Inhibition of axl/gas6 signaling in the treatment of liver fibrosis

Abstract

Compositions and methods are provided for alleviating endometriosis, kidney disease, inflammatory disease and/or transplant rejection in a mammal by administering a therapeutic dose of a pharmaceutical composition that inhibits AXL, MER or Tyro3 protein activity, for example by competitive or non-competitive inhibition of the binding interaction between AXL, MER or Tyro3 and its ligand GAS6.

What is claimed is: 
     
         1 . A method of treating, reducing, or preventing endometriosis, kidney disease, inflammatory disease and/or transplant rejection in a mammalian patient, the method comprising:
 administering one or more inhibitor agents selected from the group consisting of (a) an inhibitor of AXL, MER and/or Tyro3 activity (b) an inhibitor of GAS6 activity; and (c) an inhibitor of AXL, MER or Tyro3-GAS6 interaction.   
     
     
         2 . A method of determining the susceptibility of a subject to endometriosis, kidney disease, inflammatory disease and/or transplant rejection, said method comprising:
 detecting the level of AXL, MER or Tyro3 activity in a biological sample from a subject; and   comparing the level of the AXL, MER or Tyro3 activity in the biological sample to a predetermined level,   wherein an increase over the predetermined level is indicative of a predisposition of the subject to develop endometriosis, kidney disease, inflammatory disease and/or transplant rejection (GVHD).   
     
     
         3 . A method of determining the susceptibility of a subject to endometriosis, kidney disease, inflammatory disease and/or transplant rejection, said method comprising:
 detecting the level of GAS6 activity in a biological sample from a subject; and   comparing the level of the GAS6 activity in the biological sample to a predetermined level,   wherein an increase over the predetermined level is indicative of a predisposition of the subject to develop endometriosis, kidney disease, inflammatory disease and/or transplant rejection (GVHD).   
     
     
         4 . A method of determining treatment efficacy of administering an inhibitor agent comprising detecting the level of AXL, MER, Tyro3 and/or GAS6 activity in a biological sample from a subject treated with said inhibitor agent, wherein a decrease in AXL, MER, Tyro3 and/or GAS6 activity is indicative of the efficacy of treatment with the inhibitor agent. 
     
     
         5 . A method for determining the treatment regimen of an inhibitor agent comprising detecting the level of AXL, MER, Tyro3 and/or GAS6 activity in a biological sample from a subject treated with said inhibitor agent, and determining a treatment regimen of the inhibitor agent based on an increase or decrease in the level of AXL, MER, Tyro3 and/or GAS6 activity. 
     
     
         6 . The method of any of the preceding claims wherein the AXL, MER or Tyro3 activity level is measured by the level of AXL, MER or Tyro3 mRNA expression or the level of AXL, MER or Tyro3 protein expression. 
     
     
         7 . The method of any of the preceding claims wherein the GAS6 activity level is measured by the level of GAS6 mRNA expression or the level of GAS6 protein expression. 
     
     
         8 . The method of any of the preceding claims wherein the kidney disease is diabetic nephropathy or progressive glomerulonephritis. 
     
     
         9 . The method of any of the preceding claims wherein the inflammatory disease is liver inflammation, steatohepatitis or liver fibrosis. 
     
     
         10 . The method of any of the preceding claims wherein the biological sample is selected form the group consisting of a tissue sample, a blood sample, a serum sample, a cerebrospinal fluid (CSF) sample, an ascite fluid sample, and a cell culture sample. 
     
     
         11 . The method of any of the preceding claims wherein the inhibitor is a polypeptide, a polynucleotide, a small molecule, an antibody, an antibody fragment or antibody drug-conjugate. 
     
     
         12 . The method of any of the preceding claims wherein the inhibitor agent is capable of binding to GAS6 with increased affinity compared to wild-type AXL, MER or Tyro3. 
     
     
         13 . The method of any of the preceding claims wherein the inhibitor agent is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said polypeptide lacks the AXL, MER or Tyro3 transmembrane domain and comprises at least one amino acid modification relative to the wild-type AXL, MER or Tyro3 sequence, and wherein said change increases the affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6. 
     
     
         14 . The method of any of the preceding claims wherein the inhibitor agent binds to two or more epitopes on a single GAS6 
     
     
         15 . The method of any of the preceding claims wherein the inhibitor agent is capable of binding to the major and minor AXL, MER or Tyro3 binding sites on a single GAS6. 
     
     
         16 . The method of any of the preceding claims wherein the inhibitor agent is capable of binding the major AXL, MER or Tyro3 binding site of GAS6 and one or more additional GAS6 epitopes on a single GAS6. 
     
     
         17 . The method of any of the preceding claims wherein the inhibitor agent is capable of binding to the minor AXL, MER or Tyro3 binding site on GAS6 and one or more additional epitopes on a single GAS6. 
     
     
         18 . The method of any of the preceding claims wherein the inhibitor agent is capable of binding two or more epitopes on a single GAS6. 
     
     
         19 . The method of any of the preceding claims wherein the inhibitor agent is capable of antagonizing the major and/or minor GAS6/receptor binding interaction, and wherein the receptor is selected from AXL, MER and Tyro3. 
     
     
         20 . The method of any of the preceding claims wherein the inhibitor agent of any of the preceding claims wherein the inhibitor agent is capable of antagonizing the major GAS6/receptor binding interaction, and wherein the receptor is selected from AXL, MER and Tyro3. 
     
     
         21 . The method of any of the preceding claims wherein the inhibitor agent is capable of antagonizing the minor GAS6/receptor binding interaction, and wherein the receptor is selected from AXL, MER and Tyro3. 
     
     
         22 . The method of any of the preceding claims wherein the inhibitor agent is a polypeptide, a polypeptide-carrier fusion, a polypeptide-Fc fusion, polypeptide-conjugate, a polypeptide-drug conjugate, an antibody, a bispecific antibody, an antibody drug conjugate, an antibody fragment, an antibody-related structure, or a combination thereof. 
     
     
         23 . The method of any of the preceding claims wherein the inhibitor agent is a natural or synthetic polypeptide. 
     
     
         24 . The method of any of the preceding claims wherein the inhibitor agent is a non-antibody polypeptide. 
     
     
         25 . The method of any of the preceding claims wherein the inhibitor agent is a darpin, an avimer, an adnectin, an anticalin, an affibody, a maxibody or a combination thereof. 
     
     
         26 . The method of any of the preceding claims wherein the inhibitor agent is a polypeptide-conjugate or an antibody-conjugate. 
     
     
         27 . The method of any of the preceding claims wherein the inhibitor agent comprises a polypeptide-polymer conjugate, and wherein the polymer is a PEG, a PEG-containing polymer, a degradable polymer, a biocompatible polymer or a hydrogel. 
     
     
         28 . The method of any of the preceding claims wherein the inhibitor agent is a polypeptide, wherein the polypeptide comprises a soluble AXL variant polypeptide wherein the AXL polypeptide lacks the AXL transmembrane domain and has at least one mutation relative to wild-type that increases affinity of the AXL polypeptide binding to GAS6 compared to wild-type AXL. 
     
     
         29 . The method of any of the preceding claims wherein the inhibitor agent is a polypeptide, wherein the polypeptide comprises a soluble MER variant polypeptide wherein said MER polypeptide lacks the MER transmembrane domain and has at least one mutation relative to wild-type that increases affinity of the MER polypeptide binding to GAS6 compared to wild-type MER. 
     
     
         30 . The method of any of the preceding claims wherein the inhibitor agent is a polypeptide, wherein said polypeptide comprises a soluble Tyro3 variant polypeptide wherein said Tyro3 polypeptide lacks the Tyro3 transmembrane domain and has at least one mutation relative to wild-type that increases affinity of the Tyro3 polypeptide binding to GAS6 compared to wild-type Tyro3. 
     
     
         31 . The method of any of the preceding claims wherein the AXL, MER or Tyro3 variant polypeptide lacks a functional fibronectin (FN) domain and/or wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         32 . The method of any of the preceding claims wherein the AXL, MER or Tyro3 variant polypeptide lacks the transmembrane domain, has more than one Ig1 domain and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         33 . The method of any of the preceding claims wherein the polypeptide has two Ig1 domains. 
     
     
         34 . The method of any of the preceding claims wherein the polypeptide has three Ig1 domains. 
     
     
         35 . The method of any of the preceding claims wherein the soluble AXL, MER or Tyro3 variant polypeptide lacks the transmembrane domain, has more than one Ig2 domain and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         36 . The method of any of the preceding claims wherein the polypeptide has two Ig2 domains. 
     
     
         37 . The method of any of the preceding claims wherein the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER or Tyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, has more than one Ig1 domain, more than one Ig2 domain, and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         38 . The method of any of the preceding claims wherein the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER or Tyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks a functional fibronectin (FN) domain, has more than one Ig1 domain, more than one Ig2 domain, and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         39 . The method of any of the preceding claims wherein the soluble AXL, MER or Tyro3 variant polypeptide has two Ig1 domains and two Ig2 domains. 
     
     
         40 . The method of any of the preceding claims wherein the immunoglobulin domains are connected directly. 
     
     
         41 . The method of any of the preceding claims wherein the immunoglobulin domains are connected indirectly. 
     
     
         42 . The method of any of the preceding claims wherein the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, is capable of binding both the major and minor binding site of a single GAS6 and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6. 
     
     
         43 . The method of any of the preceding claims wherein the polypeptide has one Ig1 domain and lacks a functional Ig2 domain. 
     
     
         44 . The method of any of the preceding claims wherein the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER or Tyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, has one Ig1 domain, lacks a functional Ig2 domain and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         45 . The method of any of the preceding claims wherein the polypeptide is a soluble AXL, MER or Tyro3 variant polypeptide, wherein said soluble AXL, MER or Tyro3 variant polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, lacks a functional fibronectin (FN) domain, has one Ig1 domain, lacks a functional Ig2 domain and wherein said AXL, MER or Tyro3 variant polypeptide exhibits increased affinity of the AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to wild-type AXL, MER or Tyro3. 
     
     
         46 . The method of any of the preceding claims wherein the AXL, MER or Tyro3 variant polypeptide is a fusion protein comprising an Fc domain. 
     
     
         47 . The method of any of the preceding claims wherein the variant polypeptide lacks the AXL, MER or Tyro3 intracellular domain. 
     
     
         48 . The method of any of the preceding claims wherein the soluble AXL, MER or Tyro3 variant polypeptide further lacks a functional fibronectin (FN) domain and wherein said variant polypeptide exhibits increased affinity of the polypeptide binding to GAS6. 
     
     
         49 . The method of any of the preceding claims wherein the soluble AXL, MER or Tyro3 variant polypeptide comprises at least one amino acid modification relative to the wild-type AXL, MER or Tyro3 sequence. 
     
     
         50 . The method of any of the preceding claims wherein the soluble AXL variant polypeptide comprises at least one amino acid modification within a region selected from the group consisting of 1) between 15-50, 2) between 60-120, and 3) between 125-135 of the wild-type AXL sequence (SEQ ID NO:1). 
     
     
         51 . The method of any of the preceding claims wherein the soluble AXL variant polypeptide comprises at least one amino acid modification at 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, or 127 of the wild-type AXL sequence (SEQ ID NO: 1) or a combination thereof. 
     
     
         52 . T The method of any of the preceding claims wherein the soluble AXL variant polypeptide comprises at least one amino acid modification selected from the group consisting of 1) A19T, 2) T23M, 3) E26G, 4) E27G or E27K 5) G32S, 6) N33S, 7) T381, 8) T44A, 9) H61Y, 10) D65N, 11) A72V, 12) S74N, 13) Q78E, 14) V79M, 15) Q86R, 16) D87G, 17) D88N, 18) 190M or 190V, 19) V92A, V92G or V92D, 20) 197R, 21) T98A or T98P, 22) T105M, 23) Q109R, 24) V112A, 25) F113L, 26) H116R, 27) T118A, 28) G127R or G127E, and 29) G129E and a combination thereof. 
     
     
         53 . The method of any of the preceding claims wherein the AXL variant polypeptide comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) valine 92; and (d) glycine 127. 
     
     
         54 . The method of any of the preceding claims wherein the AXL variant polypeptide comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) aspartic acid 87 and (b) valine 92. 
     
     
         55 . The method of any of the preceding claims wherein the AXL variant polypeptide comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) valine 92; (d) glycine 127 and (e) alanine 72. 
     
     
         56 . The method of any of the preceding claims wherein the AXL variant polypeptide comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following position: alanine 72. 
     
     
         57 . The method of any of the preceding claims wherein in the AXL variant polypeptide glycine 32 residue is replaced with a serine residue, aspartic acid 87 residue is replaced with a glycine residue, valine 92 residue is replaced with an alanine residue, or glycine 127 residue is replaced with an arginine residue or a combination thereof. 
     
     
         58 . The method of any of the preceding claims wherein in the AXL variant polypeptide aspartic acid 87 residue is replaced with a glycine residue or valine 92 residue is replaced with an alanine residue or a combination thereof. 
     
     
         59 . The method of any of the preceding claims wherein in the AXL variant polypeptide alanine 72 residue is replaced with a valine residue. 
     
     
         60 . The method of any of the preceding claims wherein in the AXL variant polypeptide glycine 32 residue is replaced with a serine residue, aspartic acid 87 residue is replaced with a glycine residue, valine 92 residue is replaced with an alanine residue, glycine 127 residue is replaced with an arginine residue or an alanine 72 residue is replaced with a valine residue or a combination thereof. 
     
     
         61 . The method of any of the preceding claims wherein said AXL variant comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions:
 (a) glutamic acid 26; (b) valine 79; (c) valine 92; and (d) glycine 127.   
     
     
         62 . The method of any of the preceding claims wherein in the AXL variant polypeptide glutamic acid 26 residue is replaced with a glycine residue, valine 79 residue is replaced with a methionine residue, valine 92 residue is replaced with an alanine residue, or glycine 127 residue is replaced with an arginine residue or a combination thereof. 
     
     
         63 . The method of any of the preceding claims, wherein the AXL variant polypeptide comprises at least an amino acid region selected from the group consisting of amino acid region 19-437, 130-437, 19-132, 21-121, 26-132, 26-121 and 1-437 of the wild-type AXL polypeptide (SEQ ID NO: 1), and wherein one or more amino acid modifications occur in said amino acid region. 
     
     
         64 . The method of any of the preceding claims, wherein the AXL variant polypeptide comprises amino acid changes relative to the wild-type AXL sequence (SEQ ID NO: 1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and valine 92. 
     
     
         65 . The method of any of the preceding claims wherein in the AXL variant polypeptide glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, and valine 92 is replaced with an alanine residue, or a combination thereof. 
     
     
         66 . The method of any of the preceding claims wherein in the soluble AXL polypeptide is a fusion protein comprising an Fc domain and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92. 
     
     
         67 . The method of any of the preceding claims wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, and valine 92 is replaced with an alanine residue, or a combination thereof. 
     
     
         68 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127. 
     
     
         69 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, valine 92 is replaced with an alanine residue, and glycine 127 is replaced with an arginine residue or a combination thereof. 
     
     
         70 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain, lacks a functional FN domain, and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; and (d) valine 92. 
     
     
         71 . The method of any of the preceding claims, wherein said soluble AXL variant is a fusion protein comprising an Fc domain, lacks a functional FN domain, and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, and valine 92 is replaced with an alanine residue, or a combination thereof. 
     
     
         72 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain, lacks a functional FN domain, and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127. 
     
     
         73 . The method of any of the preceding claims, wherein said soluble AXL variant is a fusion protein comprising an Fc domain, lacks a functional FN domain, and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, valine 92 is replaced with an alanine residue, and glycine 127 is replaced with an arginine residue or a combination thereof. 
     
     
         74 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain, lacks a functional FN domain, lacks an Ig2 domain, and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72 and (d) valine 92. 
     
     
         75 . The method of any of the preceding claims, wherein said soluble AXL variant is a fusion protein comprising an Fc domain, lacks a functional FN domain, lacks an Ig2 domain and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, and valine 92 is replaced with an alanine residue or a combination thereof. 
     
     
         76 . The method of any of the preceding claims, wherein the soluble AXL polypeptide is a fusion protein comprising an Fc domain, lacks a functional FN domain, lacks an Ig2 domain, and wherein said AXL variant comprises amino acid changes relative to wild-type AXL sequence (SEQ ID NO:1) at the following positions: (a) glycine 32; (b) aspartic acid 87; (c) alanine 72; (d) valine 92; and (e) glycine 127. 
     
     
         77 . The method of any of the preceding claims, wherein said soluble AXL variant is a fusion protein comprising an Fc domain, lacks a functional FN domain, lacks an Ig2 domain and wherein glycine 32 is replaced with a serine residue, aspartic acid 87 is replaced with a glycine residue, alanine 72 is replaced with a valine residue, valine 92 is replaced with an alanine residue, and glycine 127 is replaced with an arginine residue or a combination thereof. 
     
     
         78 . The method of any of the preceding claims, wherein said soluble AXL variant polypeptide has an affinity of at least about 1×10 −8  M,1×10 −9  M, 1×10 −10  M, 1×10 −11  M or 1×10 −12  M for GAS6. 
     
     
         79 . The method of any of the preceding claims, wherein said soluble AXL variant polypeptide exhibits an affinity to GAS6 that is at least about 5-fold stronger, at least about 10-fold stronger or at least about 20-fold stronger than the affinity of the wild-type AXL polypeptide. 
     
     
         80 . The method of any of the preceding claims wherein the soluble AXL, MER or Tyro3 variant polypeptide further comprises a linker. 
     
     
         81 . The method of any of the preceding claims, wherein said linker comprises one or more (GLY) 4 SER units. 
     
     
         82 . The method of the any of the preceding claims, wherein said linker comprises 1, 2, 3 or 5 (GLY) 4 SER units. 
     
     
         83 . The method of any of the preceding claims, wherein said soluble AXL variant polypeptide inhibits binding between wild-type AXL, MER and/or Tyro3 polypeptide and a GAS6 protein in vivo or in vitro. 
     
     
         84 . The method of any of the preceding claims, wherein said soluble AXL variant polypeptide is a fusion polypeptide comprising an Fc domain.