).
Assays with the reporter. Reporter plasmid MMP-2 under the control of the promoter of MMP-2 in p.about. was received as a gift. The luciferase activity was determined using Dual-Glo Luciferase Assay reagent (Promega) in shSCRM and shAXL SKOV3ip.1 cells and were measured in a Monolight 2010 luminometer (analytical Laboratory of luminescence). The activity of Firefly luciferase was normalized to Renilla activity. Analyses were conducted in triplicate and were repeated twice.
Temporary and retroviral transfection. Temporary transfection DMA was conducted by lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 0.1 μg of MMP-2 cDNA (OpenBiosystems) was transfected in 6 cups.
miRNAs (small interfering RNA): miRNAs sequences targeting AXL or control, were purchased from Dharmacon. All miRNAs transfection was performed using Dharmacon Smart Pools with Dharmafect 1 transfection reagent according to the manufacturer's Protocol (Dharmacon, Lafayette, CO).
MSRC: Oligonucleotides for the specific degradation of AXL RNA 5'-GATTTGGAGAACACACTGA-3' were synthesized as described above. A random sequence (Scrmble) was used as control Msrdc 5'-AATTGTACTACACAAAAGTAC-3'. These oligonucleotides were cloned into RNAi-Ready pSiren RetroQ (BD Bioscience) vectors and SKOV3ip.1, MDA-231 and OVCAR-8 cells using the retroviral transduction of these vectors. Infected cells were selected in puromycin (Sigma), and polyclonal populations were tested for the reduction of the expression level of AXL Western blot analysis.
The plasmids. Ectodomain AXL corresponding to amino acids 1-451, were amplified from cDNA of human AXL (Open Biosystems) and cloned into CMV-driven pADD2 adenoviral Shuttle vector. Temporary DNA transfection with a control vector or AXL 1-451 conducted by lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions in NT cells. Conditioned medium was collected 48-72 hours after transfection.
The adhesion tests. SKOV3ip.1 shSCRM and shAXL cells fluorescently labeled Sum CMFDA (Molecular Probes). Cells were washed and separated using a nonenzymatic cell dissociation buffer (Gibco). Cells (Hae) were seeded in 96 cells pre-coated with 50 μg/ml of collagen type I (BD Bioscience). After 60 minute incubation at 37°C, cells were thoroughly washed with 5 times. Fluorescent activity (excitation 494 nm, emission 517 nm) was measured using a fluorescence spectrophotometer.p>
SKOV3ip.1 Adhesion to Collagen type 1. SKOV3ip.1 shSCRM and shAXL cells fluorescently labeled with 5 μm CMFDA (Molecular Probes). Cells were washed and separated with the help�d nonenzymatic cell dissociation buffer (Gibco). Cells (Hae) were placed in triplicate in 96-cell tablet, which pre-coated with 50 μg/ml of collagen type I (BD Bioscience). After 60 minute incubation at 37°C, cells were thoroughly washed with 5 times. Fluorescent activity (excitation 494 nm, emission 517 nm) was measured using a fluorescence spectrophotometer.p>
MDA-231 Adhesion to ECM proteins. MDA-231 shSCRM and shAXL (0.5×106cells were placed in triplicate into the wells containing the panel ECM proteins, including laminin, collagen I and IV, fibronectin and fibrinogen. Cells were incubated at 37°C for 1 hour and washed in PBS. Associated cells were stained and quantitatively determined at OD 560 in accordance with the manufacturer's Protocol (CellBiolabs).
Analyses of migration. Cell migration was studied in vitro as described above. In short, cells were deprived of serum for 24 hours and seeded (2.5 x 104cells) in triplicate on uncoated inserts (BD Biosciences), transferred into a chamber containing FBS as a chemoattractant and incubated for 24 hours. After removal of cells that have not penetrated through the membrane, the cells at the bottom of the membrane were fixed, stained and counted. Five fields were considered for each membrane. The percentage of migration was determined as follows: (average number of cells that migrate to shAXL cells/mean number of cells that mehrerau� in shSCRM cells) X 100. The experiments were conducted in triplicate and repeated three times.
Analysis of invasion in collagen. Analyses of invasion in collagen were carried out as described above. Briefly, 533 cells were seeded on collagen type I 48-well plate. Cells were cultured in standard medium or medium with the addition of a conditional control, sAXL or conditioned medium for 5-7 days and the photos were taken. Invasion through collagen was calculated quantitatively by determining the percentage of tumor cells that show the branching phenotype in a 20x field. Three fields in the sample were counted. The experiments were conducted in triplicate and was repeated 2 times.
Zymography on gelatin substrate. SKOV3ip.1 shSCRM and shAXL cells were subjected to serum starvation for 48 hours. 25,000 cells were seeded in 96 cells and conditioned medium was collected after 24 hours. Equal volumes of conditioned media were placed at non reducing conditions on 10% zymography gel (Invitrogen). After electrophoresis, the gel was washed with 2.5% (volume/volume) Triton X-100 to remove SDS, and washed with 50 mm Tris-HCl, 5 mm CaCI2and 0.1% Triton X-100 (pH of 7.8), and incubated overnight at 37°C. Zymogram were stained for 30 minutes with 0.25% (W/V) Coomassie Brilliant Blue R250 dissolved in 40% methanol and 10% glacial acetic acid. Gels were washed from the day� in 40% methanol and 10% glacial acetic acid. The experiments were conducted in duplicate and repeated three times.
Tests of cell proliferation. For monolayer growth curves of cells (50000) were seeded in 60 mm Cup in triplicate. Every three days the cells were treated with trypsin, counted using a cell counter of Coulter and 50,000 cells were again seeded and counted.
The survival analysis of HTT. Cell viability is measured HTT analysis as described above. In short, cultured with or without feeding cells (0, 3, 6 and 7 days) were incubated with phenol red in the medium for selection with 0.3 mg/ml HTT and 2.65 ág/ml N-methyldibenzothiophene sulfate. 96-well plates were returned to the incubator at 37°C during 1 to 2 hours. Metabolism of HTT was quantitatively measured by adsorption at 450.
Protein expression and Western blot analysis. Protein lysates were collected in 9 M urea, of 0.075 M Tris buffer (pH 7.6). Protein lysates were quantitatively determined using analysis by Bradford, and performed SDS-PAGE in reducing conditions according to standard methods. The resulting blots were probed with antibodies against AXL (RandD Systems), alpha-tubulin (Fitzgerald Antibodies), AKT (Cell Signaling), phospho-AKT (Cell Signaling).
For GAS6 stimulation cells were cultured in serum-free medium for 24 hours. Cells were treated with 25 μm of PI3K inhibitor (Ly294002, Mol Bio Bio Research Laboratory) or 100 I konditionierung� environment containing the AXL ectodomain 4 hours before treatment with 400 ng/ml GAS6 for 15 minutes.
For the analysis of sAXL expression in mice serum and 1.5 I of the serum from each sample was analyzed using gel electrophoresis.
The creation and production of adenovirus. Ectodomain AXL corresponding to amino acids 1-451 was amplified from AXL cDNA (Open Biosystems) and cloned into the field E1 from E1 ' E3 ' Ad 5 strain through homologous recombination with subsequent production of adenovirus in 293 cells and purification in the CsCI gradient, as previously described. Production and purification of sAXL of adenovirus was performed as described above. The creation and production of the negative control virus expressing murine IgG2-Fc fragment of immunoglobulin, described above.
The Growth Of SKOV3ip.1 and OVCAR-8 cells, peritoneal xenografts. All procedures involving animals and their care were approved by the Committee on animal care and use Stanford University in accordance with institutional and NIH guidelines.
Control and AXL SKOV3ip.1 and OVCAR-8 cells were injected intraperitoneally 1×106and 5×106cells, respectively, in 0.5 ml of PBS in naked female mice. After scarification ascites was quantified, metastatic lesions were counted and all visible injuries were uncovered and removed to determine the weight of the tumor./p>
Parent cell SKOV3ip.1 and OVCAR-8 were injected intraperitoneally with 1×106and 5×106cells, respectively, in 0.5 ml of PBS in naked female mice. After seven (SKOV3ip.1) or 14 (OVCAR-8) days after injection of tumor cells, the mice were injected sAXL or control of 1.9×108adenoviral FIGHT in 0.1 ml PBS into the tail vein. After scarification ascites was quantified, metastatic lesions were counted and all visible injuries were uncovered and removed to determine the weight of the tumor.
The study of tissue toxicity. Parent cell SKOV3ip.1 were injected intraperitoneally with 1×106and 5×106cells, respectively, in 0.5 ml of PBS in naked female mice. Seven days after injection of tumor cells, the mice were injected sAXL or control of 1.9×108adenoviral FIGHT in 0.1 ml PBS into the tail vein. On day 28 mice were scarificial. The blood was collected, and at the Department of comparative medicine Stanford University was held blood chemistry and CBC analysis. Tissue samples were collected from all major organs, including liver, kidney, brain, spleen, which was fixed in 10% formalin, embedded in paraffin, made cuts, and contrasting stained with hematoxylin and eosin.
In vivo analysis of metastasis. Control and AXL MSRC MDA-231 cells were injected intravenously with 5×105cells in 0.1 ml PBS into the tail vein th�Oh mouse. Four weeks after injection the mice were scarificial. Microscopic evaluation of the lesions in the lungs was conducted on a representative cross-section (slice) of the lung is fixed in formalin and waxed, which were stained with hematoxylin and eosin. Correct identification of lesions in the lungs with no less than four human cells with large nuclei and positive for the expression of AXL was confirmed by a Board certified pathologist. Tumor load in the lungs of mice was determined quantitatively by analysis of the expression of human GAPDH and AXL were assessed by analyzing RNA isolated from whole lung using real-time PCR.
The growth of MDA-231 cells as orthotopic tumors. MDA-231 cells were grown as subcutaneous orthotopic tumor in a six-week naked (nu/nu) mice with intradermal injections of 107cells in 0.1 ml PBS into the fatty tissue. Tumors were measured by caliper on a 38-day course. The volume was calculated by the following formula: width2× length × 0.5.
The Growth Of SKOV3ip.1 cells as subcutaneous tumors. Five million cells in 0.1 ml PBS were implanted subcutaneously into the flanks are bare (nu/nu) six-week female mice. The tumor was measured by caliper for 45 days. The volume was calculated by the following formula: width2× length × 0.5.
RNA and PCR analysis in Rea�Lincoln time. RNA was isolated from cells and tissues using trizol according to the manufacturer's protocols (Invitrogen). cDNA was synthesized from 2 µg RNA, treated with Dnazol (Invitrogen) using Superscript first-strand synthesis system for reverse transcription-PCR (Invitrogen). One μl of cDNA was subjected to amplification using SYBR Green PCR Master Mix (Applied Biosystems). The following sets of primers were used for amplification of specific target genes: 18S FWD: 5-GCCCGAAGCGTTTACTTTGA-3 REV:5-TCCATTATTCCTAGCTGCGGTATC-3; AXL FWD: 5-GTGGGCAACCCAGGGAATATC-3 REV: 5-GTACTGTCCCGTGTCGGAAAG; GAPDH 5-ATGGGGAAGGTGAAGGTCG-3 REV: 5-GGGGTCATTGATGGCAACAATA-3, MMP-2 FWD: 5 GCCCCAGACAGGTGATCTTG-REV 3 5 - GCTTGCGAGGGAAGAAGTTGT-3. PCR amplification was performed on a Prism 7900 Sequence Detection System (Applied Biosystems). We used the following cycle parameters: denaturation at 50°C for 2 minutes and at 95°C for 10 minutes, then a cycle of denaturation at 95°C for 15 sec and at 60°C for 1 minute. 18S was used as the standard mRNA. The relative expression level of mRNA was determined using the method of relative standard curve in accordance with the instructions of the manufacturer (Applied Biosystems).
Statistical analysis. Tests for communication between AXL expression and tumor formation and metastasis was performed using Fisher's exact test. All other statistical tests were performed using t-student criterion. Values of p<0.05 was considered statistically Val�of each other.
Abbreviations: GAS6 - growth retardation-specific gene 6, MMP-2 - matrix metalloproteinase; EOC - epithelial ovarian cancer; ECM - extracellular matrix; AKT - V-act homologue of the viral oncogene murine thymoma.
|Statistical analysis of AXL staining to estimate the parameters of a tumor|
|Infil duct carcinoma|
|Pearson's x2 P value = Metastatic infil carcinoma of the ducts of the Lymph nodes||0(0)||1(11)||4(44)||4(44)||9|
|Pearson's x2 P value = Metastatic serous adenocarcinoma Gland||3(9)||5(16)||6(19)||18(56)||32|
Values are presented as n (%). For tumor cell membrane staining was evaluated as 0 - absent; 1 - it is impossible to calculate; 2 - from 5 to 60% positive; 3 - from 61 to 100% positive.
We showed that the inhibition of binding of ligand GAS6 to cell by AXL overexpression of soluble AXL in wild-type mice using an adenoviral expression system resulted in reduction of tumors�eve load, the evaluation which was carried out by measuring the number and size of the tumor, compared with the untreated control, further emphasizing the importance of GAS6 and AXL, as critical goals and effective strategies to suppress the progression of metastasis in a preclinical mouse models.
Developed soluble variants of the extracellular domain of AXL, as specified herein, which have a high affinity to the ligand GAS6, allowing them to absorb the ligand and reduce endogenous AXL signaling. Developed options significantly improved the affinity to GAS6 compared to wild-type AXL.
AXL extracellular domain consists of two IgG-like domains and two fibronectin-like domains. The main GAS6 binding site is located in the Ig1 domain, and minor GAS6 binding site is located in the Ig2 area.
In order to further improve the affinity of the primary binding site, we've designed the Ig1 domain with break points corresponding provisions 19-132 in AXL SwissProt P30530. The mutant library was generated by performing PCR amplification of the decreased accuracy of the Ig1 domain of the AXL receptor using standard methods of molecular biology. The library is expressed through surface display on yeast and carefully selected fluorescence-activated sorting of cells (FACS) to isolate mutants with improved affinity binding to soluble GAS6. In our approach library screening, a library of mutant protein was subjected to several rounds of sorting, in which each successive round reduces the size of the library, while she simultaneously enriched in content of mutant proteins with the desired property, in this case, high affinity binding to GAS6.
To get AXL mutants with significantly higher affinity to GAS6, in the later rounds of the sort used to sort by the rate of dissociation. To sort by the rate of dissociation of the library of mutants of the protein is first incubated with soluble GAS6, and then washed with buffer to remove unbound GAS6 from solution. Further, the mutant library were incubated in the presence of excess soluble competitor for 2, 4, 6, 12 or 24 hours at room temperature. The excess of the competitor is used to capture GAS6, which is separated from AXL on the surface of yeast, which makes the separation step is irreversible. Mutant AXL proteins that retain binding to GAS6, are collected using FACS. Analysis of the binding of GAS6 on the combined sample of 5 products after dissociating steps after 0, 4 and 6 hours shows that these products have considerable improvement compared with the wild-type AXL in conditions of persistent binding to GAS6 (see figure 12). The histogram of the number�governmental data with dot plots shows significant improvement of library members. Sequencing of these products revealed several mutations in the domain of the Axl Ig1, which give enhanced affinity to GAS6 observed for the pooled 5 products (figure 12 and table 2). The sixth round of sorting gives a further enrichment to 3 specific clones from the selected 5 products. Table 2 shows the unique mutations of amino acids in the sequence AXL contained in selected products 5 and 6 rounds. In this table, the number of the residue in the upper line corresponds to the amino acid residue in the wild-type AXL. The second line indicates a residue that is AXL wild-type at this position. In the subsequent lines contain amino acid mutations present in the mutant. No for a specific amino acid residue in the mutant (e.g., a blank or empty cell in table 2) indicates that this amino acid residue was not mutated in the wild type residue. Standard one-letter designation of the amino acid residues used because it is well understood by those who are expert in this area.
Shows unique sequence selected from 5 and 6 products, as well as required properties of the combined clones compared to wild-type AXL, demonstrating a significant improvement in binding to GAS6 for pooled samples of 5 products.
Mutants that have been isolated p�the power of this approach, directed evolution, comprising amino acid substitutions are shown in table 3.
|Mutants selected using directed evolution|
In accordance with the crystal structure of the complex GAS6-AXL, as reported by Sasaki et al. (EMBO J 2006), all mutations that have been shown above, except E26G, G32S, N33S and G127R/E, located on the surface of the binding between AXL and GAS6.
Individual mutants AXL S6-1 and AXL S6-2, starting from the sixth round of sorting were selected for further study. The study of the equilibrium binding of wild-type AXL, AXL S6-1 and AXL S6-2 were conducted to compare the affinity of interaction with GAS6 in wild type or mutant proteins AXL. The data is laid down on a four-point sigmoidal curve, and the midpoint was taken as the equilibrium binding constants, KD. Mutants AXL S6-1 and AXL S6-2 show a significant improvement in the affinity of binding to GAS6 compared to wild-type AXL (figure 13 and table 4). Wild-type AXL has a binding affinity (KDwith GAS6 2,4±1,2×10-9M; AXL S6-2 has a binding affinity (KDwith GAS6 1,89±0,37×10-10M with GAS6; and AXL S6-1 has a binding affinity (KD) Of 1.12±0,23×10-10M with GAS6. For AXL S6-1 and AXL S6-2 it 22 times and 12.8 times the strength of GAS6 binding affinity, respectively, compared to di�them type AXL (table 4).
|The binding affinity (KD) wild-type and mutant proteins with AXL GAS6|
|Balance GAS6 binding|
|KD(M)||+/- (M)||The excess over wt|
|wt AXL||Of 2.4×10-9||Of 1.2×10-9||-----|
We also investigated thermal stability of wild type and mutant proteins AXL using a scanning circular dichroism at variable temperature. This method keeps track of the unfolding of the secondary structural elements of the coiled protein depending on the temperature. The ellipticity of each protein is controlled depending on the temperature, and the data correspond to the standard two-zone curve expander. Temperatures� melting point (T m) is the middle point of the curve expander. For wild-type AXL exhibited a melting point 41,3±0.6°C; for AXL S6-1 exhibited a melting point 54,0±0,9°C (approximately 13°C higher thermal stability than the wild-type AXL) Axl S6-2 exhibited a melting point 41,55±0,02°C (approximately the same thermal stability of wild-type AXL) (table 5).
|Thermal stability of wild type and mutant proteins AXL, as defined by the scanning circular dichroism at variable temperature|
|Average Tm(°C)||+/- (°C)||The increase compared with wt (°C)|
Soluble Axl options inhibit metastatic about�massirovanie tumors in vivo.
GAS6-AXL signaling has been implicated in the progression of many aggressive forms of solid tumors, including breast, lung and colon, and was found in the present work, the ovaries. While a clear correlation is observed between the expression of AXL and stage of the disease, and the prognosis for the patient, the use of AXL as a therapeutic target for the treatment of metastasis has not been studied. In example 1 it is shown that AXL really is a marker of metastasis in patients with breast cancer and ovarian cancer, levels of AXL expression in the primary tumors correlated with disease severity. These results indicate that antagonism of signaling pathways GAS6-AXL may offer a therapeutic window for the treatment of metastatic disease. As indicated in example 1, to test the potential of AXL as a therapeutic target, a soluble form of wild-type extracellular domain of AXL was injected using an adenoviral delivery in a mouse model of aggressive ovarian cancer. We showed that tumor metastases were significantly reduced in mice that received treatment with the soluble AXL in comparison with the control group. These data showed that the antagonism of signaling GAS6-AXL in tumor cells with soluble AXL can prevent metal�critical to the progression of the disease. Based on these results, we showed that engineering AXL mutants with higher affinity to GAS6 show greater efficacy as anti-metastatic agents, and more appropriate therapeutic mode of delivery of soluble AXL gives more significant results.
In this study we used the model of human ovarian cancer, as in experiment 1 and options intraperitoneal introduction of purified soluble AXL (sAXL) mice with existing metastatic disease. We tested wild-type AXL and engineering created a mutant AXL S6-1 with high affinity, compared with a form of AXL, E59R/T77R that is not associated with GAS6. Our results demonstrate that increased affinity AXL S6-1 leads to greater therapeutic efficacy, as was a significant reduction in tumor load, which is measured by the number and total weight of all metastatic lesions compared to wild-type AXL and negative control AXL E59R/T77R. These data are further checked on AXL and GAS6 as therapeutic targets for inhibiting metastasis, and confirm that the constructed mutant S6-1 with high affinity for AXL is a potent antagonist of the signaling system GAS6-AXL.
While example 1 shows that adenoviral delivery sAXL gave therapeutic efficiency�, this method of delivery is not of clinical significance and, thus, we confirmed that the delivery of purified sAXL will give similar results. Wild-type AXL, AXL S6-1 and AXL E59R/T77R were combined in a fragment crystallized region (Fc) of rat IgG2a in order to improve pharmacokinetics. The only difference between these options triple AXL hybridization (AXL-FC) is detected mutations in AXL Ig1 domain, which is shown in table 6A. DNA coded AXL-Fc protein was cloned into an adenoviral Shuttle vector pADD2c under the control of CMV using EcoRI and Sall restriction enzymes cut sites. pADD2 a plasmid encoding the three AXL mutant was independently transfected into HEK 293 cells using a set of Freestyle Expression kit from Life Technologies as described by the manufacturer. Proteins were purified from culture supernatant using Protein A affinity chromatography, and then by size using size exclusion chromatography.
|The protein name||Description|
|Wild-type AXL-Fc||Wild-type AXL extracellular domain, amino acids 19-440, fused with Fc region of mouse IgG2a.|
|AXL S6-1-FC||The merger of AL-Fc, as described above for wild-type AXL-Fc, however, the AXL Ig1 domain contains the following mutations for S6-1: G32S, D87G, V92A, G127R.|
|AXL E59R/T77R-FC||The merger AXL-Fc, as described above for wild-type AXL-Fc, however, the domain of the Axl Ig1 contains E59R and T77R mutations that significantly reduce binding to GAS6.|
To evaluate the ability of AXL-Fc mutants to suppression of metastasis in vivo, we used the same model of peritoneal xenotransplanted human ovarian cancer, as described in example 1. This model repeats peritoneal metastasis of human ovarian cancer, as mice rapidly develop very invasive disease, including ascites and a lot (>100) small metastatic lesions, four weeks after the Declaration of SKOV3ip.1 cells. This model gives a very accurate view of human ovarian cancer, as the majority of patients has a significant metastases at the time of diagnosis. The mice were injected SKOV3ip.1 cells and tumors were allowed to disseminate (metastases) within seven days. On the seventh day, we randomly divided the mice into three study groups and began an introductory treatment or AXL-Fc wild-type S6-1-Fc or E59R/T77R-Fc. Purified proteins were dissolved in phosphate buffer saline solution and injected into mice twice a week in those�Linux three weeks at a dose of 10 mg/kg, a total of six doses. On the twenty-eighth day all mice were scarification, and an autopsy was conducted to assess the overall tumor burden by measuring the amount of visible metastases and the total weight of all lesions. There was a deep difference between the two treatment groups, whose representative image is shown in figure 14. In mice treated with the negative control E59R/T77R-Fc, accounted for an average of 86.3±21,9 peritoneal metastases. For the mice that received wild-type AXL-Fc, this number decreased to 48.1±6,9, and for mice in engineering AXL group, S6-1-Fc, only 8.3±1,6 metastatic lesion was observed on average (figure 15 (top panel)). All visible damage were cut and collectively weighed for each mouse to measure the total mass of metastatic tumors. Treatment group engineering AXL (S6-1-Fc) again showed the most profound response, so in E59R/T77R-Fc, wild-type Fc and S6-1-Fc groups defined tumor mass amounted to 567±92,430±36 and 188±55 mg, respectively, in figure 15 (bottom panel).
Together, these results further verified AXL as a therapeutic target in the treatment of metastasis and demonstrated that the neutralization of the ligand for AXL, GAS6 is an effective antimetastatic treatment strategy. It is important to note that protein comprising a hybrid AXL-Fc, which shows no trace�wivenhoe binding to GAS6 (AXL E59R/T77R-Fc), not preventing metastasis of a tumor; a protein comprising a hybrid AXL-Fc, which binds to GAS6 with moderate affinity (wild-type AXL-FC), shows a slight inhibition of metastasis of the tumor; protein comprising a hybrid AXL-Fc, with a very high affinity to GAS6 (AXL S6-1-Fc), shows significant inhibition of metastasis of the tumor. All together, this indicates that the epitope of the interaction of GAS6 and AXL is crucial in tumor metastasis and is a powerful inhibitor of this epitope on via GAS6 AXL S6-1-Fc protein significantly inhibits metastasis of tumors. Thus, AXL S6-1-Fc protein or a protein that powerfully inhibits GAS6-Axl interaction is a promising therapeutic candidate for metastatic disease. In addition, we also demonstrated that the direct introduction of purified soluble protein AXL is a successful method of treatment by examining this approach clinically.
Methods for example 3
The cell lines. Cells SKOV3ip ovarian.1 were cultured in appropriate medium with 10% fetal calf serum and 1% penicillin and streptomycin at 37°C in 5% CO2the incubator.
AXL-Fc hybridization. Full AXL mutants, amino acids 19-440 directly merged with a region of mouse IgG2a Fc was cloned into an adenoviral Shuttle vector pADD2 under the control of CMV. Temporary DNA transfection of embryonic� cells of human kidney (HEK) 293 was carried out using a set of Freestyle Expression kit from Life Technologies, as described by the manufacturer. Fc-hybrid proteins were purified from culture supernatant after five days using Protein A affinity chromatography and size exclusive chromatography (gel filtration). Purified proteins were placed in phosphate buffer saline without additives or carriers.
SKOV3ip.1 peritoneal xerography. All procedures involving animals and their care were approved by the Committee on animal care and use Stanford University in accordance with institutional and NIH guidelines. Six female Nude mice were injected with 1×106SKOV3ip.1 cells intraperitoneally. Seven days after injection of cells, mice were randomly divided into three groups for treatment: S6-1-Fc, wild-type AXL-Fc or E59R/T77R-Fc. Purified soluble protein AXL-Fc was administered by injection intraperitoneally twice weekly at a dose of 10 mg/kg Dosing was continued for three weeks, after which mice were scarificator. An autopsy was conducted, in which metastatic lesions were counted, and then cut and collectively weighed. Tumor load was determined as the total number of lesions and the total weight of all patients tissue for each mouse.
Statistical analysis: t-student test was used and the error messages are standard oshie�kami of the mean (SEM). Values with p value <0.01 was considered significant.
Although the above invention has been described in more detail with the help of illustrations and examples for clarity of understanding, but it will be clear to any person skilled in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without deviation from the spirit or scope of the attached claims.
1. A soluble variant of the AXL polypeptide, where the polypeptide does not have the transmembrane domain of AXL and contains at least one amino acid modification compared to the sequence of wild-type AXL (SEQ ID NO: 1) where the modification increases the affinity of binding of the polypeptide with AXL specifically retarding growth protein 6 (GAS6), which is at least about 2 times stronger than the affinity of the polypeptide wild-type AXL,
where this modification is in position n, where n is selected from 32, 72, 87, 92, or 127, or a combination thereof, where n+7 corresponds to the numbering of SEQ ID NO: 1;
where a soluble variant of the AXL polypeptide inhibits the interaction between AXL and GAS-6.
2. A soluble variant of the AXL polypeptide according to claim 1, characterized in that AXL variant comprises the amino acid substitution compared to the sequence of wild-type AXL for the following positions: (a) glycine 32; (b) aspartic acid 87; (C) valine 92 and (d) glycine 127, or where specified �Ariant AXL comprises the amino acid substitution compared to the sequence of wild-type AXL for the following positions: (a) glycine 32; (b) alanine 72; (C) aspartic acid 87; (d) valine 92 and (y) glycine 127.
3. A soluble variant of the AXL polypeptide, where the polypeptide does not have a transmembrane domain AXL contains at least one amino acid modification compared to the sequence of wild-type AXL (SEQ ID NO: 1) where the modification increases the affinity of binding of the polypeptide with AXL specifically retarding growth protein 6 (GAS6), which is at least about 2 times stronger than the affinity of the AXL polypeptide of the wild type, characterized in that AXL variant includes a number of amino acid substitutions compared to the sequence of wild-type AXL (SEQ ID NO: 1), selected from the following groups: (1) Gly32Ser, Asp87Gly, Val92Ala and Gly127Arg; (2) Glu26Gly, Val79Met, Val92Ala and Gly127Glu; (3) Asn33Ser, Ser74Asn, Asp87Gly and Val92Ala; (4) Ala72Val, Ile97Arg and His116Arg; (5) Gln78Glu; (6) Ala72Val; (7) Gln86Arg, Ile90Val and Val92Ala; (8) Ala72Val and Val92Asp; (9) Asp65Asn and Asp87Gly; (10) Asp87Gly and Val92Ala; (11) Glu27Lys, His61Tyr, Ala72Val, Asp88Asn, Val92Ala and Thr98Ala; (12) Val92Ala and Gln109Arg; (13) Thr44Ala, Ala72Val, Ile90Val, Thr105Met and Glu129Lys; (14) Val92Gly; (15)Val92Ala, Val112Ala, Phe113Leu and Thr118Ala; (16) Val92Ala, Thr98Pro; (17) Glu27Gly and Asp87Gly; (18) Thr38Ile and Val92Ala; (19) Asp87Gly; (20) Thr23Met and Val92Ala; (21) Ala72Val and Phe113Leu; (22) Gln86Arg and Val92Ala; (23) Ala19Thr, Glu26Gly, Glu27Gly and Val92Ala; (24) Ile90Met and Val92Ala; (25) Gly32Ser and Asp87Gly; (26) Gly32Ser and Val92Ala; (27) Gly32Ser and Glyl27Arg; (28) Asp87Gly and Gly127Arg; (29) Val92Ala and Gly127Arg; (30) Asp87Gly, Val92Ala and Gly127Arg; (31) Gly32Ser, Val92Ala and Gly127Arg; (32) Gly32Ser, Asp87Gly and Gly127Arg; (33) Gly32Ser, Asp87Gly and Val92Ala; (34) Gly32Ser, Ala72Val, Asp87Gly, Val92Ala and Gly127Arg and (35) Ala72Val,
where the specified modificationdate in position n, where n+7 corresponds to the numbering of SEQ ID NO: 1,
where a soluble variant of the AXL polypeptide inhibits the interaction between AXL and GAS6.
4. A hybrid protein containing the Fc domain and a soluble variant of the AXL polypeptide,
where the specified variant AXL has no transmembrane domain of AXL and contains at least one amino acid modification compared to the sequence of wild-type AXL (SEQ ID NO: 1) where the modification increases the affinity of binding of the polypeptide with AXL specifically retarding growth protein 6 (GAS6), which is at least about 2 times stronger than the affinity of the polypeptide wild-type AXL,
where this modification is in position n, where n is selected from 32, 72, 87, 92, 97 or 127, or a combination thereof, where n+7 corresponds to the numbering of SEQ ID NO: 1;
where a soluble variant of the AXL polypeptide inhibits the interaction between AXL and GAS-6.
5. Pharmaceutical composition for inhibiting AXL/GAS6 ways, containing a therapeutically effective amount of one or more variants of soluble AXL polypeptide according to any of claims. 1-3, or a hybrid protein according to claim 4, or its pharmaceutically acceptable salt.
6. Pharmaceutical composition for inhibiting AXL/GAS6 ways, containing a therapeutically effective amount of one or more variants of soluble AXL polypeptide according to any of claims. 1-3 or hybrid belcaro p. 4, and further comprising an effective dose of at least one cytotoxic agent, or pharmaceutically acceptable excipient, or a combination thereof.
7. The use of a soluble version of the AXL polypeptide according to any of claims. 1-3, or a hybrid protein according to claim 4, or a composition according to claim 5 or 6 in the method of treating, reducing, or preventing the metastasis or tumor invasion of the patient related to mammals.
8. The use according to claim 7, characterized in that the tumor selected from tumors, a member of the group consisting of ovarian tumors, breast tumors, lung tumors, liver tumors, tumors of the colon, tumors of the gallbladder, pancreatic tumor, prostate tumor, and glioblastoma.
SUBSTANCE: invention relates to a set of oligonucleotide primers and fluorescently-labelled probe for identification of Burkholderia pseudomallei and differentiation of the actinobacillus mallei by the method of polymerase chain reaction with fluorescence detection.
EFFECT: invention enables in a short time with high sensitivity and specificity to detect the melioidosis agent and differentiate it from the actinobacillus mallei in samples of pure cultures and biological material.
1 dwg, 1 tbl, 3 ex
SUBSTANCE: invention refers to medicine, molecular biology and biotechnology. What is presented is a method for determining the polymorphism of human GP6 gene coding glycoprotein VI by the polymorphous position rs 1613662 based on recording melting curves with fluorescence-labelled allele-specific oligonucleotide tests.
EFFECT: due to the more reliable determination of variable positions and a possibility to detect in one test tube with the use of standard equipment, the method can be effectively used to diagnose the individual's inherited predisposition with the recording the real-time PCR results.
SUBSTANCE: invention relates to field of biotechnology, namely to internalisation of therapeutic molecules into cell, and can be applied in medicine. Obtained is composition for delivering molecules of nucleic acids into cells, containing at least one peptide with at least 92% identity to GAAEAAARVYDLGLRRLRQRRRLRRERVRA (SEQ ID NO: 2); IREIMEKFGKQPVSLPARRLKLRGRKRRQR (SEQ ID NO: 3); or YLKVVRKHHRVIAGQFFGHHHTDSFRMLYD (SEQ ID NO: 4), bound to one or several molecules of nucleic acids.
EFFECT: invention makes it possible to increase efficiency of delivery of molecules of nucleic acids into mammalian cell due to peptide, capable of internalisation into mammalian cell with efficiency, constituting at least 200% of efficiency of internalisation of peptide TAT, which has amino acid sequence GRKKRRQRRRPPQ (SEQ ID NO: 1).
8 cl, 16 dwg, 1 tbl, 8 ex
SUBSTANCE: invention refers to biotechnology, namely to a method for analysing the applicability of RNA extracted from a tissue or a cell (cells) fixed by a fixative for analysing gene expression. The method involves performing electrophoresis with the above RNA. The method implies stating, if the above RNA complies with the following equation: B/A≤1, wherein A represents the mass ratio (%) of RNA falling within the range from 1,000 to 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis, while B represents the mass ratio (%) of RNA falling within the range from more than 4,000 nucleotides to the total mass of RNA that is determined by electrophoresis. If the above RNA extracted from the tissue or cell (cells) complies with the above equation, it is considered to be applicable for analysing gene expression.
EFFECT: presented invention enables the fast and high-effective determination of RNA applicability for analysing gene expression.
7 cl, 3 dwg, 11 tbl, 7 ex
SUBSTANCE: group of inventions relates to cell lysis. A method for selective lysis of animal cells and a device for detecting microorganisms are disclosed. A method for selective lysis of animal cells in a water sample with animal cells, containing or possibly containing microorganisms, includes steps of providing a water sample with animal cells, containing or possibly containing microorganisms, adding to said sample a nonionic detergent and a buffer solution to obtain a solution with pH of about 9.5 or higher, incubating said solution for a period sufficient for lysis of animal cells. The device for detecting microorganisms in the sample consists of a lysis chamber for receiving a water sample with animal cells, a vessel with an alkaline buffer solution with pH 9.5 or higher and a nonionic detergent, or a vessel with an alkaline buffer solution with pH of about 9.5 or higher and a vessel with a nonionic detergent, a filter connected to the lysis chamber for filtering the sample after lysis of the animal cells, an indication chamber for analysing presence of DNA of microorganisms.
EFFECT: present inventions enable to process a sample without considerable dilution thereof and without using enzymatic breakdown of DNA or heat treatment, and also enables to process considerably larger sample volumes and determine lower concentrations of pathogenic microorganisms in a sample.
13 cl, 12 dwg, 8 ex
SUBSTANCE: characterised method comprises carrying out of PCR with use of specific primers to genes vc0497, vc0502 and vc0514 from the island of pandemicity VSP-II. The characterised test system comprises the components for isolation of DNA, the components for carrying out PCR, including, in particular, a primer mix VSPIIreg-F - 5'-TGGAAAGAAGAGCGTTACTGC-3', VSPIIreg-R - 5'-CCCTGTTGATGATGTGATTTG-3' to the gene vc0497, VSPIIpilin-F - 5'-CTGTGATTCGGGCTTTATCGG-3', VSPIIpilin-R - 5'-GCGTAAACTGAGCCAATAAGC-3' to the gene vc0502, VSPIIchem-F - 5'-CTTGATGGAGCGGAGAAAAC-3', VSPIIchem-R - 5'-CGATGAATAGCCTGTTGAAC-3' to the gene vc0514, taken in the ratio 1:1:1:1:1:1, respectively.
EFFECT: inventions enable to differentiate quickly and reliably the toxigenic genetically modified strains to genovariants with low and high epidemic potential.
2 cl, 1 dwg, 2 tbl
SUBSTANCE: method comprises isolation of DNA from lymphocytes in peripheral blood by the method of phenol-chloroform extraction, carrying out of PCR, amplification of 18 parts of gene MYO7A, detection in the denaturing acrylamide gel and sequencing. PCR is carried out using specially selected sequences of oligonucleotides flanking regions of 18 exons of the gene MYO7A with possible content of different mutations.
EFFECT: invention enables to simplify the method and to improve the accuracy of determining mutations of the gene MYO7A, to reduce the time of the study.
3 dwg, 1 ex
SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.
EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.
SUBSTANCE: characterised oligonucleotide primers are complementary to a specific region of the mig-gene of Mycobacterium avium and have the following base composition: 5'-CGT CAA AAG CGA ACT GCA-3' and 5'-TAA TTC GTT GCC CGA CTC-3'. The method of detecting DNA of Mycobacterium avium comprises DNA isolation, DNA amplification using oligonucleotide primers, transfer of amplification product on the gel followed by detection of the analysis results on the transilluminator. In case of positive reaction a fragment is synthesised, corresponding to the size of 157 bps.
EFFECT: inventions can be used in veterinary diagnostic, scientific and practical laboratories for detection of genetic material of Mycobacterium avium in samples.
2 cl, 1 dwg, 4 ex
SUBSTANCE: set is used to recognise mutations of a coding part of NKX2.5, CFC1, GATA4 genes associated with an orphan single-gene pathology underlying familiar congenital heart disease. The mutations are recognised by identifying a nucleotide sequence of the coding part of NKX2.5, CFC1, GATA4 genes. The coding part of NKX2.5, CFC1, GATA4 genes is amplified by means of 15 synthetic base pairs at the same temperature and annealing time; that is followed by sequencing the amplification products by means of one pair of universal primers.
EFFECT: invention enables recognising the mutations of the above genes sensitively and specifically, reducing the amplification reaction time, the number of manipulations, the agent addition time for the sequencing reaction and decreasing a probability of the reaction error.
3 cl, 1 dwg, 4 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to immunology. What is presented is a completely human monoclonal antibody, which binds insulin-like growth factor-II (IGF-II) and has a cross responsiveness to IGF-I, as well as its antigen-binding fragment. There are disclosed a nucleic acid molecule coding an antibody according to the invention, a vector and a host cell for the expression of the antibody according the invention. There are described a pharmaceutical composition, as well as conjugates for treating and diagnosing malignant tumour, using the antibody according to the invention in preparing the therapeutic agent and a method for determining IGF-II and IGF-I levels in a patient's sample.
EFFECT: present invention can find further application in cancer therapy.
16 cl, 27 ex, 18 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to biotechnology and represents anti-nerve growth factor (NGF) antibodies. The present invention also discloses a pharmaceutical composition for relieving pain associated with a disease or a condition, wherein pain progression or persistence is mediated by NGF, containing the above antibodies, as well as a kit for treating a HGF-related disease, such as e.g. osteoarthritis, nucleic acids coding a heavy or light chain of the antibody, an expression vector, a host cell for preparing the above antibodies, a method for expressing the above anti-NGF antibodies, as well as using the above antibodies in managing pain and for preparing a therapeutic agent for managing pain associated with the disease or condition, wherein pain progression or persistence is mediated by NGF.
EFFECT: present invention enables producing the anti-NGF antibodies characterised by high stability in vivo.
16 cl, 7 dwg, 13 tbl, 8 ex
SUBSTANCE: invention refers to biotechnology, more specifically to biospecific antibodies, and can be used in medicine. Constructed is an antibody containing one of the following groups of six hypervariable region (HVR) sequences: (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2, containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NIKDTY; (e) HVR-H2 containing the sequence RIYPTNGYTR; and (f) HVR-H3 containing the sequence WGGDGFYAMD; or (a) HVR-L1 containing the sequence NIAKTISGY; (b) HVR-L2 containing the sequence WGSFLY; (c) HVR-L3 containing the sequence HYSSPP; (d) HVR-H1 containing the sequence NISGTY; (e) HVR-H2 containing the sequence RIYPSEGYTR; and (f) HVR-H3 containing the sequence WVGVGFYAMD. The produced antibody specifically binds human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor (VEGF) The invention also refers to a recovered Fab fragment of the above antibody, a polynucleotide coding it, to an expression vector, a host cell, a method for producing it, as well as to using it for treating HER2-mediated diseases.
EFFECT: present invention enables producing the bispecific high-affinity antibody able to bind VEGF and HER2 simultaneously.
14 cl, 65 dwg, 16 tbl, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology. What is presented is a recovered human antibody or its antigen-binding fragment, which specifically binds to human angiopoietin-2 (hAng-2), but does not substantially binds to hAng-1 characterised by the presence of CDR variable heavy and light chain domains. What is described is a pharmaceutical composition on the basis of a therapeutically effective amount of the antibody. Disclosed are: variants of the recovered antibody or its antigen-binding fragment in producing drug preparation for treating a patient suffering various diseases including a tumour. Described are the versions of the methods of treating various diseases.
EFFECT: using this invention provides the antibody highly specific to human angiopoietin-2 (hAng-2) with an affinity constant of approximately 10-11, which does not substantially bind to hAng-1, that can find application in treating various diseases related to hAng-2 hyperactivity.
19 cl, 35 tbl, 3 dwg, 13 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology.
EFFECT: bispecific anti-human vascular endothelial growth factor VEGF and human angiopoietin-2 ANG-2 antibodies, methods for producing them, pharmaceutical compositions containing the above antibodies, and using them are described.
13 cl, 26 dwg, 15 tbl, 19 ex
SUBSTANCE: present invention refers to biotechnology, more specifically to granulocyte-macrophage colony-stimulating factor (GM-CSF) antagonists, and can be used in medicine. The invention consisting in using the GM-CSF specific antibody in treating or preventing multiple sclerosis in the patients with multiple sclerosis.
EFFECT: invention enables delaying the onset of multiple sclerosis recurrences.
9 cl, 5 dwg, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to immunology and biotechnology. There are presented version recombinant human VEGF antibodies containing variable regions of heavy and light chains containing complementarity-determining regions (CDR) of rabbit antibodies. There are also presented: recovered molecules of nucleic acids coding the above antibodies; an expression vector containing the above molecule of nucleic acid; and a host cell for expression of the antibody according to the invention, containing the above expression vector. What is disclosed is a pharmaceutical composition containing a therapeutically effective amount of the above antibody and a pharmaceutically acceptable carrier.
EFFECT: invention enables extending the range of human VEGF antibodies recovered from a rabbit.
24 cl, 15 dwg, 12 tbl, 7 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biotechnology and immunology. Described are versions of antibodies, binding the GRM molecule, as well as their antigen-binding fragments, amino acid sequences of variable parts of which are presented in the claim materials. Nucleic acid, coding the said antibodies, is presented. Claimed is a method of obtaining the RGM-binding protein, which includes cultivation of a host cell in a culture medium under conditions suitable for obtaining the binding protein, capable of binding with RGM, where the host cell contains an expression vector, containing the separated nucleic acid, coding the said antibody. Described is a pharmaceutical composition for treating a disease, in which the SGM A activity produces a negative impact, which contains a therapeutically efficient quantity of the said antibody and a pharmaceutically acceptable carrier. Claimed is an application of the said antibody for obtaining a medication, used for a) reduction of hRGM A binding with a patient's Neogenin receptor; or b) for reduction of hRGM A binding with BMP-2 and BMP-4 in the patient.
EFFECT: invention makes it possible to obtain antibodies against GRM, which are used for treating diseases, associated with excessive interaction of RGM with the Neogenin receptor, BMP-2 and BMP-4.
13 cl, 16 dwg, 10 tbl, 11 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and immunology. What is presented is an antibody representing a neutralising VEGFR-2/KDR antibody with its hypervariable regions being identical to the hypervariable regions of TTAC 0001 of VEGFR-2/KDR antibody fused with a binding domain of angiopoietin 2 which is Tie-2 ligand for treating cancer by angiogenesis inhibition. A DNA coding the above antibody, an expression vector containing the above DNA, and a CHO host cell transformed by the above vector for preparing the antibody are also described. What is also presented is a method for preparing the antibody involving: host cell incubation, and the antibody recovery from a culture fluid of CHO cell. What is described is a pharmaceutical composition for treating an angiogenesis-related disease, containing an effective amount of the above antibody and at least one pharmaceutically acceptable carrier.
EFFECT: invention enables preparing the VEGFR-2/KDR antibody fused with the binding domain of angiopoietin 2 which may be used for effective treatment of a disease related to excessive angiogenesis.
13 cl, 10 dwg, 8 ex
SUBSTANCE: invention relates to the field of immunology, namely to enzyme-immunoassay, in particular to a method of detecting forms of vascular endothelial growth factor (VEGF) with a size more than 110 amino acids in a biological sample. The method includes the following stages: contact and incubation of the biological sample with an uptake reagent, immobilised on a solid substrate, where the uptake reagent contains a monoclonal antibody, which recognises and specifically binds with residues, in quantity more than 110, from human VEGF; separation of the biological sample from the immobilised uptake reagents; contact of the immobilised molecular complex of the reagent of the uptake-target with detected antibody, which binds with VEGF domains, responsible for binding with KDR and/or FLT1 receptor, or which binds with an epitope in VEGF1-110; measurement of the level of VEGF110+, bound with reagents of the uptake, with application of means of detection for the detected antibody. Set of immune assay reagents for detection of VEGF110+ forms in the biological sample. An antibody 5C3, obtained from hybridoma 5C3.1.1 with a depositary number PTA-7737, with the said antibody 5C3 binding VEGF110+ forms, including VEGF121+. Hybridoma 5C3.1.1, deposited in ATCC with the depositary number PTA-7737, to obtain the monoclonal antibody 5C3.
EFFECT: application of the claimed invention makes it possible to increase accuracy of detecting VEGF isoforms, which must not include isoform VEGF110 and must obligatory include isoform VEGF121.
25 cl, 3 dwg, 2 tbl, 1 ex
SUBSTANCE: extracted polypeptide represents a fragment of extracted peptide T101 and consists of amino-acid sequence WWTFFLPSTLWERK, in which at least one amino acid is replaced by the corresponding D-amino acid. The proposed invention can be used in medicine for treatment of autoimmune and/or inflammatory diseases.
EFFECT: invention allows obtaining a polypeptide capable of inducing proliferation of lymphocytes of peripheral blood of a human being, inhibiting growth of a tumour and modulating an immune system.
4 cl, 15 dwg, 1 tbl, 11 ex