U.S. patent application number 17/465203 was filed with the patent office on 2022-07-14 for modified axl peptides and their use in inhibition of axl signaling in anti-metastatic therapy.
The applicant listed for this patent is Aravive Biologics, Inc., The Board of Trustees of the Leland Stanford Junior University. Invention is credited to Jennifer R. Cochran, Katherine Cynthia Fuh, Amato J. Giaccia, Susan Hershenson, Douglas Jones, Mihalis Kariolis, Yu Miao, Erinn Bruno Rankin.
Application Number | 20220220458 17/465203 |
Document ID | / |
Family ID | 1000006229042 |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220220458 |
Kind Code |
A1 |
Giaccia; Amato J. ; et
al. |
July 14, 2022 |
Modified AXL Peptides and Their Use in Inhibition of AXL Signaling
in Anti-Metastatic Therapy
Abstract
Compositions and methods are provided for alleviating cancer in
a mammal by administering a therapeutic dose of a pharmaceutical
composition that inhibits activity of 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.
Inventors: |
Giaccia; Amato J.;
(Stanford, CA) ; Rankin; Erinn Bruno; (Redwood
City, CA) ; Cochran; Jennifer R.; (Stanford, CA)
; Jones; Douglas; (Newton, MA) ; Kariolis;
Mihalis; (San Mateo, CA) ; Fuh; Katherine
Cynthia; (St. Louis, MO) ; Miao; Yu;
(Sunnyvale, CA) ; Hershenson; Susan; (Palo Alto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the Leland Stanford Junior University
Aravive Biologics, Inc. |
Stanford
Houston |
CA
TX |
US
US |
|
|
Family ID: |
1000006229042 |
Appl. No.: |
17/465203 |
Filed: |
September 2, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15783850 |
Oct 13, 2017 |
11136563 |
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17465203 |
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14650854 |
Jun 9, 2015 |
9822347 |
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PCT/US2013/074786 |
Dec 12, 2013 |
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15783850 |
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61737276 |
Dec 14, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/32 20130101;
A61K 45/06 20130101; C07K 16/46 20130101; C12Y 207/10001 20130101;
C07K 2319/30 20130101; C12N 9/12 20130101 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C07K 16/46 20060101 C07K016/46; A61K 45/06 20060101
A61K045/06 |
Claims
1-80. (canceled)
81. An inhibitor agent selected from the group consisting of (a) an
inhibitor of AXL, MER or Tyro3-GAS6 interaction, (b) an inhibitor
of AXL, MER and/or Tyro3 activity, and (c) an inhibitor of GAS6
activity, and wherein the inhibitor agent binds to GAS6 with
increased affinity compared to wild-type AXL, MER or Tyro3.
82. The inhibitor agent of claim 81 wherein the inhibitor agent
binds to two or more epitopes on a single GAS6, optionally where at
least one of the epitopes is the major or minor AXL, MER or Tyro3
binding site of GAS6.
83. The inhibitor agent of claim 82, wherein the inhibitor
comprises a soluble AXL variant polypeptide, wherein said soluble
AXL variant polypeptide: lacks the AXL transmembrane domain; lacks
both AXL fibronectin (FN) domains; has a set of amino acid
substitutions relative to SEQ ID NO: 1, selected from the group
consisting of: 1) Ala72Val; 2) Asp87Gly; 3) Val92Ala, Val92Gly or
Val92Asp; 4) Ala19Thr; 5) Thr23Met; 6) Glu26Gly; 7) Glu27Gly or
Glu27Lys; 8) Gly32Ser; 9) Asn33Ser; 10) Thr38lle; 11) Thr44Ala; 12)
His61Tyr; 13) Asp65Asn; 14) Ser74Asn; 15) Gln78Glu; 16) Val79Met;
17) Gln86Arg; 18) Asp88Asn; 19) lle90Met or lle90Val; 20) lle97Arg;
21) Thr98Ala or Thr98Pro; 22) Thr105Met; 23) Gln109Arg; 24)
Val112Ala; 25) Phe113Leu; 26) His116Arg; 27) Thr118Ala; 28)
Gly127Arg or Gly127Glu; 29) Glu129Lys; 30) Glu26Gly, Val79Met;
Val92Ala and Gly127Glu; 31) Asp87Gly, Val92Ala and Gly127Arg; 32)
Gly32Ser, Val92Ala and Gly127Arg; 33) Gly32Ser, Asp87Gly and
Gly127Arg; 34) Gly32Ser, Asp87Gly, and Val92Ala; and 35) Asp87Gly
and Val92Ala; and wherein the inhibitor further comprises an Fc
domain linked to the soluble AXL variant polypeptide by a linker
comprising from 1 to 5 (GLY)4SER (SEQID NO: 10) units.
84. A pharmaceutical formulation comprising an inhibitor of claim
81 and a pharmaceutically acceptable excipient.
85. A method of reducing growth or metastasis of a tumor that
expresses GAS6, the method comprising administering to a patient
with a tumor that expresses GAS6 an effective dose of the
pharmaceutical formulation of claim 84.
Description
CROSS REFERENCE
[0001] This application claims benefit and is a Continuation of
application Ser. No. 15/783,850, filed Oct. 13, 2017, which is a
Continuation of application Ser. No. 14/650,854 filed Jun. 9, 2015,
now U.S. Pat. No. 9,822,347,issue Nov. 21, 2017, which is a 371
application and claims the benefit of PCT Application No.
PCT/US2013/074786, filed Dec. 12, 2013, which claims benefit of
U.S. Provisional Patent Application No. 61/737,276, filed Dec. 14,
2012, which applications are incorporated herein by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to tumor invasion and
metastasis, e.g., treatments or diagnoses of tumor invasion or
metastasis via pathways related to AXL, MER and Tyro3 and/or
GAS6.
BACKGROUND OF THE INVENTION
[0003] Invasion and metastasis are the most insidious and
life-threatening aspects of cancer. While tumors with minimal or no
invasion may be successfully removed, once the neoplasm becomes
invasive, it can disseminate via the lymphatics and/or vascular
channels to multiple sites, and complete removal becomes very
difficult. Invasion and metastases kill hosts through two
processes: local invasion and distant organ colonization and
injury. Local invasion can compromise the function of involved
tissues by local compression, local destruction, or prevention of
normal organ function. The most significant turning point in
cancer, however, is the establishment of distant metastasis. The
patient can no longer be cured by local therapy alone at this
point.
[0004] The process of metastasis is a cascade of linked sequential
steps involving multiple host-tumor interactions. This complex
process requires the cells to enter into the vascular or lymphatic
circulation, arrest at a distant vascular or lymphatic bed,
actively extravasate into the organ interstitium and parenchyma,
and proliferate as a secondary colony. Metastatic potential is
influenced by the local microenvironment, angiogenesis,
stroma-tumor interactions, elaboration of cytokines by the local
tissue, and by the molecular phenotype of the tumor and host
cells.
[0005] Local microinvasion can occur early, even though distant
dissemination may not be evident or may not yet have begun. Tumor
cells penetrate the epithelial basement membrane and enter the
underlying interstitial stroma during the transition from in situ
to invasive carcinoma. Once the tumor cells invade the underlying
stroma, they gain access to the lymphatics and blood vessels for
distant dissemination while releasing matrix fragments and growth
factors. General and widespread changes occur in the organization,
distribution, and quantity of the epithelial basement membrane
during the transition from benign to invasive carcinoma.
[0006] Therapeutic efforts in cancer prevention and treatment are
being focused at the level of signaling pathways or selective
modulatory proteins. Protein kinase activities, calcium
homeostasis, and oncoprotein activation are driving signals and
therefore may be key regulatory sites for therapeutic intervention.
Kinases in signaling pathways regulating invasion and angiogenesis
may be important regulators of metastasis. One of the largest
classes of biochemical molecular targets is the family of receptor
tyrosine kinases (RTKs). The most common receptor tyrosine kinase
molecular targets to date are the EGF and vascular endothelial
growth factor (VEGF) receptors. Newer kinase molecular targets
include the type III RTK family of c-kit, and abl. Inhibitors of
these molecules have been administered in combination with classic
chemotherapeutics.
[0007] Metastases ultimately are responsible for much of the
suffering and mortality from cancer. A need exists to identify and
target molecular and functional markers that identify metastatic
cancer cells and to generate reagents for their specific
inhibition.
[0008] Publications in this field include, inter alia, Li et al.
Oncogene. (2009) 28(39):3442-55; United States Patent Application,
20050186571 by Ullrich et al.; United States Patent Application
20080293733 by Bearss et al.; Sun et al. Oncology. 2004;
66(6):450-7; Gustafsson et al. Clin Cancer Res. (2009)
15(14):4742-9; Wimmel et al. Eur J Cancer. 2001 37(17):2264-74;
Koorstra et al. Cancer Biol Ther. 2009 8(7):618-26; Tai et al.
Oncogene. (2008) 27(29):4044-55
[0009] The receptor tyrosine kinase AXL (also known as Ufo and
Tyro7) belongs to a family of tyrosine receptors that includes
Tyro3 (Sky) and Mer (Tyro12). A common ligand for AXL family is
GAS6 (Growth arrest-specific protein 6). Human AXL is a 2,682-bp
open reading frame capable of directing the synthesis of an
894-amino acid polypeptide. Two variant mRNAs have been
characterized, transcript variant 1 may be accessed at Genbank,
NM_021913.3 and transcript variant 2 may be accessed at
NM_001699.4. The polypeptide sequence of the native protein is
provided as SEQ ID NO:1, and specific reference may be made to the
sequence with respect to amino acid modifications. Important
cellular functions of GAS6/AXL include cell adhesion, migration,
phagocytosis, and inhibition of apoptosis. GAS6 and AXL family
receptors are highly regulated in a tissue and disease specific
manner.
[0010] AXL, MER and Tyro3 are each characterized by a unique
molecular structure, in that the intracellular region has the
typical structure of a receptor tyrosine kinase and the
extracellular domain contains fibronectin III and Ig motifs similar
to cadherin-type adhesion molecules. During development, AXL, MER
and Tyro3 are expressed in various organs, including the brain,
suggesting that this RTK is involved in mesenchymal and neural
development. In the adult, AXL, MER and Tyro3 expression is low but
returns to high expression levels in a variety of tumors. GAS6 is,
so far, the single, activating ligand for AXL, MER and Tyro3.
[0011] Receptor tyrosine kinases (RTK) are generally activated by
ligands that promote receptor dimerization and, in turn,
autophosphorylation of tyrosine residues within the cytosolic
domain. Binding of signaling proteins to these phosphorylated
tyrosine residues then leads to downstream signaling. AXL, MER and
Tyro3 family of RTKs are unique in that they are activated by GAS6,
members of the vitamin K-dependent protein family that resembles
blood coagulation factors rather than typical growth factors.
SUMMARY OF THE INVENTION
[0012] The present invention is based in part on the discovery that
AXL, MER and Tyro3 and/or GAS6 related pathways are related to
tumor invasion and/or metastasis. Accordingly, the present
invention provides compositions and methods useful for treating
tumor invasion and/or metastasis, e.g., via inhibition of AXL, MER
and/or Tyro3 and/or GAS6 related pathways. In addition, the present
invention provides reagents and methods useful for determining the
susceptibility or likelihood of a tumor to become invasive and/or
metastatic, e.g., via detecting the level of activity of AXL, MER,
Tyro3 and/or GAS6.
[0013] In some embodiments, the agent useful for treating tumor
invasion and/or metastasis, e.g., via inhibition of AXL, MER and
Tyro3 and/or GAS6 related pathways is an inhibitor agent. In some
embodiments, the inhibitor agent is selected from the group
consisting of (a) an inhibitor of AXL, MER and/or Tyro3 activity,
(b) an inhibitor of GAS6 activity and (c) and inhibitor of AXL, MER
and/or Tyro3-GAS6 interaction, wherein the inhibitor agent is
capable of binding to GAS6 with increased affinity compared to
wild-type AXL, MER or Tyro3.
[0014] In some embodiments, the inhibitor agent binds to two or
more epitopes on a single GAS6.
[0015] In some embodiments, at least one of the epitopes is the
major or minor AXL, MER or Tyro3 binding site on GAS6.
[0016] In some embodiments, the inhibitor agent is capable of
binding to the major and minor AXL, MER or Tyro3 binding sites on a
single GAS6.
[0017] In some embodiments, the inhibitor agent is capable of
binding to the major AXL, MER or Tyro3 binding site of GAS6 and one
or more additional GAS6 epitopes on a single GAS6.
[0018] In some embodiments, 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.
[0019] In some embodiments, the inhibitor agent is capable of
binding two or more epitopes on a single GAS6.
[0020] In some embodiments, the inhibitor agent is capable of
antagonizing the major and/or minor GAS6/receptor binding
interaction, where the receptor is selected from AXL, MER and
Tyro3.
[0021] In some embodiments, the inhibitor agent is capable of
antagonizing the major GAS6/receptor binding interaction, where the
receptor is selected from AXL, MER and Tyro3.
[0022] In some embodiments, the inhibitor agent is capable of
antagonizing the minor GAS6/receptor binding interaction, where the
receptor is selected from AXL, MER and Tyro3.
[0023] In some embodiments, the inhibitor agent is a polypeptide, a
polypeptide-carrier fusion, a polypeptide-Fc fusion, a
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.
[0024] In some embodiments, the inhibitor agent is a natural or
synthetic polypeptide.
[0025] In some embodiments, the inhibitor agent is a non-antibody
polypeptide.
[0026] In some embodiments, the inhibitor agent of the present
invention can include, for example but is not limited to a darpin,
an avimer, an adnectin, an anticalin, an affibody, a maxibody, or
other protein structural scaffold, or a combination thereof.
[0027] In some embodiments, the inhibitor agent is a
polypeptide-conjugate or antibody-conjugate.
[0028] In some embodiments, the inhibitor agent is a
polypeptide-polymer conjugate, where the polymer is selected from
PEG, PEG-containing polymers, degradable polymers, biocompatible
polymers, hydrogels, and other polymer structures or a combination
thereof.
[0029] In some embodiments, the inhibitor agent is a polypeptide,
wherein said polypeptide comprises a soluble AXL variant
polypeptide wherein said AXL variant 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.
[0030] In some embodiments, the inhibitor agent is a polypeptide,
wherein said polypeptide comprises a soluble MER variant
polypeptide wherein said MER variant 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.
[0031] In some embodiments, the inhibitor agent is a polypeptide,
wherein said polypeptide comprises a soluble Tyro3 variant
polypeptide wherein said Tyro3 variant 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.
[0032] In some embodiments, the inhibitor is an AXL, MER or Tyro3
variant polypeptide that inhibits binding between a wild-type AXL,
MER and/or Tyro3 polypeptide and a GAS6 protein in vivo or in
vitro.
[0033] In some embodiments, the polypeptide lacks a functional
fibronectin (FN) domain and/or exhibits increased affinity of the
AXL, MER or Tyro3 variant polypeptide binding to GAS6 compared to
wild-type AXL, MER or Tyro3.
[0034] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the transmembrane domain, has more than one Ig1
domain and exhibits increased affinity of the AXL, MER or Tyro3
variant polypeptide binding to GAS6 as compared to wild-type AXL,
MER or Tyro3.
[0035] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide has two Ig1 domains. In some embodiments, the
polypeptide has three Ig1 domains.
[0036] In some embodiments, the AXL, MER or Tyro3 polypeptide lacks
the transmembrane domain, has more than one Ig2 domain and exhibits
increased affinity of the AXL, MER or Tyro3 polypeptide binding to
GAS6 as compared to wild-type AXL, MER or Tyro3.
[0037] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide has two Ig2 domains.
[0038] In some embodiments, the polypeptide is a soluble AXL, MER
or Tyro3 variant polypeptide, wherein said soluble AXL variant
polypeptide lacks the AXL, MER or Tyro3 transmembrane domain, has
more than one Ig1 domain, more than one Ig2 domain and exhibits
increased affinity of the AXL, MER or Tyro3 variant polypeptide
binding to GAS6 as compared to wild-type AXL, MER or Tyro3.
[0039] In some embodiments, 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.
[0040] In some embodiments, the soluble AXL, MER or Tyro3 variant
polypeptide has two Ig1 domains and two Ig2 domains.
[0041] In some embodiments, the soluble AXL, MER or Tyro3 variant
polypeptide has the immunoglobulin domains connected directly.
[0042] In some embodiments, the soluble AXL, MER or Tyro3 variant
polypeptide has the immunoglobulin domains connected
indirectly.
[0043] In some embodiments, the soluble AXL, MER or Tyro3 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 as compared to wild-type AXL, MER or Tyro3.
[0044] In some embodiments, the polypeptide has one Ig1 domain and
lacks a functional Ig2 domain.
[0045] In some embodiments, 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.
[0046] In some embodiments, 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.
[0047] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide is a fusion protein comprising an Fc domain.
[0048] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide further comprises a linker. In some embodiments, the
linker comprises one or more (GLY).sub.4SER units.
[0049] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the AXL, MER or Tyro3 intracellular domain.
[0050] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks a functional fibronectin (FN) domain and wherein
said AXL, MER or Tyro3 variant polypeptide exhibits increased
affinity of the polypeptide binding to GAS6 as compared to
wild-type AXL, MER or Tyro3.
[0051] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide comprises at least one amino acid modification relative
to the wild-type AXL, MER or Tyro3 sequence.
[0052] In some embodiments, 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).
[0053] In some embodiments, 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.
[0054] In some embodiments, 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) 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 T98P, 22)
T105M, 23) Q109R, 24) V112A, 25) F113L, 26) H116R, 27) T118A, 28)
G127R or G127E, and 29) G129E and a combination thereof.
[0055] In some embodiments, the soluble 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.
[0056] In some embodiments, the soluble 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.
[0057] In some embodiments, the soluble 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.
[0058] In some embodiments, the soluble AXL variant polypeptide
comprises amino acid changes relative to the wild-type AXL sequence
(SEQ ID NO: 1) at the following position: alanine 72.
[0059] In some embodiments, in the soluble AXL variant polypeptide
the 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.
[0060] In some embodiments, in the soluble 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.
[0061] In some embodiments, in the soluble AXL variant polypeptide
alanine 72 residue is replaced with a valine residue.
[0062] In some embodiments, in the soluble 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.
[0063] In some embodiments, the soluble AXL variant polypeptide
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.
[0064] In some embodiments, in the soluble 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.
[0065] In some embodiments, the soluble 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.
[0066] In some embodiments, the soluble 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.
[0067] In some embodiments, in the soluble 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.
[0068] In some embodiments, the soluble AXL variant 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.
[0069] In some embodiments, the soluble AXL variant polypeptide 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.
[0070] In some embodiments, the soluble AXL variant 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.
[0071] In some embodiments, the soluble AXL variant 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.
[0072] In some embodiments, the soluble AXL variant 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.
[0073] In some embodiments, the soluble AXL variant polypeptide 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.
[0074] In some embodiments, the soluble AXL variant 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.
[0075] In some embodiments, the soluble AXL variant polypeptide 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.
[0076] In some embodiments, the soluble AXL variant 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.
[0077] In some embodiments, the soluble AXL variant polypeptide 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.
[0078] In some embodiments, the soluble AXL variant 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.
[0079] In some embodiments, the soluble AXL variant polypeptide 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.
[0080] In some embodiments, the soluble AXL variant polypeptide of
any of the preceding claims, wherein said soluble AXL variant
polypeptide has an affinity of at least about 1.times.10.sup.-8 M,
1.times.10.sup.-9 M, 1.times.10.sup.-10 M, 1.times.10.sup.-11 M or
1.times.10.sup.-12 M for GAS6.
[0081] In some embodiments, the 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.
[0082] In some embodiments, the soluble AXL variant polypeptide
comprises one or more (GLY)4SER (SEQ ID NO:10) units. In some
embodiments, the linker comprises 1, 2, 3 or 5 (GLY)4SER (SEQ ID
NO:10) units.
[0083] In some embodiments, the soluble AXL variant polypeptide
inhibits binding between wild-type AXL, MER and/or Tyro3
polypeptide and a GAS6 protein in vivo or in vitro.
[0084] In some embodiments, the soluble AXL variant polypeptide is
a fusion polypeptide comprising an Fc domain.
[0085] In some embodiments, the present invention provides a
pharmaceutical composition comprising a therapeutically effective
amount of one or more soluble AXL, MER or Tyro3 variant
polypeptides.
[0086] In some embodiments, the pharmaceutical composition further
comprises at least one cytotoxic agent or a pharmaceutically
acceptable excipient or a combination thereof.
[0087] In some embodiments, the present invention also provides
methods of treating, reducing, or preventing the metastasis or
invasion of a tumor in a mammalian patient, the method comprising:
administering to said patient an effective dose of the inhibitor
agent of the present invention. In some embodiments, the inhibitor
agent is an AXL, MER or Tyro3 variant polypeptide of any of the
preceding claims.
[0088] In some embodiments, the tumor for treatment is a tumor
selected from the group consisting of an ovarian tumor, a breast
tumor, a lung tumor, a liver tumor, a colon tumor, a gallbladder
tumor, a pancreatic tumor, a prostate tumor, and glioblastoma.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIG. 1. Describes the four domains of AXL and some
embodiments of the various combinations of AXL-Fc constructs that
have been made and tested.
[0090] FIG. 2. Describes some embodiments of the various
combinations of monovalent AXL-Fc constructs.
DEFINITIONS
[0091] In the description that follows, a number of terms
conventionally used in the field of cell culture are utilized
extensively. In order to provide a clear and consistent
understanding of the specification and claims, and the scope to be
given to such terms, the following definitions are provided.
[0092] "Inhibitors," "activators," and "modulators" of 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 for receptor or ligand
binding or signaling, e.g., ligands, receptors, agonists,
antagonists, and their homologs and mimetics.
[0093] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of two or more amino
acid residues. The terms apply to amino acid polymers in which one
or more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. The terms "antibody" and "antibodies" are used
interchangeably herein and refer to a polypeptide capable of
interacting with and/or binding to another molecule, often referred
to as an antigen. Antibodies can include, for example
"antigen-binding polypeptides" or "target-molecule binding
polypeptides." Antigens of the present invention can include for
example any polypeptides described in the present invention.
[0094] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, gamma-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refer to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid. All single
letters used in the present invention to represent amino acids are
used according to recognized amino acid symbols routinely used in
the field, e.g., A means Alanine, C means Cysteine, etc. An amino
acid is represented by a single letter before and after the
relevant position to reflect the change from original amino acid
(before the position) to changed amino acid (after position). For
example, A19T means that amino acid alanine at position 19 is
changed to threonine.
[0095] The terms "subject," "individual," and "patient" are used
interchangeably herein to refer to a mammal being assessed for
treatment and/or being treated. In an embodiment, the mammal is a
human. The terms "subject," "individual," and "patient" thus
encompass individuals having cancer, including without limitation,
adenocarcinoma of the ovary or prostate, breast cancer,
glioblastoma, etc., including those who have undergone or are
candidates for resection (surgery) to remove cancerous tissue.
Subjects may be human, but also include other mammals, particularly
those mammals useful as laboratory models for human disease, e.g.
mouse, rat, etc.
[0096] The term "tumor," as used herein, refers to all neoplastic
cell growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues.
[0097] The terms "cancer," "neoplasm," and "tumor" are used
interchangeably herein to refer to cells which exhibit autonomous,
unregulated growth, such that they exhibit an aberrant growth
phenotype characterized by a significant loss of control over cell
proliferation. In general, cells of interest for detection,
analysis, classification, or treatment in the present application
include precancerous (e.g., benign), malignant, pre-metastatic,
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, renal cancer, carcinoma, melanoma, head and neck
cancer, and brain cancer.
[0098] The "pathology" of cancer includes all phenomena that
compromise the well-being of the patient. This includes, without
limitation, abnormal or uncontrollable cell growth, metastasis,
interference with the normal functioning of neighboring cells,
release of cytokines or other secretory products at abnormal
levels, suppression or aggravation of inflammatory or immunological
response, neoplasia, premalignancy, malignancy, invasion of
surrounding or distant tissues or organs, such as lymph nodes,
etc.
[0099] As used herein, the terms "cancer recurrence" and "tumor
recurrence," and grammatical variants thereof, refer to further
growth of neoplastic or cancerous cells after diagnosis of cancer.
Particularly, recurrence may occur when further cancerous cell
growth occurs in the cancerous tissue. "Tumor spread," similarly,
occurs when the cells of a tumor disseminate into local or distant
tissues and organs; therefore tumor spread encompasses tumor
metastasis. "Tumor invasion" occurs when the tumor growth spread
out locally to compromise the function of involved tissues by
compression, destruction, or prevention of normal organ
function.
[0100] As used herein, the term "metastasis" refers to the growth
of a cancerous tumor in an organ or body part, which is not
directly connected to the organ of the original cancerous tumor.
Metastasis will be understood to include micrometastasis, which is
the presence of an undetectable amount of cancerous cells in an
organ or body part which is not directly connected to the organ of
the original cancerous tumor. Metastasis can also be defined as
several steps of a process, such as the departure of cancer cells
from an original tumor site, and migration and/or invasion of
cancer cells to other parts of the body. Therefore, the present
invention contemplates a method of determining the risk of further
growth of one or more cancerous tumors in an organ or body part
which is not directly connected to the organ of the original
cancerous tumor and/or any steps in a process leading up to that
growth.
[0101] Depending on the nature of the cancer, an appropriate
patient sample is obtained. As used herein, the phrase "cancerous
tissue sample" refers to any cells obtained from a cancerous tumor.
In the case of solid tumors which have not metastasized, a tissue
sample from the surgically removed tumor will typically be obtained
and prepared for testing by conventional techniques.
[0102] The definition encompasses blood and other liquid samples of
biological origin, solid tissue samples such as a biopsy specimen
or tissue cultures or cells derived therefrom and the progeny
thereof. The definition also includes samples that have been
manipulated in any way after their procurement, such as by
treatment with reagents; washed; or enrichment for certain cell
populations, such as cancer cells. The definition also includes
sample that have been enriched for particular types of molecules,
e.g., nucleic acids, polypeptides, etc. The term "biological
sample" encompasses a clinical sample, and also includes tissue
obtained by surgical resection, tissue obtained by biopsy, cells in
culture, cell supernatants, cell lysates, tissue samples, organs,
bone marrow, blood, plasma, serum, and the like. A "biological
sample" includes a sample obtained from a patient's cancer cell,
e.g., a sample comprising polynucleotides and/or polypeptides that
is obtained from a patient's cancer cell (e.g., a cell lysate or
other cell extract comprising polynucleotides and/or polypeptides);
and a sample comprising cancer cells from a patient. A biological
sample comprising a cancer cell from a patient can also include
non-cancerous cells.
[0103] The term "diagnosis" is used herein to refer to the
identification of a molecular or pathological state, disease or
condition, such as the identification of a molecular subtype of
breast cancer, prostate cancer, or other type of cancer.
[0104] The term "prognosis" is used herein to refer to the
prediction of the likelihood of cancer-attributable death or
progression, including recurrence, metastatic spread, and drug
resistance, of a neoplastic disease, such as ovarian cancer. The
term "prediction" is used herein to refer to the act of foretelling
or estimating, based on observation, experience, or scientific
reasoning. In one example, a physician may predict the likelihood
that a patient will survive, following surgical removal of a
primary tumor and/or chemotherapy for a certain period of time
without cancer recurrence.
[0105] As used herein, the terms "treatment," "treating," and the
like, refer to administering an agent, or carrying out a procedure
(e.g., radiation, a surgical procedure, etc.), for the purposes of
obtaining an effect. The effect may be prophylactic in terms of
completely or partially preventing a disease or symptom thereof
and/or may be therapeutic in terms of effecting a partial or
complete cure for a disease and/or symptoms of the disease.
"Treatment," as used herein, covers any treatment of any metastatic
tumor in a mammal, particularly in a human, and includes: (a)
preventing the disease or a symptom of a disease from occurring in
a subject which may be predisposed to the disease but has not yet
been diagnosed as having it (e.g., including diseases that may be
associated with or caused by a primary disease; (b) inhibiting the
disease, i.e., arresting its development; and (c) relieving the
disease, i.e., causing regression of the disease. In tumor (e.g.,
cancer) treatment, a therapeutic agent may directly decrease the
metastasis of tumor cells.
[0106] Treating may refer to any indicia of success in the
treatment or amelioration or prevention of an cancer, including any
objective or subjective parameter such as abatement; remission;
diminishing of symptoms or making the disease condition more
tolerable to the patient; slowing in the rate of degeneration or
decline; or making the final point of degeneration less
debilitating. The treatment or amelioration of symptoms can be
based on objective or subjective parameters; including the results
of an examination by a physician. Accordingly, the term "treating"
includes the administration of the compounds or agents of the
present invention to prevent or delay, to alleviate, or to arrest
or inhibit development of the symptoms or conditions associated
with neoplasia, e.g., tumor or cancer. The term "therapeutic
effect" refers to the reduction, elimination, or prevention of the
disease, symptoms of the disease, or side effects of the disease in
the subject.
[0107] "In combination with", "combination therapy" and
"combination products" refer, in certain embodiments, to the
concurrent administration to a patient of a first therapeutic and
the compounds as used herein. When administered in combination,
each component can be administered at the same time or sequentially
in any order at different points in time. Thus, each component can
be administered separately but sufficiently closely in time so as
to provide the desired therapeutic effect.
[0108] According to the present invention, the first therapeutic
can be any suitable therapeutic agent, e.g., cytotoxic agents. One
exemplary class of cytotoxic agents are chemotherapeutic agents,
e.g., they can be combined with treatment to inhibit AXL or GAS6
signaling. Exemplary 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 alpha, etoposide, filgrastim, fludarabine,
fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin,
ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole,
leucovorin, megestrol, mesna, methotrexate, metoclopramide,
mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron,
paclitaxel (Taxol.TM.), pilocarpine, prochloroperazine, rituximab,
saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,
vinblastine, vincristine and vinorelbine tartrate. For ovarian
cancer treatment, a preferred chemotherapeutic agent with which an
AXL or GAS6 signaling inhibitor can be combined is paclitaxel
(Taxol.TM.).
[0109] Other combination therapies are radiation, surgery, and
hormone deprivation (Kwon et al., Proc. Natl. Acad. Sci U.S.A., 96:
15074-9, 1999). Angiogenesis inhibitors can also be combined with
the methods of the invention.
[0110] "Concomitant administration" of a known cancer therapeutic
drug with a pharmaceutical composition of the present invention
means administration of the drug and AXL inhibitor at such time
that both the known drug and the composition of the present
invention will have a therapeutic effect. Such concomitant
administration may involve concurrent (i.e. at the same time),
prior, or subsequent administration of the drug with respect to the
administration of a compound of the present invention. A person of
ordinary skill in the art would have no difficulty determining the
appropriate timing, sequence and dosages of administration for
particular drugs and compositions of the present invention.
[0111] As used herein, the phrase "disease-free survival," refers
to the lack of such tumor recurrence and/or spread and the fate of
a patient after diagnosis, with respect to the effects of the
cancer on the life-span of the patient. The phrase "overall
survival" refers to the fate of the patient after diagnosis,
despite the possibility that the cause of death in a patient is not
directly due to the effects of the cancer. The phrases, "likelihood
of disease-free survival", "risk of recurrence" and variants
thereof, refer to the probability of tumor recurrence or spread in
a patient subsequent to diagnosis of cancer, wherein the
probability is determined according to the process of the
invention.
[0112] As used herein, the term "correlates," or "correlates with,"
and like terms, refers to a statistical association between
instances of two events, where events include numbers, data sets,
and the like. For example, when the events involve numbers, a
positive correlation (also referred to herein as a "direct
correlation") means that as one increases, the other increases as
well. A negative correlation (also referred to herein as an
"inverse correlation") means that as one increases, the other
decreases.
[0113] "Dosage unit" refers to physically discrete units suited as
unitary dosages for the particular individual to be treated. Each
unit can contain a predetermined quantity of active compound(s)
calculated to produce the desired therapeutic effect(s) in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms can be dictated by (a) the
unique characteristics of the active compound(s) and the particular
therapeutic effect(s) to be achieved, and (b) the limitations
inherent in the art of compounding such active compound(s).
[0114] "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 as well as for human
pharmaceutical use. Such excipients can be solid, liquid,
semisolid, or, in the case of an aerosol composition, gaseous.
[0115] The terms "pharmaceutically acceptable", "physiologically
tolerable" and grammatical variations thereof, as they refer to
compositions, carriers, diluents and reagents, are used
interchangeably and represent that the materials are capable of
administration to or upon a human without the production of
undesirable physiological effects to a degree that would prohibit
administration of the composition.
[0116] A "therapeutically effective amount" means the amount that,
when administered to a subject for treating a disease, is
sufficient to effect treatment for that disease.
DETAILED DESCRIPTION
[0117] AXL, MER and Tyro3 are the three receptor protein tyrosine
kinases whose ligand is GAS6. As such, the present invention is
based in part on the discovery of inhibitor agents that inhibit
and/or antagonize the interaction of the wild-type AXL, MER and/or
Tyro3 receptor with the GAS6 ligand.
[0118] According to the present invention, such an inhibitor agent
can be selected from (a) an inhibitor of AXL, MER and/or Tyro3
activity, (b) an inhibitor of GAS6 activity and (c) an inhibitor of
AXL, MER and/or Tyro3-GAS6 interaction, wherein the inhibitor agent
is capable of binding to GAS6 with increased affinity compared to
wild-type AXL, MER and/or Tyro3.
[0119] In some embodiments, the inhibitor agent binds to two or
more epitopes on a single GAS6 molecule. The two or more epitopes
can include at least one of the major and/or minor AXL, MER and/or
Tyro3 binding site on GAS6. In some embodiments, the epitopes are
separate or distinct epitopes. In some embodiments the epitopes
overlap. In some embodiments, the epitopes do not overlap. In some
embodiments, the epitopes are adjacent. In some embodiments, the
epitopes are not adjacent. In some embodiments, the epitopes
include the major and/or minor AXL, MER and/or Tyro3 binding site
on GAS6. These GAS6 epitopes of the present invention, and to which
the inhibitor agents of the present invention bind, can be located
on one or more GAS6 molecules. In some embodiments, the epitopes
are located on a single GAS6 molecule.
[0120] In some embodiments, the inhibitor agent is capable of
binding to the major and/or minor AXL, MER and/or Tyro3 binding
sites on a single GAS6. In some embodiments, the inhibitor agent is
capable of binding the major AXL, MER and/or Tyro3 binding site of
GAS6 and one or more additional GAS6 epitopes. In other
embodiments, the inhibitor agent is capable of binding to the AXL,
MER and/or Tyro3 minor binding site on GAS6 and one or more
additional epitopes. In some other embodiments, the inhibitor agent
is capable of binding two or more distinct epitopes on GAS6. The
additional GAS6 epitopes can include any epitopes on GAS6 which
lead to increased affinity and/or increased avidity of the
inhibitor agent binding to GAS6 as compared to wild-type AXL, MER
and/or Tyro3. In some embodiments, the AXL, MER and/or Tyro3
variant polypeptides of the present invention bind two epitopes on
a single GAS6 molecule. In some embodiments, the two epitopes are
the major and minor AXL, MER and/or Tyro3 binding sites.
[0121] According to the invention, GAS6 receptors include AXL, MER
and Tyro3. The inhibitor agents of the present invention can also
in some embodiments antagonize the major and/or minor GAS6/receptor
interaction. In some embodiments, the inhibitor agent is capable of
antagonizing the major and/or minor GAS6/receptor binding
interaction. In other embodiments, the inhibitor agent is capable
of antagonizing the major GAS6/receptor binding interaction (e.g.,
interfering with and/or inhibiting the major GAS6/receptor binding
interaction). In some embodiments, the inhibitor agent is capable
of antagonizing the minor GAS6/receptor binding interaction (e.g.,
interfering with and/or inhibiting the minor GAS6/receptor binding
interaction).
[0122] Inhibitor agents can also include for example protein
scaffolds (i.e., smaller proteins that are capable of achieving
comparable affinity and specificity using molecular structures that
can be for example one-tenth the size of full antibodies).
[0123] The inhibitor agents can also include non-antibody
polypeptides. In some embodiments, the inhibitor agent is a
non-antibody polypeptide. In some embodiments, the non-antibody
polypeptide can include but is not limited to peptibodies, darpins,
avimers, adnectins, anticalins, affibodies, maxibodies, or other
protein structural scaffold, or a combination thereof.
[0124] In some embodiments the inhibitor agent provided by the
present invention is an AXL, MER and/or Tyro3 variant polypeptide,
e.g., an AXL, MER and/or Tyro3 variant polypeptide that has a
binding activity to GAS6 that is substantially equal to or better
than the binding activity of a wild-type AXL, MER and/or Tyro3
polypeptide. In some embodiments of the present invention, the AXL,
MER and/or Tyro3 variant polypeptides are utilized as therapeutic
agents.
[0125] The AXL protein, with reference to the native sequence of
SEQ ID NO: 1, comprises an immunoglobulin (Ig)-like domain from
residues 27-128, a second Ig-like domain from residues 139-222,
fibronectin type 3 domains from residues 225-332 and 333-427,
intracellular domain from residues 473-894 including tyrosine
kinase domain. The tyrosine residues at 779, 821 and 866 become
autophosphorylated upon receptor dimerization and serve as docking
sites for intracellular signaling molecules. The native cleavage
site to release the soluble form of the polypeptide lies at
residues 437-451.
[0126] For the purposes of the invention, a soluble form of AXL
(sAXL) is the portion of the polypeptide that is sufficient to bind
GAS6 at a recognizable affinity, e.g., high affinity, which
normally lies between the signal sequence and the transmembrane
domain, i.e. generally from about SEQ ID NO: 1 residue 19-437, but
which may comprise or consist essentially of a truncated version of
from about residue 19, 25, 30, 35, 40, 45, 50 to about residue 132,
450, 440, 430, 420, 410, 400, 375, 350, to 321, e.g., residue
19-132. According to the methods of the present invention, SEQ ID
NO:1 can be used interchangeably with amino acids 8-894 of SEQ ID
NO: 1, both of which refer to the wild-type AXL sequence. In some
embodiments, a soluble form of AXL lacks the transmembrane domain,
and optionally the intracellular domain.
[0127] In some embodiments, the inhibitor agent is a soluble AXL
variant polypeptide that 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 as compared to wild-type
GAS6.
[0128] The MER protein, with reference to the native SEQ ID NO:2,
comprises an immunoglobulin (Ig)-like domain from residues 81-186,
a second Ig-like domain from residues 197-273, fibronectin type 3
domains from residues 284-379 and 383-482, intracellular domain
from residues 527-999 including tyrosine kinase domain. The
tyrosine residues at 749, 753, 754 and 872 become
autophosphorylated upon receptor dimerization and serve as docking
sites for intracellular signaling molecules.
[0129] For the purposes of the invention, a soluble form of MER
(sMER) is the portion of the polypeptide that is sufficient to bind
GAS6 at a recognizable affinity, e.g., high affinity, which
normally lies between the signal sequence and the transmembrane
domain, i.e. generally from about SEQ ID NO: 2 residue 21-526, but
which may comprise or consist essentially of a truncated version In
some embodiments, a soluble form of MER lacks the transmembrane
domain (i.e., generally from about SEQ ID NO: 2 residue 506-526),
and optionally the intracellular domain (i.e., generally from about
SEQ ID NO: 2 residue 527-999).
[0130] In some embodiments, the inhibitor agent is 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 as compared to wild-type MER.
[0131] The Tyro3 protein, with reference to the native SEQ ID NO:3,
comprises an immunoglobulin (Ig)-like domain from residues 41-128,
a second Ig-like domain from residues 139-220, fibronectin type 3
domains from residues 225-317 and 322-413, intracellular domain
from residues 451-890 including tyrosine kinase domain. The
tyrosine residues at 681, 685, 686 and 804 become
autophosphorylated upon receptor dimerization and serve as docking
sites for intracellular signaling molecules.
[0132] For the purposes of the invention, a soluble form of Tyro3
(sTyro3) is the portion of the Tyro3 polypeptide that is sufficient
to bind GAS6 at a recognizable affinity, e.g., high affinity, which
normally lies between the signal sequence and the transmembrane
domain, i.e. generally from about SEQ ID NO: 3 residue 41-450, but
which may comprise or consist essentially of a truncated version In
some embodiments, a soluble form of AXL lacks the transmembrane
domain (i.e., generally from about SEQ ID NO: 3 residue 430-450),
and optionally the intracellular domain (i.e., generally from about
SEQ ID NO: 3 residue 451-890).
[0133] In some embodiments, the inhibitor agent is a soluble Tyro3
variant polypeptide wherein said Tyro3 polypeptide lacks the Tyro3
transmembrane domain and has at least one mutation relative to
wild-type Tyro3 that increases affinity of the Tyro3 polypeptide
binding to GAS6 as compared to wild-type Tyro3.
[0134] In some embodiments, the AXL, MET or Tyro3 variant
polypeptide lacks the AXL, MET or Tyro3 transmembrane domain and is
a soluble variant polypeptide, e.g., sAXL, sMER or sTyro3 variant
polypeptide.
[0135] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the AXL, MER or Tyro3 intracellular domain.
[0136] In some embodiments, the inhibitor agent of the present
invention inhibits binding between a wild-type AXL, MER and/or
Tyro3 polypeptide and a GAS6 protein in vivo or in vitro. In some
embodiments, the AXL, MER or Tyro3 variant polypeptide inhibits
binding between a wild-type AXL, MER and/or Tyro3 polypeptide and a
GAS6 protein in vivo or in vitro.
[0137] The inhibitor agents of the present invention can also
exhibit an enhanced or better pharmacokinetic profile. In some
embodiments, the enhanced or better pharmacokinetic profile
includes for example but is not limited to a better absorption
profile, better distribution profile, better metabolism profile,
better excretion profile, better liberation profile, increased
half-life, decrease half-life, faster rate of action, longer
duration of effect as compared to AXL, MER and/or Tyro3 wild-type
polypeptides which do not lack a transmembrane domain. One of skill
in the art would understand preferred pharmacokinetic profile
parameters for particular needs including for example treatment
regimens, and how to appropriately implement such parameters in
treatment regimens.
[0138] The wild-type AXL, MER and Tyro3 all contain two fibronectin
domains. In some embodiments, the AXL, MER and Tyro3 polypeptides
of the invention lack a functional fibronectin (FN) domain. Lacks
or lacking a functional fibronectin domain can include but is not
limited to deletion of one or both fibronectin domains and/or
introducing mutations that inhibit, reduce or remove the
functionality of one or both fibronectin domains, where such
mutations can include for example but are not limited to
substitution, deletion and insertion mutations. In some
embodiments, the polypeptides of the invention have fibronectin 1
(FN1) deleted, fibronectin 2 (FN2) deleted, or FN1 and FN2 both
deleted. In some embodiments, the polypeptides of the invention
have portions of FN1 mutated and/or deleted, FN2 mutated and/or
deleted, or FN1and FN2 mutated and/or deleted.
[0139] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks a functional AXL, MER or Tyro3 fibronectin (FN)
domain. In some embodiments, the AXL, MER or Tyro3 variant
polypeptide exhibits increased affinity of the polypeptide binding
to GAS6 as compared to wild-type AXL, MER and/or Tyro3. In some
embodiments, the AXL, MER or Tyro3 variant polypeptide lacks a
functional fibronectin (FN) domain also exhibits increased affinity
of the polypeptide binding to GAS6 as compared to wild-type AXL,
MER and/or Tyro3.
[0140] In some embodiments, the lack of a functional fibronectin
domain results in increased affinity of the AXL, MER or Tyro3
polypeptide binding to GAS6. In some embodiments, the lack of a
functional fibronectin domain results in an enhanced or better
pharmacokinetic profile, including for example but not limited to a
better absorption profile, better distribution profile, better
metabolism profile, better excretion profile, better liberation
profile, increased half-life, decreased half-life, faster rate of
action, longer duration of effect as compared to other wild-type
polypeptides or other polypeptides which do not lack a functional
fibronectin domain. One of skill in the art would understand
preferred pharmacokinetic profile parameters for particular needs
including for example treatment regimens, and how to appropriately
implement such parameters in treatment regimens.
[0141] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the transmembrane domain and has more than one
Ig1 domain and exhibits increased affinity of the AXL, MER or Tyro3
polypeptide binding to GAS6 as compared to wild-type AXL, MER
and/or Tyro3. In some embodiments, the AXL, MER or Tyro3
polypeptide has two Ig1 domains. In some embodiments, the AXL, MER
or Tyro3 polypeptide has three Ig1 domains. In some embodiments,
the AXL, MER or Tyro3 polypeptide has more than one Ig1 domain
and/or more than one Ig2 domain. In some embodiments, the AXL, MER
or Tyro3 polypeptide has two Ig2 domains. In some embodiments, the
AXL, MER or Tyro3 polypeptide has two Ig1 domains and 2 Ig2
domains. In some embodiments, the AXL, MER or Tyro3 polypeptide
includes for example but is not limited to one of the following Ig
domain configurations, as well as any combinations or variations
thereof: [0142] Ig1 [0143] Ig1-Ig2 [0144] Ig1-Ig1 [0145]
Ig1-Ig1-Ig1 [0146] Ig1-Ig2-Ig1 [0147] Ig1-Ig2-Ig 1-Ig2
[0148] In some embodiments, the AXL, MER or Tyro3 polypeptide also
lacks the AXL, MER or Tyro3 transmembrane domain and/or exhibits
increased affinity of the AXL, MER or Tyro3 polypeptide binding to
GAS6. In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the transmembrane domain, has more than one Ig1
domain, has more than one Ig2 domain and exhibits increased
affinity of the AXL, MER or Tyro3 polypeptide binding to GAS6 as
compared to wild-type AXL, MER and/or Tyro3.
[0149] In some embodiments, the AXL, MER or Tyro3 has the
immunoglobulin domains connected directly to one another. In some
embodiments, the AXL, MER or Tyro3 has the immunoglobulin domains
connected indirectly, e.g., through a linker molecule including for
example any amino acid linker known in the art.
[0150] In some embodiments, the one or more AXL, MER or Tyro3 Ig1
and/or 1 or more AXL, MER or Tyro3 Ig2 domains result in an
enhanced or better pharmacokinetic profile, including for example
but not limited to a better absorption profile, better distribution
profile, better metabolism profile, better excretion profile,
better liberation profile, increased half-life, decreased
half-life, faster rate of action, longer duration of effect as
compared to other wild-type polypeptides or other polypeptides
which do not lack a functional fibronectin domain. One of skill in
the art would understand preferred pharmacokinetic profile
parameters for particular needs including for example treatment
regimens, and how to appropriately implement such parameters in
treatment regimens.
[0151] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide lacks the AXL, MER or Tyro3 transmembrane domain and is
capable of binding two or more epitopes on a single GAS6. In some
embodiments, the AXL, MER or Tyro3 variant polypeptide lacks the
AXL, MER or Tyro3 transmembrane domain and is capable of binding
both the major and minor AXL, MER and/or Tyro3 binding sites on a
single GAS6. In some embodiments, the binding of both the major and
minor AXL, MER and/or Tyro3 binding is simultaneous. In some
embodiments, the binding of both the major and minor AXL, MER
and/or Tyro3 binding sites is simultaneous on a single GAS6.
[0152] The present invention also provides AXL, MER or Tyro3
variant polypeptides that do not bind two epitopes on a single GAS6
molecule. The present invention also provides AXL, MER or Tyro3
variant polypeptides that do not bind two epitopes on a single GAS6
molecule simultaneously. In some embodiments, the AXL, MER and/or
Tyro3 variant polypeptide is not capable of binding two epitopes on
a single GAS6, this includes for example monomeric AXL, MER and/or
Tyro3 variant polypeptides. In some embodiments, the monomeric AXL,
MER or Tyro3 variant polypeptide comprises one Ig1 domain. In some
embodiments, the monomeric AXL, MER and/or Tyro3 variant
polypeptide is an Fc fusion polypeptide that does not bind to more
than one site on a singe Gas6 molecule simultaneously. In some
embodiments, the monomeric AXL, MER and/or Tyro3 variant
polypeptide that is not capable of binding two epitopes on a single
GAS6 comprises two AXL, MER and/or Tyro3 variant polypeptides each
of which are not capable of binding two epitopes on a single GAS6
simultaneously. In some embodiments, the monomeric AXL, MER and/or
Tyro3 variant polypeptide that is not capable of simultaneously
binding two epitopes on a single GAS6 has one Ig1 domain. In some
embodiments, the monomeric AXL, MER and/or Tyro3 variant
polypeptide that is not capable of simultaneously binding two
epitopes on a single GAS6 has an altered half-life when compared to
AXL, MER and/or Tyro3 variant polypeptides that are capable of
binding two epitopes on a single GAS6. In some embodiments, the
polypeptide has one Ig1 domain and lacks a functional Ig2 domain.
In some embodiments, the Ig1 domain comprises amino acids 1-131 of
AXL (SEQ ID NO:1; or in some embodiments 8-138 of SEQ ID NO:1). In
some embodiments, 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. In some embodiments, the polypeptide
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.
[0153] The wild-type AXL, MER and Tyro3 all contain an Ig2 domain.
In some embodiments, the AXL, MER and Tyro3 polypeptides of the
invention lack a functional Ig2 domain. Lacks or lacking a
functional Ig2 domain can include but is not limited to deletion of
the Ig2 domain and/or introduction of mutations that inhibit,
reduce or remove the functionality of the Ig2 domain, where such
mutations can include for example but are not limited to
substitution, deletion and insertion mutations. In some
embodiments, the polypeptides of the invention lack a functional
Ig2 domain. In some embodiments, the polypeptides of the invention
lack a functional Ig2 domain and have a wild-type AXL, MER and/or
Tyro3 Ig1 domain. In some embodiments, the polypeptides of the
invention lack a functional Ig2 domain and have one or more
mutations in the Ig1 domain relative to the wild-type AXL, MER
and/or Tyro3 Ig1 domain.
[0154] In some embodiments, the AXL, MER and/or Tyro3 variant
polypeptide includes a linker. A wide variety of linkers are known
in the art and any known linker can be employed with the methods of
the present invention. In some embodiments, the AXL, MER or Tyro3
variant polypeptide includes one or more linkers or linker units.
In some embodiments, the linker is an amino acid linker, including
an amino acid sequence of 2, 3, 4 or 5 amino acids which are
different that the wild-type AXL, MER and/or Tyro3 sequences. In
some embodiments, the linker has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or
more units. In some embodiments, the linker is (GLY)4SER (SEQ ID
NO:10). In some embodiments, the linker has 1, 2, 3, 4, 5, 6, 7, 8,
9, 10 or more (GLY)4SER (SEQ ID NO:10) units. In some embodiments,
the linker has 1, 2, 3 or 5 (GLY)4SER (SEQ ID NO:10) units. In some
embodiments, the linkers are between the AXL, MER or Tyro3 variant
polypeptide and the Fc portion of a fusion polypeptide. In some
embodiments, the linkers are between the AXL, MER or Tyro3 variant
polypeptide and the Fc portion of a fusion polypeptide and the AXL,
MER or Tyro3 variant polypeptide lacks a functional fibronectin
domain.
[0155] In some embodiments, AXL, MER and/or Tyro3 variant
polypeptides of the present invention also include one or more
amino acid modifications within the soluble form of wild-type AXL,
MER and/or Tyro3, e.g., one or more amino acid modifications that
increase its affinity for GAS6. According to the present invention,
amino acid modifications include any naturally occurring or
man-made amino acid modifications known or later discovered in the
field. In some embodiments, amino acid modifications include any
naturally occurring mutation, e.g., substitution, deletion,
addition, insertion, etc. In some other embodiments, amino acid
modifications include replacing existing amino acid with another
amino acid, e.g., a conservative equivalent thereof. In yet some
other embodiments, amino acid modifications include replacing one
or more existing amino acids with non-natural amino acids or
inserting one or more non-natural amino acids. In still some other
embodiments, amino acid modifications include at least 1, 2, 3, 4,
5, or 6 or 10 amino acid mutations or changes.
[0156] In some exemplary embodiments, one or more amino acid
modifications can be used to alter properties of the soluble form
of AXL, MER and/or Tyro3 e.g., affecting the stability, binding
activity and/or specificity, etc. Techniques for 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). Methods for site specific 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 (1992); Jones and Winistorfer,
Biotechniques 12:528-30 (1992); Barton et al., Nucleic Acids Res
18:7349-55 (1990); Marotti and Tomich, Gene Anal Tech 6:67-70
(1989); and Zhu Anal Biochem 177:120-4 (1989).
[0157] In some embodiments, AXL variant polypeptides of the present
invention include one or more amino acid modifications within one
or more regions of residue 18 to 130, residue 10 to 135, residue 15
to 45, residue 60 to 65, residue 70 to 80, residue 85 to 90,
residue 91 to 99, residue 104 to 110, residue 111 to 120, residue
125 to 130, residue 19 to 437, residue 130 to 437, residue 19 to
132, residue 21 to 132, residue 21 to 121, residue 26 to 132, or
residue 26 to 121 of wild-type AXL (SEQ ID NO: 1). In some other
embodiments, AXL variant polypeptides of the present invention
include one or more amino acid modifications within one or more
regions of residue 20 to 130, residue 37 to 124 or residue 141 to
212 of wild-type AXL (SEQ ID NO: 1). In yet some other embodiments,
AXL polypeptide variants of the present invention include one or
more amino acid modifications at one or more positions of 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
wild-type AXL (SEQ ID NO 1).
[0158] In yet some other embodiments, AXL polypeptide variants of
the present invention include one or more amino acid modifications
including without any limitation 1) A19T, 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
T98P, 22) T105M, 23) Q109R, 24) V112A, 25) F113L, 26) H116R, 27)
T118A, 28) G127R or G127E, and 29) E129K and a combination
thereof.
[0159] In yet some other embodiments, AXL variant polypeptides of
the present invention include one or more amino acid modifications
at position 32, 87, 92, or 127 of wild-type AXL (SEQ ID NO: 1) or a
combination thereof, e.g., G32S; D87G; V92A and/or G127R. In yet
some other embodiments, AXL polypeptide variants of the present
invention include one or more amino acid modifications at position
26, 79, 92, 127 of wild-type AXL (SEQ ID NO: 1) or a combination
thereof, e.g., E26G, V79M; V92A and/or G127E. In yet some other
embodiments, AXL variant polypeptides of the present invention
include one or more amino acid modifications at position 32, 87,
92, 127 and/or 72 of wild-type AXL (SEQ ID NO: 1) or a combination
thereof, e.g., G32S; D87G; V92A; G127R and/or A72V. In yet some
other embodiments, AXL variant polypeptides of the present
invention include one or more amino acid modifications at position
87, 92 and/or 127 of wild-type AXL (SEQ ID NO: 1) or a combination
thereof, e.g., D87G; V92A; and/or G127R. In yet some other
embodiments, AXL variant polypeptides of the present invention
include one or more amino acid modifications at position 32, 92,
and/or 127 of wild-type AXL (SEQ ID NO: 1) or a combination
thereof, e.g., G32S; V92A; and/or G127R. In yet some other
embodiments, AXL variant polypeptides of the present invention
include one or more amino acid modifications at position 32, 87
and/or 127 of wild-type AXL (SEQ ID NO: 1) or a combination
thereof, e.g., G32S; D87G; and/or G127R. In yet some other
embodiments, AXL polypeptide variants of the present invention
include one or more amino acid modifications at position 32, 87
and/or 92 of wild-type AXL (SEQ ID NO: 1) or a combination thereof,
e.g., G32S; D87G; and/or V92A. In yet some other embodiments, AXL
variant polypeptides of the present invention include one or more
amino acid modifications at position 26, 79, 92, 127 of wild-type
AXL (SEQ ID NO: 1) or a combination thereof, e.g., E26G, V79M; V92A
and/or G127E. In yet some other embodiments, AXL variant
polypeptides of the present invention include one or more amino
acid modifications at position 87 and 92 of wild-type AXL (SEQ ID
NO: 1) or a combination thereof, e.g., D87G and V92A. In yet some
other embodiments, AXL variant polypeptides of the present
invention include at least one amino acid modification at position
72 of wild-type AXL (SEQ ID NO: 1), e.g., A72V.
[0160] According to the present invention, the inhibitor agent can
include but is not limited to 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.
[0161] The inhibitor agents of the present invention can include
peptides or polypeptides. The peptides and polypeptides of the
present invention can include natural and/or synthetic
polypeptides. Synthetic polypeptides and methods of making
synthetic polypeptides are well known in the art and any known
methods for making synthetic polypeptides can be employed with the
methods of the present invention. In some embodiments, the
inhibitor agent is a natural or synthetic polypeptide. In some
embodiments, the inhibitor agent is a natural or synthetic
polypeptide-fusion. In some embodiments, the inhibitor agent is a
natural or synthetic polypeptide-Fc fusion. In some embodiments the
natural or synthetic polypeptide-fusion is a fusion with another
protein structural class or scaffold or a natural or synthetic
polypeptide-fusion with a polymer or hydrogel or related
structure.
[0162] According to the present invention, the AXL, MER or Tyro3
variant polypeptides of the present invention can be further
modified, e.g., joined to a wide variety of other oligopeptides or
proteins for a variety of purposes. For instance, various
post-translation or post-expression modifications can be carried
out with respect to AXL, MER or Tyro3 variant polypeptides of the
present invention. For example, by employing the appropriate coding
sequences, one may provide farnesylation or prenylation. In some
embodiments, the AXL, MER or Tyro3 variant polypeptides of the
present invention can be PEGylated, where the polyethyleneoxy group
provides for enhanced lifetime in the blood stream. The AXL, MER or
Tyro3 variant polypeptides of the present invention can also be
combined with other proteins, such as the Fc of an IgG isotype,
which can be complement binding, with a toxin, such as ricin,
abrin, diphtheria toxin, or the like, or with specific binding
agents that allow targeting to specific moieties on a target cell.
The inhibitor agents of the present invention can include
polypeptide conjugates and antibody-conjugates. In some
embodiments, the inhibitor agent is a polypeptide-conjugate or
antibody-conjugate. In some embodiments, the polypeptide conjugate
is a drug conjugate. In some embodiments, the peptide or
polypeptide conjugate is an antibody-drug conjugates. In some
embodiments, the polypeptide conjugate is a polymer conjugate.
Polymers of the present invention include but are not limited to
PEG, PEG-containing polymers, degradable polymers, biocompatible
polymers, hydrogels, as well as other polymer structures that could
be conjugated to a polypeptide, and can include combinations
thereof.
[0163] In some embodiments, the AXL, MER or Tyro3 variant
polypeptide of the present invention is a fusion protein, e.g.,
fused in frame with a second polypeptide. In some embodiments, the
second polypeptide is capable of increasing the size of the fusion
protein, e.g., so that the fusion protein will not be cleared from
the circulation rapidly. In some other embodiments, the second
polypeptide is part or whole of Fc region. In some other
embodiments, the second polypeptide is any suitable polypeptide
that is substantially similar to Fc, e.g., providing increased size
and/or additional binding or interaction with Ig molecules. In yet
some other embodiments, the second polypeptide is part or whole of
an albumin protein, e.g., a human serum albumin protein. In some
embodiments, the second polypeptide is a protein or peptide that
binds to albumin.
[0164] In some other embodiments, the second polypeptide is useful
for handling the AXL, MER or Tyro3 variant polypeptides, e.g.,
purification of AXL, MER or Tyro3 variant polypeptides or for
increasing stability in vitro or in vivo. For example, AXL, MER or
Tyro3 variant polypeptides of the present invention can be combined
with parts of the constant domain of immunoglobulins (IgG),
resulting in chimeric or fusion polypeptides. These fusion proteins
facilitate purification and show an increased half-life in vivo.
One reported example describes chimeric proteins consisting of the
first two domains of the human CD4-polypeptide and various domains
of the constant regions of the heavy or light chains of mammalian
immunoglobulins. EP A 394,827; Traunecker et al., Nature, 331:
84-86, 1988. Fusion proteins having 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 alone. Fountoulakis et al., J. Biochem.
270: 3958-3964, 1995.
[0165] In yet some other embodiments, the second polypeptide is a
marker sequence, such as a peptide which facilitates purification
of the fused polypeptide. For example, the marker amino acid
sequence can be a hexa-histidine peptide, such as the tag provided
in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,
Calif., 91311), among others, many of which are commercially
available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA
86: 821-824, 1989, for instance, hexa-histidine provides for
convenient purification of the fusion protein. Another peptide tag
useful for purification, the "HA" tag, corresponds to an epitope
derived from the influenza hemagglutinin protein. Wilson et al.,
Cell 37: 767, 1984.
[0166] In still some other embodiments, the second polypeptide is
an entity useful for improving the characteristics of AXL, MER or
Tyro3 polypeptide variants of the present invention. For instance,
a region of additional amino acids, particularly charged amino
acids, may be added to the N-terminus of the polypeptide to improve
stability and persistence during purification from the host cell or
subsequent handling and storage. Also, peptide moieties may be
added to the AXL, MER or Tyro3 polypeptide variants of the present
invention to facilitate purification and subsequently removed prior
to final preparation of the polypeptide. The addition of peptide
moieties to facilitate handling of polypeptides are familiar and
routine techniques in the art.
[0167] In still yet some embodiments, AXL, MER or Tyro3 variant
polypeptides of the present invention have a binding activity to
GAS6 that is at least equal or better than the wild-type AXL, MER
or Tyro3. In some other embodiments, AXL, MER or Tyro3 variant
polypeptides of the present invention has a binding activity or
affinity to GAS6 that is at least 1-fold, 2-fold, 3-fold, 4-fold,
5-fold, or 6-fold greater than that of the wild-type AXL, MER or
Tyro3. In some other embodiments, AXL, MER or Tyro3 polypeptide
variant of the present invention has a binding activity or affinity
to GAS6 of at least about 1.times.10.sup.-6, 1.times.10.sup.-7,
1.times.10.sup.-8 or 1.times.10.sup.-9 M 1.times.10.sup.-10M,
1.times.10.sup.-11M or 1.times.10.sup.-12M. In yet some other
embodiments, sAXL polypeptides of the present invention is capable
of inhibiting, inhibit or compete with wild-type AXL binding to
GAS6 either in vivo, in vitro or both. In yet some other
embodiments, sAXL polypeptides of the present invention inhibit or
compete with the binding of AXL S6-1, AXL S6-2, and/or AXL S6-5 (as
described in WO2011/091305). In yet some other embodiments, sAXL
polypeptides of the present invention inhibit or compete with the
binding of any sAXL variant as described in WO2011/091305.
[0168] The inhibitor agents of the present invention bind to GAS6
with increased affinity. In some embodiments, the AXL, MER or Tyro3
variant polypeptide exhibits increased affinity of the AXL, MER or
Tyro3 polypeptide binding to GAS6 as compared to wild-type AXL, MER
or Tyro3. In some embodiments, AXL, MER or Tyro3 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, 50-fold stronger, 100-fold stronger or at least
200-fold stronger, etc. than the affinity of the wild-type AXL, MER
or Tyro3 polypeptide. In some embodiments, the soluble AXL has a
about a 115-fold stronger affinity to GAS6 than the affinity of the
wild-type AXL polypeptide.
[0169] The ability of a molecule to bind to GAS6 can be determined,
for example, by the ability of the putative ligand to bind to GAS6
coated on an assay plate. In one embodiment, the binding activity
of AXL, MER or Tyro3 variant polypeptides of the present invention
to a GAS6 can be assayed by either immobilizing the ligand, e.g.,
GAS6 or the AXL, MER or Tyro3 variant polypeptides. For example,
the assay can include immobilizing GAS6 fused to a His tag onto
Ni-activated NTA resin beads. Agents can be added in an appropriate
buffer and the beads incubated for a period of time at a given
temperature. After washes to remove unbound material, the bound
protein can be released with, for example, SDS, buffers with a high
pH, and the like and analyzed.
[0170] In still yet other embodiments, AXL, MER or Tyro3 variant
polypeptides of the present invention has a better thermal
stability than the thermal stability of a wild-type AXL. In some
embodiments, the melting temperature of AXL, MER or Tyro3 variant
polypeptides of the present invention is at least 5.degree. C.,
10.degree. C., 15.degree. C., or 20.degree. C. higher than the
melting temperature of a wild-type AXL.
[0171] According to the present invention, AXL, MER or Tyro3
variant polypeptides of the present invention can also include one
or more modifications that do not alter primary sequences of the
AXL, MER or Tyro3 variant polypeptides of the present invention.
For example, such modifications can include chemical derivatization
of polypeptides, e.g., acetylation, amidation, carboxylation, etc.
Such modifications can also include modifications of glycosylation,
e.g. those made by modifying the glycosylation patterns of a
polypeptide during its synthesis and processing or in further
processing steps; e.g. by exposing the polypeptide to enzymes which
affect glycosylation, such as mammalian glycosylating or
deglycosylating enzymes. In some embodiments, AXL, MER or Tyro3
polypeptide variants of the present invention include AXL, MER or
Tyro3 variant polypeptides having phosphorylated amino acid
residues, e.g. phosphotyrosine, phosphoserine, or
phosphothreonine.
[0172] In some other embodiments, AXL, MER or Tyro3 variant
polypeptides of the present invention include AXL, MER or Tyro3
variant polypeptides further modified to improve their resistance
to proteolytic degradation or to optimize solubility properties or
to render them more suitable as a therapeutic agent. For example,
AXL, MER or Tyro3 polypeptide variants of the present invention
further include analogs of AXL, MER or Tyro3 variant polypeptides
containing residues other than naturally occurring L-amino acids,
e.g. D-amino acids or non-naturally occurring synthetic amino
acids. D-amino acids may be substituted for some or all of the
amino acid residues.
[0173] In yet some other embodiments, AXL, MER or Tyro3 variant
polypeptides of the present invention include at least two same or
different AXL, MER or Tyro3 variant polypeptides linked covalently
or non-covalently. For example, in some embodiments, AXL, MER or
Tyro3 polypeptide variants of the present invention include two,
three, four, five, or six same or different AXL, MER or Tyro3
variant polypeptides linked covalently, e.g., so that they will
have the appropriate size, but avoiding unwanted aggregation.
[0174] According to the present invention, AXL, MER or Tyro3
variant polypeptides of the present invention can be produced by
any suitable means known or later discovered in the field, e.g.,
produced from eukaryotic or prokaryotic cells, synthesized in
vitro, etc. Where the protein is produced by prokaryotic cells, it
may be further processed by unfolding, e.g. heat denaturation, DTT
reduction, etc. and may be further refolded, using methods known in
the art.
[0175] The AXL, MER or Tyro3 variant polypeptides may be prepared
by in vitro synthesis, using conventional methods as known in the
art. Various commercial synthetic apparatuses are available, for
example, automated synthesizers by Applied Biosystems, Inc., Foster
City, Calif., Beckman, etc. By using synthesizers, naturally
occurring amino acids may be substituted with unnatural amino
acids. The particular sequence and the manner of preparation will
be determined by convenience, economics, purity required, and the
like.
[0176] The AXL, MER or Tyro3 variant polypeptides may also be
isolated and purified in accordance with conventional methods of
recombinant synthesis. A lysate may be prepared of the expression
host and the lysate purified using HPLC, exclusion chromatography,
gel electrophoresis, affinity chromatography, or other purification
technique. For the most part, the compositions which are used will
comprise at least 20% by weight of the desired product, more
usually at least about 75% by weight, preferably at least about 95%
by weight, and for therapeutic purposes, usually at least about
99.5% by weight, in relation to contaminants related to the method
of preparation of the product and its purification. Usually, the
percentages will be based upon total protein.
[0177] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing coding sequences
and appropriate transcriptional/translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination. Alternatively, RNA capable of encoding the
polypeptides of interest may be chemically synthesized. One of
skill in the art can readily utilize well-known codon usage tables
and synthetic methods to provide a suitable coding sequence for any
of the polypeptides of the invention. Direct chemical synthesis
methods include, for example, the phosphotriester method of Narang
et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method
of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the
diethylphosphoramidite method of Beaucage et al. (1981) Tetra.
Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No.
4,458,066. Chemical synthesis produces a single stranded
oligonucleotide. This can be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. While
chemical synthesis of DNA is often limited to sequences of about
100 bases, longer sequences can be obtained by the ligation of
shorter sequences. Alternatively, subsequences may be cloned and
the appropriate subsequences cleaved using appropriate restriction
enzymes.
[0178] The nucleic acids may be isolated and obtained in
substantial purity. Usually, the nucleic acids, either as DNA or
RNA, will be obtained substantially free of other
naturally-occurring nucleic acid sequences, generally being at
least about 50%, usually at least about 90% pure and are typically
"recombinant," e.g., flanked by one or more nucleotides with which
it is not normally associated on a naturally occurring chromosome.
The nucleic acids of the invention can be provided as a linear
molecule or within a circular molecule, and can be provided within
autonomously replicating molecules (vectors) or within molecules
without replication sequences. Expression of the nucleic acids can
be regulated by their own or by other regulatory sequences known in
the art. The nucleic acids of the invention can be introduced into
suitable host cells using a variety of techniques available in the
art, 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, protoplast fusion, viral infection,
electroporation, gene gun, calcium phosphate-mediated transfection,
and the like.
[0179] In some embodiments, the present invention provides
expression vectors for in vitro or in vivo expression of one or
more AXL, MER and/or Tyro3 polypeptide variants of the present
invention, either constitutively or under one or more regulatory
elements. In some embodiments, the present invention provides a
cell population comprising one or more expression vectors for
expressing AXL, MER and/or Tyro3 polypeptide variants of the
present invention, either constitutively or under one or more
regulatory elements.
[0180] According to another aspect of the invention, it provides
isolated antibodies or fragments thereof which specifically bind to
a GAS6 protein. GAS6 (growth arrest-specific 6) belongs
structurally to the family of plasma vitamin K-dependent proteins.
GAS6 has a high structural homology with the natural anticoagulant
protein S, sharing the same modular composition and having 40%
sequence identity. GAS6 has growth factor-like properties through
its interaction with receptor tyrosine kinases of the TAM family;
Tyro3, AXL and MER. Human GAS6 is a 678 amino acid protein that
consists of a gamma-carboxyglutamate (Gla)-rich domain that
mediates binding to phospholipid membranes, four epidermal growth
factor-like domains, and two laminin G-like (LG) domains. The
sequence of the transcript variants of human GAS6 may be accessed
at Genbank at NM_001143946.1; NM_001143945.1; and NM_000820.2,
respectively.
[0181] GAS6 employs a unique mechanism of action, interacting
through its vitamin K-dependent GLA (gamma-carboxyglutamic acid)
module with phosphatidylserine-containing membranes and through its
carboxy-terminal LamG domains with the TAM membrane receptors.
[0182] According to the present invention, isolated antibodies of
the present invention include any isolated antibodies with a
recognizable binding specificity against GAS6. In some embodiments,
isolated antibodies are partially or fully humanized antibodies. In
some other embodiments, isolated antibodies are monoclonal or
polyclonal antibodies. In yet some other embodiments, isolated
antibodies are chimeric antibodies, e.g., with consistent regions,
variable regions and/or CDR3 or a combination thereof from
different sources. In yet some other embodiments, isolated
antibodies are a combination of various features described
herein.
[0183] According to the present invention, fragments of the
isolated antibodies of the present invention include a polypeptide
containing a region of the antibody (either in the context of an
antibody scaffold or a non-antibody scaffold) that is sufficient or
necessary for a recognizable specific binding of the polypeptide
towards GAS6. In some embodiments, fragments of the isolated
antibodies of the present invention include variable light chains,
variable heavy chains, one or more CDRs of heavy chains or light
chains or combinations thereof, e.g., Fab, Fv, etc. In some
embodiments, fragments of the isolated antibodies of the present
invention include a polypeptide containing a single chain antibody,
e.g., ScFv. In yet some embodiments, fragments of the isolated
antibodies of the present invention include variable regions only
or variable regions in combination with part of Fc region, e.g.,
CH1 region. In still some embodiments, fragments of the isolated
antibodies of the present invention include minibodies, e.g.,
VL-VH-CH3 or diabodies.
[0184] In some embodiments, isolated antibodies of the present
invention bind to an epitope comprised in or presented by one or
more amino acid regions that interact with AXL, MER and/or Tyro3.
In some other embodiments, isolated antibodies of the present
invention bind to an epitope comprised in or presented by one or
more amino acid regions of GAS6, e.g., L295-T317, E356-P372,
R389-N396, D398-A406, E413-H429, and W450-M468 of GAS6.
[0185] In yet some other embodiments, isolated antibodies of the
present invention bind to an epitope comprised in or presented by
one or more amino acid regions, e.g., LRMFSGTPVIRLRFKRLQPT (SEQ ID
NO: 4), EIVGRVTSSGP (SEQ ID NO: 5), RNLVIKVN (SEQ ID NO: 6),
DAVMKIAVA (SEQ ID NO: 7), ERGLYHLNLTVGIPFH (SEQ ID NO: 8), and
WLNGEDTTIQETVVNRM (SEQ ID NO: 9).
[0186] In yet some other embodiments, isolated antibodies of the
present invention bind to an epitope comprised in or presented by
at least one, two, three, four, five, or six amino acids in a
region of L295-T317, E356-P372, R389-N396, D398-A406, E413-H429,
and W450-M468 of GAS6. In yet some other embodiments, isolated
antibodies of the present invention bind to an epitope comprised in
or presented by at least one, two, three, four, five or six amino
acids in a region of LRMFSGTPVIRLRFKRLQPT (SEQ ID NO:4),
EIVGRVTSSGP (SEQ ID NO:5), RNLVIKVN (SEQ ID NO: 6), DAVMKIAVA (SEQ
ID NO: 7), ERGLYHLNLTVGIPFH (SEQ ID NO: 8), and WLNGEDTTIQETVVNRM
(SEQ ID NO: 9).
[0187] In still some other embodiments, isolated antibodies of the
present invention is capable of inhibiting, inhibits or competes
with the binding between wild-type AXL, MER and/or Tyro3 or AXL,
MER and/or Tyro3 polypeptide variants of the present invention and
GAS6.
[0188] According to the present invention, the AXL, MER or Tyro3
variant polypeptides and isolated antibodies of the present
invention can be provided in pharmaceutical compositions suitable
for therapeutic use, e.g., for human treatment. In some
embodiments, pharmaceutical compositions of the present invention
include one or more therapeutic entities of the present invention,
e.g., AXL polypeptide variants and/or isolated antibodies against
GAS6 or pharmaceutically acceptable salts, esters or solvates
thereof or any prodrug thereof. In some other embodiments,
pharmaceutical compositions of the present invention include one or
more therapeutic entities of the present invention in combination
with another cytotoxic agent, e.g., another anti-tumor agent. In
yet some other embodiments, pharmaceutical compositions of the
present invention include one or more therapeutic entities of the
present invention in combination with another pharmaceutically
acceptable excipient.
[0189] In still some other embodiments, therapeutic entities of the
present invention are often administered as pharmaceutical
compositions comprising an active therapeutic agent, i.e., and a
variety of other pharmaceutically acceptable components. (See
Remington's Pharmaceutical Science, 15.sup.th ed., Mack Publishing
Company, Easton, Pa., 1980). The preferred form depends on the
intended mode of administration and therapeutic application. The
compositions can also include, depending on the formulation
desired, pharmaceutically-acceptable, non-toxic carriers or
diluents, which are defined as vehicles commonly used to formulate
pharmaceutical compositions for animal or human administration. The
diluent is selected so as not to affect the biological activity of
the combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0190] In still some other embodiments, pharmaceutical compositions
of the present invention can also include large, slowly metabolized
macromolecules such as proteins, polysaccharides such as chitosan,
polylactic acids, polyglycolic acids and copolymers (such as latex
functionalized Sepharose.TM., agarose, cellulose, and the like),
polymeric amino acids, amino acid copolymers, and lipid aggregates
(such as oil droplets or liposomes). Additionally, these carriers
can function as immunostimulating agents (i.e., adjuvants).
[0191] According to yet another aspect of the invention, it
provides methods for treating, reducing or preventing tumor
metastasis or tumor invasion by inhibiting the AXL, MER or Tyro3
signaling pathway and/or GAS6 signaling pathway. In some
embodiments, methods of the present invention include inhibiting
the activity of AXL, MER, Tyro3 and/or GAS6, or the interaction
between AXL, MER and/or Tyro3 and GAS6. For example, the activity
of AXL, MER, Tyro3 and/or GAS6 can be inhibited at the gene
expression level, mRNA processing level, translation level,
post-translation level, protein activation level, etc. In some
other examples, the activity of AXL, MER, Tyro3 or GAS6 can be
inhibited by small molecules, biological molecules, e.g.,
polypeptides, polynucleotides, antibodies, antibody drug
conjugates, etc. In some other examples, the activity of AXL, MER,
Tyro3 or GAS6 can be inhibited by one or more AXL, MER or Tyro3
variant polypeptides or isolated antibodies of the present
invention.
[0192] In yet other embodiments, methods of the present invention
include administering to a subject in need of treatment a
therapeutically effective amount or an effective dose of a
therapeutic entity (e.g., inhibitor agent) of the present
invention, e.g., an inhibitor of AXL, MER and/or Tyro3 activity or
GAS6 activity or an inhibitor of interaction between AXL, MER
and/or Tyro3 and GAS6. In some embodiments, effective doses of the
therapeutic entity of the present invention, e.g. for the treatment
of metastatic cancer, described herein vary depending upon many
different factors, including means of administration, target site,
physiological state of the patient, whether the patient is human or
an animal, other medications administered, and whether treatment is
prophylactic or therapeutic. Usually, the patient is a human but
nonhuman mammals including transgenic mammals can also be treated.
Treatment dosages need to be titrated to optimize safety and
efficacy.
[0193] In some embodiments, the dosage may range from about 0.0001
to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body
weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg
body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per every two weeks or
once a month or once every 3 to 6 months. Therapeutic entities of
the present invention are usually administered on multiple
occasions. Intervals between single dosages can be weekly, monthly
or yearly. Intervals can also be irregular as indicated by
measuring blood levels of the therapeutic entity in the patient.
Alternatively, therapeutic entities of the present invention can be
administered as a sustained release formulation, in which case less
frequent administration is required. Dosage and frequency vary
depending on the half-life of the polypeptide in the patient.
[0194] In prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period
of time. Some patients continue to receive treatment for the rest
of their lives. In therapeutic applications, a relatively high
dosage at relatively short intervals is sometimes required until
progression of the disease is reduced or terminated, and preferably
until the patient shows partial or complete amelioration of
symptoms of disease. Thereafter, the patent can be administered a
prophylactic regime.
[0195] In still other embodiments, methods of the present invention
include treating, reducing or preventing tumor metastasis or tumor
invasion of ovarian cancer, breast cancer, lung cancer, liver
cancer, colon cancer, gallbladder cancer, pancreatic cancer,
prostate cancer, and/or glioblastoma.
[0196] In still yet some other embodiments, for prophylactic
applications, pharmaceutical compositions or medicaments are
administered to a patient susceptible to, or otherwise at risk of a
disease or condition in an amount sufficient to eliminate or reduce
the risk, lessen the severity, or delay the outset of the disease,
including biochemical, histologic and/or behavioral symptoms of the
disease, its complications and intermediate pathological phenotypes
presenting during development of the disease.
[0197] In still yet some other embodiments, for therapeutic
applications, therapeutic entities of the present invention are
administered to a patient suspected of, 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
intermediate pathological phenotypes in development of the disease.
An amount adequate to accomplish therapeutic or prophylactic
treatment is defined as a therapeutically- or
prophylactically-effective dose. In both prophylactic and
therapeutic regimes, agents are usually administered in several
dosages until a sufficient response has been achieved. Typically,
the response is monitored and repeated dosages are given if there
is a recurrence of the cancer.
[0198] According to the present invention, compositions for the
treatment of metastatic cancer can be administered by parenteral,
topical, intravenous, intratumoral, oral, subcutaneous,
intraarterial, intracranial, intraperitoneal, intranasal or
intramuscular means. The most typical route of administration is
intravenous or intratumoral although other routes can be equally
effective.
[0199] For parenteral administration, 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 that can be a sterile liquid such as
water, oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. I n general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies and/or
polypeptides can be administered in the form of a depot injection
or implant preparation which can be formulated in such a manner as
to permit a sustained release of the active ingredient. An
exemplary composition comprises polypeptide at 1 mg/mL, formulated
in aqueous buffer consisting of 10 mM Tris, 210 mM sucrose, 51 mM
L-arginine, 0.01% polysorbate 20, adjusted to pH 7.4 with HCl or
NaOH.
[0200] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above. Langer, 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 preparation which can be formulated in such a manner as to
permit a sustained or pulsatile release of the active
ingredient.
[0201] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0202] For suppositories, binders and carriers include, for
example, polyalkylene glycols or triglycerides; such suppositories
can be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0203] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins.
Glenn et al., Nature 391: 851, 1998. Co-administration can be
achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein.
[0204] Alternatively, transdermal delivery can be achieved using a
skin patch or using transferosomes. Paul et al., Eur. J. Immunol.
25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368:
201-15, 1998.
[0205] The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
[0206] Preferably, a therapeutically effective dose of the antibody
compositions described herein will provide therapeutic benefit
without causing substantial toxicity.
[0207] Toxicity of the proteins described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. The data obtained from
these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the proteins described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1).
[0208] Also within the scope of the invention are kits comprising
the compositions (e.g., AXL, MER or Tyro3 variant polypeptides and
formulations thereof) of the invention and instructions for use.
The kit can further contain a least one additional reagent. Kits
typically include a label indicating the intended use of the
contents of the kit. The term label includes any writing, or
recorded material supplied on or with the kit, or which otherwise
accompanies the kit.
[0209] According to yet another aspect of the invention, it
provides methods for determining the ability of a tumor to undergo
tumor invasion and/or metastasis by detecting and/or determining
the level of AXL, MER and/or Tyro3 activity or GAS6 activity in a
biological sample from a subject of interest. In some embodiment,
the level of AXL, MER and/or Tyro3 activity or GAS6 activity is
measured by the level of mRNA expression, the level of protein
expression, the level of protein activation or any suitable
indicator corresponding to the activity of AXL, MER and/or Tyro3 or
GAS6 either directly or indirectly. In some embodiments, the level
of AXL, MER and/or Tyro3 activity or GAS6 activity in a biological
sample is further compared to a predetermined level, e.g., standard
level obtained by establishing normal levels or ranges of AXL, MER
and/or Tyro3 activity or GAS6 activity based on a population of
samples from tumors that do not develop tumor invasion or tumor
metastasis or from normal tissues. For example, an increase of AXL,
MER and/or Tyro3 activity or GAS6 activity over the predetermined
level or standard level is indicative of a predisposition of the
tumor to undergo tumor invasion or tumor metastasis.
[0210] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0211] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible. In the following, examples
will be described to illustrate parts of the invention. It is also
understood that the terminology used herein is for the purposes of
describing particular embodiments.
EXPERIMENTAL
Example 1--Affinities of Various AXL FC Constructs
[0212] FIG. 1 shows the four domains of AXL and the various
combinations of AXL Fc constructs made and tested.
[0213] The following AXL Fc constructs were made:
[0214] a. Full-length wild-type Fc fusion
[0215] b. Full-length AXL peptide 1 Fc fusion
[0216] c. AXL peptide 1 Fn(-) Fc fusion (this is the Fn-
construct)
[0217] d. Full-length AXL peptide 1 Fc fusion with minor GAS6
binding site knocked out
[0218] e. AXL peptide 1 Fn(-) Fc fusion, 3.times. gly4ser linker
between Fc and AXL
[0219] f. AXL peptide 1 Fn(-) Fc fusion, 5.times. gly4ser linker
between Fc and AXL
[0220] g. AXL peptide 1 A72V Fn(-) Fc fusion, 3.times. gly4ser
linker between Fc and AXL
[0221] The following Table 1 outlines the affinities of the above
constructs to GAS6, with wild-type AXL as a comparison.
TABLE-US-00001 TABLE 1 Construct Affinities AXL clone Fn domains Fc
Linker K.sub.d (pM) Wild-type Ig1 - None None 32.8 .+-. 0.63 AXL
peptide 1 Ig1 - None None 2.7 .+-. 0.05 (a) Wild-type + hIgG None
9.2 .+-. 0.17 (b) AXL peptide 1 + hIgG None 0.4 .+-. 0.01 (c) AXL
peptide 1 - hIgG None 2.6 .+-. 0.05 (d) AXL peptide 1 + hIgG None
2.6 .+-. 0.10 (-) minor site (e) AXL peptide 1 - hIgG 3x
gly.sub.4ser 1.2 .+-. 0.03 (SEQ ID NO: 10) (f) AXL peptide 1 - hIgG
5x gly.sub.4ser 1.2 .+-. 0.03 (SEQ ID NO: 10) (g) AXL peptide 1 -
hIgG 3x gly.sub.4ser 0.3 .+-. 0.00 A72V (SEQ ID NO: 10)
[0222] There are several conclusions that can be drawn from the
data set in Table 1 above.
[0223] Fc-fusion constructs provide enhancements in affinity over
the monomeric forms. For example: wild-type AXL Ig1 (monomeric) has
a .about.33 pM affinity, whereas wild-type Fc fusion has an
affinity of .about.9 pM and AXL peptide 1 Ig1 (monomeric) has an
affinity of .about.3 pM, whereas AXL peptide 1-Fc fusion has an
affinity of .about.0.4 pM
[0224] Significant affinity improvements for AXL peptide 1 over
wild-type AXL. In addition, AXL peptide 1 plus the A72V mutation
has a further enhancement in affinity, construct (e) compared to
construct (g), over wild-type AXL.
[0225] The mechanism of increasing the affinity in the Fc fusion
comes from multivalent binding to a single GAS6 molecule.
Specifically, one arm of the fusion binds to the major AXL binding
site while the other binds the minor. This conclusion was based on
the following experimental data: [0226] a. AXL peptide 1 Ig1
(monomer) has the same affinity as the AXL peptide 1 Fn(-) Fc
fusion, (c) in the table above. This suggests that simply having
two copies of AXL is insufficient for providing affinity
improvement. [0227] b. Full-length AXL peptide 1 with the minor
binding site removed has the same affinity as the monomer and the
Fn(-) fusion. This shows that the minor binding site has a
definitive role in the affinity improvements. [0228] c. The Fn(-)
constructs are arranged such that the minor binding site is
inaccessible to the large GAS6 molecule. The addition of linkers
between AXL and the Fc provide additional flexibility and space,
and with that a two-fold improvement in affinity is obtained. This
further supports the idea that the minor binding site is
important.
[0229] Overall, Example 1 shows that Fc fusions of the AXL ECD can
have improved affinity to GAS6 compared to wild-type AXL. While not
being bound by theory, one mechanism underlying this improved
affinity is simultaneous binding of one arm of the construct to the
major AXL binding site on GAS6 and the other arm to the minor AXL
binding site on GAS6.
Example 2--Affinities of Various AXL FC Constructs
[0230] Sequence: AXL peptide 2-Fc. The AXL peptide 2 includes amino
acids 1-131 of AXL, has the Ig2 domain deleted and has both the FN
domains deleted.
[0231] This construct includes amino acids 1-131 of AXL fused to
the human IgG1, with a single Gly4Ser (SEQ ID NO:10) linker
connecting the two domains (see FIG. 2). Additionally, constructs
can include various mutations in the AXL portion of the molecule,
as described by the present invention. For example, embodiments can
include having AXL peptide 1 Ig1 Fc fusion or AXL peptide 1 plus
A72V.
[0232] The affinity of AXL peptide 1 Ig1-Fc to human GAS6 has been
measured to be 1.7 +/-0.03 pM.
[0233] Overall, Example 2 shows that Fc fusions of the AXL Ig1
domain can have improved affinity to GAS6 compared to wild-type
AXL.
[0234] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or only and is not intended to limit the
scope of the present invention which will be limited only by the
appended claims.
[0235] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
appended claims.
TABLE-US-00002 LISTING OF SEQUENCES: SEQ ID NO: 1 is the amino acid
sequence of human wild type AXL
MAWRCPRMGRVPLAWCLALCGWACMAPRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWL
RDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLPY
FLEEPEDRTVAANTPFNLSCQAQGPPEPVDLLWLQDAVPLATAPGHGPQRSLHVPGLNKTSSFSCEAHNA
KGVTTSRTATITVLPQQPRNLHLVSRQPTELEVAWTPGLSGIYPLTHCTLQAVLSNDGMGIQAGEPDPPE
EPLTSQASVPPHQLRLGSLHPHTPYHIRVACTSSQGPSSWTHWLPVETPEGVPLGPPENISATRNGSQAF
VHWQEPRAPLQGTLLGYRLAYQGQDTPEVLMDIGLRQEVTLELQGDGSVSNLTVCVAAYTAAGDGPWSLP
VPLEAWRPGQAQPVHQLVKEPSTPAFSWPWWYVLLGAVVAAACVLILALFLVHRRKKETRYGEVFEPTVE
RGELVVRYRVRKSYSRRTTEATLNSLGISEELKEKLRDVMVDRHKVALGKTLGEGEFGAVMEGQLNQDDS
ILKVAVKTMKIAICTRSELEDFLSEAVCMKEFDHPNVMRLIGVCFQGSERESFPAPVVILPFMKHGDLHS
FLLYSRLGDQPVYLPTQMLVKFMADIASGMEYLSTKRFIHRDLAARNCMLNENMSVCVADFGLSKKIYNG
DYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWSFGVTMWEIATRGQTPYPGVENSEIYDYLRQGNRLKQ
PADCLDGLYALMSRCWELNPQDRPSFTELREDLENTLKALPPAQEPDEILYVNMDEGGGYPEPPGAAGGA
DPPTQPDPKDSCSCLTAAEVHPAGRYVLCPSTTPSPAQPADRGSPAAPGQEDGA SEQ ID NO: 2
is the amino acid sequence of human wild type MER
MGPAPLPLLLGLFLPALWRRAITEAREEAKPYPLFPGPFPGSLQTDHTPLLSLPHASGYQPALMFSPTQP
GRPHTGNVAIPQVTSVESKPLPPLAFKHTVGHIILSEHKGVKFNCSISVPNIYQDTTISWWKDGKELLGA
HHAITQFYPDDEVTAIIASFSITSVQRSDNGSYICKMKINNEEIVSDPIYIEVQGLPHFTKQPESMNVTR
NTAFNLTCQAVGPPEPVNIFWVQNSSRVNEQPEKSPSVLTVPGLTEMAVFSCEAHNDKGLTVSKGVQINI
KAIPSPPTEVSIRNSTAHSILISWVPGFDGYSPFRNCSIQVKEADPLSNGSVMIFNTSALPHLYQIKQLQ
ALANYSIGVSCMNEIGWSAVSPWILASTTEGAPSVAPLNVTVFLNESSDNVDIRWMKPPTKQQDGELVGY
RISHVWQSAGISKELLEEVGQNGSRARISVQVHNATCTVRIAAVTRGGVGPFSDPVKIFIPAHGWVDYAP
SSTPAPGNADPVLIIFGCFCGFILIGLILYISLAIRKRVQETKFGNAFTEEDSELVVNYIAKKSFCRRAI
ELTLHSLGVSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQRE
IEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTL
LKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIE
SLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTD
PLDRPTFSVLRLQLEKLLESLPDVRNQADVIYVNTQLLESSEGLAQGSTLAPLDLNIDPDSIIASCTPRA
AISVVTAEVHDSKPHEGRYILNGGSEEWEDLTSAPSAAVTAEKNSVLPGERLVRNGVSWSHSSMLPLGSS
LPDELLFADDSSEGSEVLM SEQ ID NO: 3 is the amino acid sequence of
human wild type Tyro3
MALRRSMGRPGLPPLPLPPPPRLGLLLAALASLLLPESAAAGLKLMGAPVKLTVSQGQPVKLNCSVEGME
EPDIQWVKDGAVVQNLDQLYIPVSEQHWIGFLSLKSVERSDAGRYWCQVEDGGETEISQPVWLTVEGVPF
FTVEPKDLAVPPNAPFQLSCEAVGPPEPVTIVWWRGTTKIGGPAPSPSVLNVTGVTQSTMFSCEAHNLKG
LASSRTATVHLQALPAAPFNITVTKLSSSNASVAWMPGADGRALLQSCTVQVTQAPGGWEVLAVVVPVPP
FTCLLRDLVPATNYSLRVRCANALGPSPYADWVPFQTKGLAPASAPQNLHAIRTDSGLILEWEEVIPEAP
LEGPLGPYKLSWVQDNGTQDELTVEGTRANLTGWDPQKDLIVRVCVSNAVGCGPWSQPLVVSSHDRAGQQ
GPPHSRTSWVPVVLGVLTALVTAAALALILLRKRRKETRFGQAFDSVMARGEPAVHFRAARSFNRERPER
IEATLDSLGISDELKEKLEDVLIPEQQFTLGRMLGKGEFGSVREAQLKQEDGSFVKVAVKMLKADIIASS
DIEEFLREAACMKEFDHPHVAKLVGVSLRSRAKGRLPIPMVILPFMKHGDLHAFLLASRIGENPFNLPLQ
TLIRFMVDIACGMEYLSSRNFIHRDLAARNCMLAEDMTVCVADFGLSRKIYSGDYYRQGCASKLPVKWLA
LESLADNLYTVQSDVWAFGVTMWEIMTRGQTPYAGIENAEIYNYLIGGNRLKQPPECMEDVYDLMYQCWS
ADPKQRPSFTCLRMELENILGQLSVLSASQDPLYINIERAEEPTAGGSLELPGRDQPYSGAGDGSGMGAV
GGTPSDCRYILTPGGLAEQPGQAEHQPESPLNETQRLLLLQQGLLPHSSC SEQ ID NO: 4 is
the amino acid sequence region L295-T317 of human GAS6
LRMFSGTPVIRLRFKRLQPT SEQ ID NO: 5 is the amino acid sequence region
E356-P372 of human GAS6 ElVGRVTSSGP SEQ ID NO: 6 is the amino acid
sequence region R389-N396 of human GAS6 RNLVIKVN SEQ ID NO: 7 is
the amino acid sequence region D398-A406 of human GAS6 DAVMKIAVA
SEQ ID NO: 8 is the amino acid sequence region E413-H429 of human
GAS6 ERGLYHLNLTVGIPFH SEQ ID NO: 9 is the amino acid sequence
region W450-M468 of human GAS6 WLNGEDTTIQETVVNRM SEQ ID NO: 10 is
the (GLY).sub.4SER linker sequence GGGGS
Sequence CWU 1
1
101887PRTH. sapiens 1Met Gly Arg Val Pro Leu Ala Trp Cys Leu Ala
Leu Cys Gly Trp Ala1 5 10 15Cys Met Ala Pro Arg Gly Thr Gln Ala Glu
Glu Ser Pro Phe Val Gly 20 25 30Asn Pro Gly Asn Ile Thr Gly Ala Arg
Gly Leu Thr Gly Thr Leu Arg 35 40 45Cys Gln Leu Gln Val Gln Gly Glu
Pro Pro Glu Val His Trp Leu Arg 50 55 60Asp Gly Gln Ile Leu Glu Leu
Ala Asp Ser Thr Gln Thr Gln Val Pro65 70 75 80Leu Gly Glu Asp Glu
Gln Asp Asp Trp Ile Val Val Ser Gln Leu Arg 85 90 95Ile Thr Ser Leu
Gln Leu Ser Asp Thr Gly Gln Tyr Gln Cys Leu Val 100 105 110Phe Leu
Gly His Gln Thr Phe Val Ser Gln Pro Gly Tyr Val Gly Leu 115 120
125Glu Gly Leu Pro Tyr Phe Leu Glu Glu Pro Glu Asp Arg Thr Val Ala
130 135 140Ala Asn Thr Pro Phe Asn Leu Ser Cys Gln Ala Gln Gly Pro
Pro Glu145 150 155 160Pro Val Asp Leu Leu Trp Leu Gln Asp Ala Val
Pro Leu Ala Thr Ala 165 170 175Pro Gly His Gly Pro Gln Arg Ser Leu
His Val Pro Gly Leu Asn Lys 180 185 190Thr Ser Ser Phe Ser Cys Glu
Ala His Asn Ala Lys Gly Val Thr Thr 195 200 205Ser Arg Thr Ala Thr
Ile Thr Val Leu Pro Gln Gln Pro Arg Asn Leu 210 215 220His Leu Val
Ser Arg Gln Pro Thr Glu Leu Glu Val Ala Trp Thr Pro225 230 235
240Gly Leu Ser Gly Ile Tyr Pro Leu Thr His Cys Thr Leu Gln Ala Val
245 250 255Leu Ser Asn Asp Gly Met Gly Ile Gln Ala Gly Glu Pro Asp
Pro Pro 260 265 270Glu Glu Pro Leu Thr Ser Gln Ala Ser Val Pro Pro
His Gln Leu Arg 275 280 285Leu Gly Ser Leu His Pro His Thr Pro Tyr
His Ile Arg Val Ala Cys 290 295 300Thr Ser Ser Gln Gly Pro Ser Ser
Trp Thr His Trp Leu Pro Val Glu305 310 315 320Thr Pro Glu Gly Val
Pro Leu Gly Pro Pro Glu Asn Ile Ser Ala Thr 325 330 335Arg Asn Gly
Ser Gln Ala Phe Val His Trp Gln Glu Pro Arg Ala Pro 340 345 350Leu
Gln Gly Thr Leu Leu Gly Tyr Arg Leu Ala Tyr Gln Gly Gln Asp 355 360
365Thr Pro Glu Val Leu Met Asp Ile Gly Leu Arg Gln Glu Val Thr Leu
370 375 380Glu Leu Gln Gly Asp Gly Ser Val Ser Asn Leu Thr Val Cys
Val Ala385 390 395 400Ala Tyr Thr Ala Ala Gly Asp Gly Pro Trp Ser
Leu Pro Val Pro Leu 405 410 415Glu Ala Trp Arg Pro Gly Gln Ala Gln
Pro Val His Gln Leu Val Lys 420 425 430Glu Pro Ser Thr Pro Ala Phe
Ser Trp Pro Trp Trp Tyr Val Leu Leu 435 440 445Gly Ala Val Val Ala
Ala Ala Cys Val Leu Ile Leu Ala Leu Phe Leu 450 455 460Val His Arg
Arg Lys Lys Glu Thr Arg Tyr Gly Glu Val Phe Glu Pro465 470 475
480Thr Val Glu Arg Gly Glu Leu Val Val Arg Tyr Arg Val Arg Lys Ser
485 490 495Tyr Ser Arg Arg Thr Thr Glu Ala Thr Leu Asn Ser Leu Gly
Ile Ser 500 505 510Glu Glu Leu Lys Glu Lys Leu Arg Asp Val Met Val
Asp Arg His Lys 515 520 525Val Ala Leu Gly Lys Thr Leu Gly Glu Gly
Glu Phe Gly Ala Val Met 530 535 540Glu Gly Gln Leu Asn Gln Asp Asp
Ser Ile Leu Lys Val Ala Val Lys545 550 555 560Thr Met Lys Ile Ala
Ile Cys Thr Arg Ser Glu Leu Glu Asp Phe Leu 565 570 575Ser Glu Ala
Val Cys Met Lys Glu Phe Asp His Pro Asn Val Met Arg 580 585 590Leu
Ile Gly Val Cys Phe Gln Gly Ser Glu Arg Glu Ser Phe Pro Ala 595 600
605Pro Val Val Ile Leu Pro Phe Met Lys His Gly Asp Leu His Ser Phe
610 615 620Leu Leu Tyr Ser Arg Leu Gly Asp Gln Pro Val Tyr Leu Pro
Thr Gln625 630 635 640Met Leu Val Lys Phe Met Ala Asp Ile Ala Ser
Gly Met Glu Tyr Leu 645 650 655Ser Thr Lys Arg Phe Ile His Arg Asp
Leu Ala Ala Arg Asn Cys Met 660 665 670Leu Asn Glu Asn Met Ser Val
Cys Val Ala Asp Phe Gly Leu Ser Lys 675 680 685Lys Ile Tyr Asn Gly
Asp Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met 690 695 700Pro Val Lys
Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr705 710 715
720Ser Lys Ser Asp Val Trp Ser Phe Gly Val Thr Met Trp Glu Ile Ala
725 730 735Thr Arg Gly Gln Thr Pro Tyr Pro Gly Val Glu Asn Ser Glu
Ile Tyr 740 745 750Asp Tyr Leu Arg Gln Gly Asn Arg Leu Lys Gln Pro
Ala Asp Cys Leu 755 760 765Asp Gly Leu Tyr Ala Leu Met Ser Arg Cys
Trp Glu Leu Asn Pro Gln 770 775 780Asp Arg Pro Ser Phe Thr Glu Leu
Arg Glu Asp Leu Glu Asn Thr Leu785 790 795 800Lys Ala Leu Pro Pro
Ala Gln Glu Pro Asp Glu Ile Leu Tyr Val Asn 805 810 815Met Asp Glu
Gly Gly Gly Tyr Pro Glu Pro Pro Gly Ala Ala Gly Gly 820 825 830Ala
Asp Pro Pro Thr Gln Pro Asp Pro Lys Asp Ser Cys Ser Cys Leu 835 840
845Thr Ala Ala Glu Val His Pro Ala Gly Arg Tyr Val Leu Cys Pro Ser
850 855 860Thr Thr Pro Ser Pro Ala Gln Pro Ala Asp Arg Gly Ser Pro
Ala Ala865 870 875 880Pro Gly Gln Glu Asp Gly Ala 8852999PRTH.
sapiens 2Met Gly Pro Ala Pro Leu Pro Leu Leu Leu Gly Leu Phe Leu
Pro Ala1 5 10 15Leu Trp Arg Arg Ala Ile Thr Glu Ala Arg Glu Glu Ala
Lys Pro Tyr 20 25 30Pro Leu Phe Pro Gly Pro Phe Pro Gly Ser Leu Gln
Thr Asp His Thr 35 40 45Pro Leu Leu Ser Leu Pro His Ala Ser Gly Tyr
Gln Pro Ala Leu Met 50 55 60Phe Ser Pro Thr Gln Pro Gly Arg Pro His
Thr Gly Asn Val Ala Ile65 70 75 80Pro Gln Val Thr Ser Val Glu Ser
Lys Pro Leu Pro Pro Leu Ala Phe 85 90 95Lys His Thr Val Gly His Ile
Ile Leu Ser Glu His Lys Gly Val Lys 100 105 110Phe Asn Cys Ser Ile
Ser Val Pro Asn Ile Tyr Gln Asp Thr Thr Ile 115 120 125Ser Trp Trp
Lys Asp Gly Lys Glu Leu Leu Gly Ala His His Ala Ile 130 135 140Thr
Gln Phe Tyr Pro Asp Asp Glu Val Thr Ala Ile Ile Ala Ser Phe145 150
155 160Ser Ile Thr Ser Val Gln Arg Ser Asp Asn Gly Ser Tyr Ile Cys
Lys 165 170 175Met Lys Ile Asn Asn Glu Glu Ile Val Ser Asp Pro Ile
Tyr Ile Glu 180 185 190Val Gln Gly Leu Pro His Phe Thr Lys Gln Pro
Glu Ser Met Asn Val 195 200 205Thr Arg Asn Thr Ala Phe Asn Leu Thr
Cys Gln Ala Val Gly Pro Pro 210 215 220Glu Pro Val Asn Ile Phe Trp
Val Gln Asn Ser Ser Arg Val Asn Glu225 230 235 240Gln Pro Glu Lys
Ser Pro Ser Val Leu Thr Val Pro Gly Leu Thr Glu 245 250 255Met Ala
Val Phe Ser Cys Glu Ala His Asn Asp Lys Gly Leu Thr Val 260 265
270Ser Lys Gly Val Gln Ile Asn Ile Lys Ala Ile Pro Ser Pro Pro Thr
275 280 285Glu Val Ser Ile Arg Asn Ser Thr Ala His Ser Ile Leu Ile
Ser Trp 290 295 300Val Pro Gly Phe Asp Gly Tyr Ser Pro Phe Arg Asn
Cys Ser Ile Gln305 310 315 320Val Lys Glu Ala Asp Pro Leu Ser Asn
Gly Ser Val Met Ile Phe Asn 325 330 335Thr Ser Ala Leu Pro His Leu
Tyr Gln Ile Lys Gln Leu Gln Ala Leu 340 345 350Ala Asn Tyr Ser Ile
Gly Val Ser Cys Met Asn Glu Ile Gly Trp Ser 355 360 365Ala Val Ser
Pro Trp Ile Leu Ala Ser Thr Thr Glu Gly Ala Pro Ser 370 375 380Val
Ala Pro Leu Asn Val Thr Val Phe Leu Asn Glu Ser Ser Asp Asn385 390
395 400Val Asp Ile Arg Trp Met Lys Pro Pro Thr Lys Gln Gln Asp Gly
Glu 405 410 415Leu Val Gly Tyr Arg Ile Ser His Val Trp Gln Ser Ala
Gly Ile Ser 420 425 430Lys Glu Leu Leu Glu Glu Val Gly Gln Asn Gly
Ser Arg Ala Arg Ile 435 440 445Ser Val Gln Val His Asn Ala Thr Cys
Thr Val Arg Ile Ala Ala Val 450 455 460Thr Arg Gly Gly Val Gly Pro
Phe Ser Asp Pro Val Lys Ile Phe Ile465 470 475 480Pro Ala His Gly
Trp Val Asp Tyr Ala Pro Ser Ser Thr Pro Ala Pro 485 490 495Gly Asn
Ala Asp Pro Val Leu Ile Ile Phe Gly Cys Phe Cys Gly Phe 500 505
510Ile Leu Ile Gly Leu Ile Leu Tyr Ile Ser Leu Ala Ile Arg Lys Arg
515 520 525Val Gln Glu Thr Lys Phe Gly Asn Ala Phe Thr Glu Glu Asp
Ser Glu 530 535 540Leu Val Val Asn Tyr Ile Ala Lys Lys Ser Phe Cys
Arg Arg Ala Ile545 550 555 560Glu Leu Thr Leu His Ser Leu Gly Val
Ser Glu Glu Leu Gln Asn Lys 565 570 575Leu Glu Asp Val Val Ile Asp
Arg Asn Leu Leu Ile Leu Gly Lys Ile 580 585 590Leu Gly Glu Gly Glu
Phe Gly Ser Val Met Glu Gly Asn Leu Lys Gln 595 600 605Glu Asp Gly
Thr Ser Leu Lys Val Ala Val Lys Thr Met Lys Leu Asp 610 615 620Asn
Ser Ser Gln Arg Glu Ile Glu Glu Phe Leu Ser Glu Ala Ala Cys625 630
635 640Met Lys Asp Phe Ser His Pro Asn Val Ile Arg Leu Leu Gly Val
Cys 645 650 655Ile Glu Met Ser Ser Gln Gly Ile Pro Lys Pro Met Val
Ile Leu Pro 660 665 670Phe Met Lys Tyr Gly Asp Leu His Thr Tyr Leu
Leu Tyr Ser Arg Leu 675 680 685Glu Thr Gly Pro Lys His Ile Pro Leu
Gln Thr Leu Leu Lys Phe Met 690 695 700Val Asp Ile Ala Leu Gly Met
Glu Tyr Leu Ser Asn Arg Asn Phe Leu705 710 715 720His Arg Asp Leu
Ala Ala Arg Asn Cys Met Leu Arg Asp Asp Met Thr 725 730 735Val Cys
Val Ala Asp Phe Gly Leu Ser Lys Lys Ile Tyr Ser Gly Asp 740 745
750Tyr Tyr Arg Gln Gly Arg Ile Ala Lys Met Pro Val Lys Trp Ile Ala
755 760 765Ile Glu Ser Leu Ala Asp Arg Val Tyr Thr Ser Lys Ser Asp
Val Trp 770 775 780Ala Phe Gly Val Thr Met Trp Glu Ile Ala Thr Arg
Gly Met Thr Pro785 790 795 800Tyr Pro Gly Val Gln Asn His Glu Met
Tyr Asp Tyr Leu Leu His Gly 805 810 815His Arg Leu Lys Gln Pro Glu
Asp Cys Leu Asp Glu Leu Tyr Glu Ile 820 825 830Met Tyr Ser Cys Trp
Arg Thr Asp Pro Leu Asp Arg Pro Thr Phe Ser 835 840 845Val Leu Arg
Leu Gln Leu Glu Lys Leu Leu Glu Ser Leu Pro Asp Val 850 855 860Arg
Asn Gln Ala Asp Val Ile Tyr Val Asn Thr Gln Leu Leu Glu Ser865 870
875 880Ser Glu Gly Leu Ala Gln Gly Ser Thr Leu Ala Pro Leu Asp Leu
Asn 885 890 895Ile Asp Pro Asp Ser Ile Ile Ala Ser Cys Thr Pro Arg
Ala Ala Ile 900 905 910Ser Val Val Thr Ala Glu Val His Asp Ser Lys
Pro His Glu Gly Arg 915 920 925Tyr Ile Leu Asn Gly Gly Ser Glu Glu
Trp Glu Asp Leu Thr Ser Ala 930 935 940Pro Ser Ala Ala Val Thr Ala
Glu Lys Asn Ser Val Leu Pro Gly Glu945 950 955 960Arg Leu Val Arg
Asn Gly Val Ser Trp Ser His Ser Ser Met Leu Pro 965 970 975Leu Gly
Ser Ser Leu Pro Asp Glu Leu Leu Phe Ala Asp Asp Ser Ser 980 985
990Glu Gly Ser Glu Val Leu Met 9953890PRTH. sapiens 3Met Ala Leu
Arg Arg Ser Met Gly Arg Pro Gly Leu Pro Pro Leu Pro1 5 10 15Leu Pro
Pro Pro Pro Arg Leu Gly Leu Leu Leu Ala Ala Leu Ala Ser 20 25 30Leu
Leu Leu Pro Glu Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala 35 40
45Pro Val Lys Leu Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys
50 55 60Ser Val Glu Gly Met Glu Glu Pro Asp Ile Gln Trp Val Lys Asp
Gly65 70 75 80Ala Val Val Gln Asn Leu Asp Gln Leu Tyr Ile Pro Val
Ser Glu Gln 85 90 95His Trp Ile Gly Phe Leu Ser Leu Lys Ser Val Glu
Arg Ser Asp Ala 100 105 110Gly Arg Tyr Trp Cys Gln Val Glu Asp Gly
Gly Glu Thr Glu Ile Ser 115 120 125Gln Pro Val Trp Leu Thr Val Glu
Gly Val Pro Phe Phe Thr Val Glu 130 135 140Pro Lys Asp Leu Ala Val
Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys145 150 155 160Glu Ala Val
Gly Pro Pro Glu Pro Val Thr Ile Val Trp Trp Arg Gly 165 170 175Thr
Thr Lys Ile Gly Gly Pro Ala Pro Ser Pro Ser Val Leu Asn Val 180 185
190Thr Gly Val Thr Gln Ser Thr Met Phe Ser Cys Glu Ala His Asn Leu
195 200 205Lys Gly Leu Ala Ser Ser Arg Thr Ala Thr Val His Leu Gln
Ala Leu 210 215 220Pro Ala Ala Pro Phe Asn Ile Thr Val Thr Lys Leu
Ser Ser Ser Asn225 230 235 240Ala Ser Val Ala Trp Met Pro Gly Ala
Asp Gly Arg Ala Leu Leu Gln 245 250 255Ser Cys Thr Val Gln Val Thr
Gln Ala Pro Gly Gly Trp Glu Val Leu 260 265 270Ala Val Val Val Pro
Val Pro Pro Phe Thr Cys Leu Leu Arg Asp Leu 275 280 285Val Pro Ala
Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn Ala Leu 290 295 300Gly
Pro Ser Pro Tyr Ala Asp Trp Val Pro Phe Gln Thr Lys Gly Leu305 310
315 320Ala Pro Ala Ser Ala Pro Gln Asn Leu His Ala Ile Arg Thr Asp
Ser 325 330 335Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Ala
Pro Leu Glu 340 345 350Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val
Gln Asp Asn Gly Thr 355 360 365Gln Asp Glu Leu Thr Val Glu Gly Thr
Arg Ala Asn Leu Thr Gly Trp 370 375 380Asp Pro Gln Lys Asp Leu Ile
Val Arg Val Cys Val Ser Asn Ala Val385 390 395 400Gly Cys Gly Pro
Trp Ser Gln Pro Leu Val Val Ser Ser His Asp Arg 405 410 415Ala Gly
Gln Gln Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 420 425
430Val Leu Gly Val Leu Thr Ala Leu Val Thr Ala Ala Ala Leu Ala Leu
435 440 445Ile Leu Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln
Ala Phe 450 455 460Asp Ser Val Met Ala Arg Gly Glu Pro Ala Val His
Phe Arg Ala Ala465 470 475 480Arg Ser Phe Asn Arg Glu Arg Pro Glu
Arg Ile Glu Ala Thr Leu Asp 485 490 495Ser Leu Gly Ile Ser Asp Glu
Leu Lys Glu Lys Leu Glu Asp Val Leu 500 505 510Ile Pro Glu Gln Gln
Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu 515 520 525Phe Gly Ser
Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe 530 535 540Val
Lys Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser545 550
555 560Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe
Asp 565 570 575His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg
Ser Arg Ala
580 585 590Lys Gly Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met
Lys His 595 600 605Gly Asp Leu His Ala Phe Leu Leu Ala Ser Arg Ile
Gly Glu Asn Pro 610 615 620Phe Asn Leu Pro Leu Gln Thr Leu Ile Arg
Phe Met Val Asp Ile Ala625 630 635 640Cys Gly Met Glu Tyr Leu Ser
Ser Arg Asn Phe Ile His Arg Asp Leu 645 650 655Ala Ala Arg Asn Cys
Met Leu Ala Glu Asp Met Thr Val Cys Val Ala 660 665 670Asp Phe Gly
Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln 675 680 685Gly
Cys Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu 690 695
700Ala Asp Asn Leu Tyr Thr Val Gln Ser Asp Val Trp Ala Phe Gly
Val705 710 715 720Thr Met Trp Glu Ile Met Thr Arg Gly Gln Thr Pro
Tyr Ala Gly Ile 725 730 735Glu Asn Ala Glu Ile Tyr Asn Tyr Leu Ile
Gly Gly Asn Arg Leu Lys 740 745 750Gln Pro Pro Glu Cys Met Glu Asp
Val Tyr Asp Leu Met Tyr Gln Cys 755 760 765Trp Ser Ala Asp Pro Lys
Gln Arg Pro Ser Phe Thr Cys Leu Arg Met 770 775 780Glu Leu Glu Asn
Ile Leu Gly Gln Leu Ser Val Leu Ser Ala Ser Gln785 790 795 800Asp
Pro Leu Tyr Ile Asn Ile Glu Arg Ala Glu Glu Pro Thr Ala Gly 805 810
815Gly Ser Leu Glu Leu Pro Gly Arg Asp Gln Pro Tyr Ser Gly Ala Gly
820 825 830Asp Gly Ser Gly Met Gly Ala Val Gly Gly Thr Pro Ser Asp
Cys Arg 835 840 845Tyr Ile Leu Thr Pro Gly Gly Leu Ala Glu Gln Pro
Gly Gln Ala Glu 850 855 860His Gln Pro Glu Ser Pro Leu Asn Glu Thr
Gln Arg Leu Leu Leu Leu865 870 875 880Gln Gln Gly Leu Leu Pro His
Ser Ser Cys 885 890420PRTH. sapiens 4Leu Arg Met Phe Ser Gly Thr
Pro Val Ile Arg Leu Arg Phe Lys Arg1 5 10 15Leu Gln Pro Thr
20510PRTH. sapiens 5Glu Val Gly Arg Val Thr Ser Ser Gly Pro1 5
1068PRTH. sapiens 6Arg Asn Leu Val Ile Lys Val Asn1 579PRTH.
sapiens 7Asp Ala Val Met Lys Ile Ala Val Ala1 5816PRTH. sapiens
8Glu Arg Gly Leu Tyr His Leu Asn Leu Thr Val Gly Ile Pro Phe His1 5
10 15917PRTH. sapiens 9Trp Leu Asn Gly Glu Asp Thr Thr Ile Gln Glu
Thr Val Val Asn Arg1 5 10 15Met105PRTArtificial SequenceSynthetic
Linker 10Gly Gly Gly Gly Ser1 5
* * * * *