U.S. patent application number 14/750368 was filed with the patent office on 2016-03-24 for compositions comprising antibodies to lingo or fragments thereof.
The applicant listed for this patent is Biogen MA Inc.. Invention is credited to Christilyn Graff, Sha Mi, R. Blake Pepinsky.
Application Number | 20160083468 14/750368 |
Document ID | / |
Family ID | 41170082 |
Filed Date | 2016-03-24 |
United States Patent
Application |
20160083468 |
Kind Code |
A1 |
Mi; Sha ; et al. |
March 24, 2016 |
Compositions Comprising Antibodies to Lingo or Fragments
Thereof
Abstract
Endogenous LINGO-1 is a negative regulator for neuronal
survival, axon regeneration, oligodendrocyte differentiation and
myelination. Molecules that block endogenous LINGO-1 function, such
anti-LINGO-1 antibodies can be used as therapeutics for the
treatment of neuron and oligodendrocyte dysfunction. The present
invention provides antibodies specific for LINGO-1, and methods of
using such antibodies as antagonists of endogenous LINGO-1
function. The invention further provides specific hybridoma and
phage library-derived monoclonal antibodies, nucleic acids encoding
these antibodies, and vectors and host cells comprising these
antibodies. The invention further provides methods of promoting
oligodendrocyte survival and myelination in a vertebrate,
comprising administering to a vertebrate in need of such treatment
an effective amount of an anti-LINGO-1 antibody
Inventors: |
Mi; Sha; (Belmont, MA)
; Pepinsky; R. Blake; (Arlington, MA) ; Graff;
Christilyn; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen MA Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
41170082 |
Appl. No.: |
14/750368 |
Filed: |
June 25, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13802503 |
Mar 13, 2013 |
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14750368 |
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13243795 |
Sep 23, 2011 |
8425910 |
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13802503 |
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12500472 |
Jul 9, 2009 |
8058406 |
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13243795 |
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61079355 |
Jul 9, 2008 |
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Current U.S.
Class: |
424/172.1 ;
435/188; 435/252.3; 435/252.31; 435/252.33; 435/254.2; 435/254.21;
435/254.23; 435/334; 435/419; 435/69.6; 530/387.3; 530/389.1;
530/391.1; 530/391.7 |
Current CPC
Class: |
A61K 45/06 20130101;
C07K 2317/35 20130101; C07K 2317/515 20130101; C07K 2317/54
20130101; A61P 21/02 20180101; C07K 2317/71 20130101; A61P 25/14
20180101; C07K 2317/92 20130101; C07K 2317/565 20130101; A61K
47/6879 20170801; A61P 25/00 20180101; C07K 2317/41 20130101; C07K
16/2803 20130101; C07K 2317/51 20130101; A61K 2039/505 20130101;
C07K 2317/55 20130101; A61P 9/10 20180101; A61P 25/28 20180101;
A61P 25/16 20180101; C07K 2319/01 20130101; A61P 43/00 20180101;
A61K 47/60 20170801; A61K 39/3955 20130101; C07K 16/28 20130101;
C07K 2317/56 20130101; C07K 2317/94 20130101; C07K 2317/76
20130101; C07K 16/18 20130101; A61K 47/6849 20170801; C07K 2317/21
20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61K 47/48 20060101
A61K047/48 |
Claims
1-221. (canceled)
222. An isolated antibody or antigen-binding fragment thereof that
specifically binds to a human LINGO-1 polypeptide, wherein the
antibody or antigen-binding fragment comprises: a VH region
comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein
the CDR1 region and the CDR2 region of the VH region are identical
to the amino acid sequences of SEQ ID NO:6 and SEQ ID NO:7,
respectively, and the CDR3 region of the VH region is identical to
the amino acid sequence of SEQ ID NO:8, except for 1-5 amino acid
substitutions; a VL region comprising a CDR1 region, a CDR2 region,
and a CDR3 region, wherein the CDR1 region, the CDR2 region and the
CDR3 region of the VL region are identical to the amino acid
sequences of SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16,
respectively.
223. The antibody or antigen-binding fragment of claim 222, wherein
the VH CDR3 region is identical to an amino acid sequence selected
from the group consisting of SEQ ID NOs:35-49.
224. The antibody or antigen-binding fragment of claim 222, wherein
the VL region comprises the amino acid sequence of SEQ ID
NO:13.
225. The antibody or antigen-binding fragment of claim 222, wherein
the VH region comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs:71-85.
226. The antibody or antigen-binding fragment of claim 224, wherein
the VH region comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs:71-85.
227. The antibody or antigen-binding fragment of claim 222, further
comprising a heterologous polypeptide fused thereto.
228. The antibody or antigen-binding fragment of claim 222, wherein
the antibody or antigen-binding fragment is conjugated to an agent
selected from the group consisting of a therapeutic agent, a
prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a
biological response modifier, a pharmaceutical agent, and
polyethylene glycol.
229. The antibody or antigen-binding fragment of claim 222, wherein
the antibody or antigen-binding fragment is fused to a brain
targeting moiety.
230. The antibody or antigen-binding fragment of claim 229, wherein
the brain targeting moiety is selected from the group consisting
of: (i) the single domain antibody FC5, (ii) mAB 83-14, (iii) the
B2 peptide, (iv) the B6 peptide, (v) the B8 peptide, and (vi) the
OX26 monoclonal antibody.
231. A pharmaceutical composition comprising the antibody or
antigen-binding fragment of claim 222 and a pharmaceutically
acceptable carrier.
232. A host cell comprising: (a) two expression vectors, wherein
the first expression vector encodes the VH region of the antibody
or antigen-binding fragment of claim 222 and the second expression
vector encodes the VL region of the antibody or antigen-binding
fragment of claim 222; or (b) a single expression vector that
encodes both the VH region of the antibody or antigen-binding
fragment of claim 222 and the VL region of the antibody or
antigen-binding fragment of claim 222.
233. An in vitro method of producing an anti-LINGO-1 antibody or
antigen-binding fragment comprising culturing the host cell of
claim 232 in a culture, and recovering the antibody or
antigen-binding fragment from the culture.
234. A method of treating a CNS injury in a human subject in need
thereof, the method comprising administering to the subject the
antibody or antigen-binding fragment of claim 222.
235. The method of claim 234, wherein the CNS injury is multiple
sclerosis.
236. The method of claim 234, wherein the CNS injury is ischemic
optic neuropathy.
237. The method of claim 234, wherein the antibody or
antigen-binding fragment is co-administered with an additional
therapeutic agent for treatment of the CNS injury.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] Related applications U.S. Ser. No. 13/243,795, filed Sep.
23, 2011, U.S. Ser. No. 12/500,472, filed Jul. 9, 2009, and U.S.
61/079,355, filed Jul. 9, 2008 are herein incorporated by reference
in their entireties.
REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: sequence listing.ascii.txt, Size: 258 kilobytes; and Date of
Creation: Jul. 9, 2009) fled with the application is incorporated
by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates to neurology, neurobiology and
molecular biology. More particularly, this invention relates to
molecules and methods for treatment of neurological diseases,
disorders and injuries such as spinal cord injury.
[0005] 2. Background of the Invention
[0006] Axons and dendrites extend from neurons. The distal tip of
an extending axon or neurite includes a specialized region, known
as the growth cone. Growth cones sense the local environment and
guide axonal growth toward a neuron's target cell. Growth cones
respond to environmental cues, for example, surface adhesiveness,
growth factors, neurotransmitters and electric fields. The growth
cones generally advance at a rate of one to two millimeters per
day. The growth cone explores the area ahead of it and on either
side, by means of elongations classified as lamellipodia and
filopodia. When an elongation contacts an unfavorable surface, it
withdraws. When an elongation contacts a favorable growth surface,
it continues to extend and guides the growth cone in that
direction. When the growth cone reaches an appropriate target cell
a synaptic connection is created.
[0007] Nerve cell function is influenced by contact between neurons
and other cells in their immediate environment (Rutishauser, et
al., 1988, Physiol. Rev. 68:819). These cells include specialized
glial cells, oligodendrocytes in the central nervous system (CNS),
and Schwann cells in the peripheral nervous system (PNS), which
sheathe the neuronal axon with myelin (Lemke, 1992, in An
Introduction to Molecular Neurobiology, Z. Hall, Ed., p. 281,
Sinauer).
[0008] CNS neurons have the inherent potential to regenerate after
injury, but they are inhibited from doing so by inhibitory proteins
present in myelin (Brittis et al., 2001, Neuron 30:11-14; Jones et
al., 2002, J. Neurosci. 22:2792-2803; Grimpe et al., 2002, J.
Neurosci.:22:3144-3160).
[0009] Several myelin inhibitory proteins found on oligodendrocytes
have been characterized. Known examples of myelin inhibitory
proteins include NogoA (Chen at al., Nature, 2000, 403, 434-439;
Grandpre et al., Nature 2000, 403, 439-444), myelin associated
glycoprotein (MAG) (McKerracher et al., 1994, Neuron 13:805-811;
Mukhopadhyay et al., 1994, Neuron 13:757-767) and oligodendrocyte
glycoprotein (OM-gp), Mikol at al., 1988, J. Cell. Biol.
106:1273-1279). Each of these proteins has been separately shown to
be a ligand for the neuronal Nogo receptor-1 (NgR1 (Wang et al.,
Nature 2002, 417, 941-944; Grandpre et al, Nature 2000, 403,
439-444; Chen et al., Nature, 2000, 403, 434-439; Domeniconi et
al., Neuron 2002, published online Jun. 28, 2002).
[0010] Nogo receptor-1 (NgR1) is a GPI-anchored membrane protein
that contains 8 leucine rich repeats (Fournier et al., 2001, Nature
409:341-346). Upon interaction with inhibitory proteins (e.g.,
NogoA, MAG and OM-gp), the NgR1 complex transduces signals that
lead to growth cone collapse and inhibition of neurite
outgrowth.
[0011] There is an unmet need for molecules and methods for
inhibiting NgR1-mediated growth cone collapse and the resulting
inhibition of neurite outgrowth. Additionally there is a need for
molecules which increase neuronal survival and axon regeneration.
Particularly for the treatment of disease, disorders or injuries
which involve axonal injury, neuronal or oligodendrocyte cell
death, demyelination or dymyelination or generally relate to the
nervous system.
[0012] Such diseases, disorders or injuries include, but are not
limited to, multiple sclerosis (MS), progressive multifocal
leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine
myelolysis (CPM), adrenoleukodystrophy, Alexander's disease,
Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy
(Krabbe's disease) and Wallerian Degeneration, optic neuritis,
transverse myelitis, amylotrophic lateral sclerosis (ALS),
Huntington's disease, Alzheimer's disease, Parkinson's disease,
spinal cord injury, traumatic brain injury, post radiation injury,
neurologic complications of chemotherapy, stroke, acute ischemic
optic neuropathy, vitamin E deficiency, isolated vitamin E
deficiency syndrome, AR, Bassen-Kornzweig syndrome,
Marchiafava-Bignami syndrome, metachromatic leukodystrophy,
trigeminal neuralgia, and Bell's palsy. Among these diseases, MS is
the most widespread, affecting approximately 2.5 million people
worldwide.
[0013] MS generally begins with a relapsing-remitting pattern of
neurologic involvement, which then progresses to a chronic phase
with increasing neurological damage. MS is associated with the
destruction of myelin, oligodendrocytes and axons localized to
chronic lesions. The demyelination observed in MS is not always
permanent and remyelination has been documented in early stages of
the disease. Remyelination of neurons requires
oligodendrocytes.
[0014] Various disease-modifying treatments are available for MS,
including the use of corticosteroids and immunomodulators such as
interferon beta and Tysabri.RTM.. In addition, because of the
central role of oligodendrocytes and myelination in MS, there have
been efforts to develop therapies to increase oligodendrocyte
numbers or enhance myelination. See, e.g., Cohen at al., U.S. Pat.
No. 5,574,009; Chang et al., N. Engl. J. Med. 346:165-73 (2002).
However, there remains an urgent need to devise additional
therapies for MS and other demyelination and dismyelination
disorders.
BRIEF SUMMARY OF THE INVENTION
[0015] The present invention is based on the discovery that certain
LINGO-1 antibodies promote survival, proliferation and
differentiation of oligodendrocytes and neuronal cells, as well as
myelination of neurons. LINGO-1, previously called Sp35, has been
described in detail in International Applications
PCT/US2006/026271, filed Jul. 7, 2006, PCT/US2004/008323, filed
Mar. 17, 2004, PCT/US2005/022881, filed Jun. 24, 2005 and
PCT/US2008/000316, filed Jan. 9, 2008, each of which is
incorporated by reference in its entirety herein. Based on these
discoveries, the invention relates generally to antibodies, antigen
binding fragments or derivatives thereof which can be used as an
antagonist of LINGO-1. Additionally, the invention generally
relates to methods for treating various diseases, disorders or
injuries associated with demyelination, dysmyelination,
oligodendrocyte/neuronal cell death or axonal injury by the
administration of a LINGO-1 antagonist antibody or antigen binding
fragment.
[0016] In certain embodiments, the invention includes an isolated
antibody or antigen binding fragment thereof which specifically
binds to the same LINGO-1 epitope as the reference monoclonal
antibody Li62 or Li81.
[0017] Certain embodiments of the invention include an isolated
polypeptide comprising an immunoglobulin heavy chain variable
region (VH) wherein the CDR1, CDR2 and CDR3 regions are selected
from the polypeptide sequences shown in Table 3 or at least 80%,
85%, 90% or 95% identical to the polypeptide sequences shown in
Table 3 or at least 80%, 85%, 90, 95% or 100% identical to the VH
CDR1, CDR2 and CDR3 regions of the immunoglobulin heavy chain of
Li62 or Li81. In some embodiments, the VH comprises the polypeptide
sequence of SEQ ID NO: 4 or SEQ ID NO:8 or any one of SEQ ID NOs:
17 to 49.
[0018] Certain embodiments of the invention include an isolated
polypeptide comprising an immunoglobulin light chain variable
region (VL) wherein the CDR1, CDR2 and CDR3 regions are selected
from the polypeptide sequences shown in Table 4 or at least 80%,
85%, 90% or 95% identical to the polypeptide sequences shown in
Table 4 or at least 80%, 85%, 90%, 95% or 100% identical to the VL
CDR1, CDR2 and CDR3 regions of the immunoglobulin light chain of
Li62 or Li81.
[0019] Certain embodiments of the invention include an isolated
polypeptide comprising an immunoglobulin heavy chain variable
region (VH) selected from the group consisting of SEQ ID NOs: 1, 5,
and 53-85 or at least 80%, 85%, 90% or 95% identical to said SEQ ID
NOs: 1, 5 and 53-85.
[0020] Certain embodiments of the invention include an isolated
polypeptide comprising an immunoglobulin light chain variable
region (VL) selected from the group consisting of SEQ ID NOs: 9 and
13, as shown in Table 4, or at least 80%, 85%, 90% or 95% identical
to said SEQ ID NOs: 9 and 13, as shown in Table 4.
[0021] Other embodiments of the invention include an isolated
polynucleotide comprising a nucleic acid encoding an immunoglobulin
heavy chain variable region (VH) selected from the group consisting
of SEQ ID NOs: 1, 5 and 53-85, or at least 80%, 85%, 90% or 95%
identical to said SEQ ID NOs: 1, 5 and 53-85. In some embodiments,
the polynucleotide comprises a nucleic acid encoding the
polypeptide sequence of SEQ ID NO: 4 or SEQ ID NO:8 or any one of
SEQ ID NOs: 17 to 49.
[0022] Other embodiments of the invention include an isolated
polynucleotide comprising a nucleic acid encoding an immunoglobulin
light chain variable region (VL) selected from the group consisting
of SEQ ID NOs: 9 and 13, as shown in Table 4, or at least 80%, 85%,
90% or 95% identical to said SEQ ID NOs: 9 and 13, as shown in
Table 4.
[0023] In certain embodiments, the invention includes compositions
comprising the antibodies or antigen binding fragments described
herein.
[0024] In additional embodiments, the invention includes methods
for treating CNS injury, ALS, Huntington's disease, Alzheimer's
disease, Parkinson's disease, diabetic neuropathy and stroke
comprising administering to an animal in need of said treatment an
effective amount of an agent selected from the group consisting of
an isolated LINGO-1 antibody or fragment thereof or compositions
comprising said antibody or fragment thereof.
[0025] In other embodiments, the invention includes methods for
treating diseases or disorders associated with inhibition of
oligodendrocyte growth or differentiation; demyelination or
dysmyelination of CNS neurons including multiple sclerosis (MS),
progressive multifocal leukoencephalopathy (PML), encephalomyelitis
(EPL), central pontine myelolysis (CPM), Wallerian Degeneration,
adrenoleukodystrophy, Alexander's disease, and Pelizaeus Merzbacher
disease (PMZ) by administering to an animal in need of said
treatment an effective amount of an agent selected from the group
consisting of an isolated LINGO-1 antibody or fragment thereof or
compositions comprising said antibody or fragment thereof.
[0026] Other embodiments of the present invention include a method
of inhibiting signal transduction by Nogo receptor 1 (NgR1),
comprising contacting the NgR1 with an effective amount of an agent
selected from the group consisting of the isolated LINGO-1 antibody
or fragment thereof or compositions comprising said antibody or
fragment thereof.
[0027] Additional embodiments of the present invention include a
method of decreasing inhibition of axonal growth of a central
nervous system (CNS) neuron, comprising contacting the neuron with
an effective amount of an agent selected from the group consisting
of the isolated LINGO-1 antibody or fragment thereof of or
compositions comprising said antibody or fragment thereof.
[0028] Other embodiments of the present invention include a method
of inhibiting growth cone collapse of a CNS neuron, comprising
contacting the neuron with an effective amount of an agent selected
from the group consisting of the isolated LINGO-1 antibody or
fragment thereof or compositions comprising said antibody or
fragment thereof.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0029] FIG. 1: Western blot of co-cultured oligodendrocyte
precursor cells and DRGs after incubation with anti-LINGO-1
antibodies (Li33 PDL, Li62 (agly) and Li81 (agly)) and control
antibody (h5C8) as described in Example 2.
[0030] FIG. 2: Western blot of MBP, MOG and .beta.-actin in rat
A2B5+ progenitor cells treated with anti-LINGO-1 antibody (Li81
(agly)) or control antibody (Ctrl) as described in Example 4.
[0031] FIG. 3: Bar graph showing the number of MBP-positive cells
in human oligodendrocyte precursor cell cultures treated with
anti-LINGO-1 antibody (Li81 (agly)) or a control antibody (hIgG1)
as described in Example 5.
[0032] FIG. 4: Bar graph showing intensity of black gold
immunostaining to mark myelination in lysolecithin ("Lyso")-treated
brain slices exposed to control antibody (5C8) or anti-LINGO-1
antibody (Li81 (agly)) at concentrations of 30 .mu.g/ml (81-30), 10
.mu.g/ml (81-10), 3 .mu.g/ml (81-3), or 1 .mu.g/ml (81-1).
Experiments were performed as described in Example 6.
[0033] FIG. 5: Graphs depicting IC50 values of an aglycosylated
anti-LINGO-1 antibody (Li81 (agly)), a control antibody (WT 5c8)
and an aglycosylated control antibody (agly 5c8) for human Fc
receptors CD16 (A), CD32a (B), CD32b (C) and CD64 (D). Experiments
were performed as described in Example 7.
[0034] FIG. 6: Graph depicting binding of anti-LINGO-1 antibodies
(Li33 ("Di33IgG1") and Li13 "Di13IgG1")), aglycosylated
anti-LINGO-1 antibody (Li81 (agly) ("Di81IgG1Agly"), and a control
antibody (huIgG1) to CD64 and CD32 as measured by the cell bridging
assay described in Example 7.
[0035] FIG. 7: Graph depicting complement activation in CHO cells
expressing LINGO-1 incubated with anti-LINGO-1 antibodies (Li81
(agly) ("Dli81"), Li33 ("Dli33IgG1"), Li13 ("Dli13IgG1")) and a
positive control antibody (LTbetaR-Ig). Experiments were performed
as described in Example 8.
[0036] FIG. 8: Images showing lesions in lysolecithin-treated
animals administered a control antibody ("Ctrl") or an anti-LINGO-1
antibody (Li81 (agly)) and a bar graph depicting the size of
demyelinated lesions in rats treated with lysolecithin and
administered control antibody or anti-LINGO-1 antibody (Li81
(agly)). Experiments were performed as described in Example 9.
[0037] FIG. 9: Graph depicting EAE score to assess paralysis in
rats administered recombinant myelin oligodendrocyte glycoprotein
and treated with control antibody ("Isotype ctr") or anti-LINGO-1
antibody (Li81). Downward arrows indicate timepoints at which
antibody treatment was administered. Experiments were performed as
described in Example 10.
[0038] FIG. 10: Graph showing the binding of Li113 Fab to LINGO-1
as measured by ELISA assay.
[0039] FIG. 11: Graph depicting efficacy of LINGO-1 monoclonal
antibodies and Fabs in an oligodendrocye differentiation assay. Bar
height represents the concentration of MBP as measured by ELISA.
Antibodies were tested at concentrations of 1 .mu.g/ml (black), 0.3
.mu.g/ml (dark grey), 0.1 .mu.g/ml (light grey) and 0.03 .mu.g/ml
(white).
[0040] FIG. 12: Image showing the size exclusion chromatography
profiles for Li33 Ig1 (agly) and Ig2. Top panel shows the elution
profile of BIO RAD gel filtration markers and shows molecular
masses.
[0041] FIG. 13: Graph depicting the denaturation of Li33 Ig1 and
Ig2 by guanidine hydrochloride. Flourescence data from the emission
spectra at 350 nm are plotted as a function of the guanidine
hydrochloride concentration and standardized using the change in
flourescence from maximum for each test condition. NEM refers to
N-ethylmaleimide and TCEP refers to
Tris(2-carboxyethyl)phosphine.
[0042] FIG. 14: Image showing the results of an analytical
ultracentrifugation evaluating the aggregation state of Li33 Ig2.
Absorbance scan data from velocity sedimentation centrifugation
studies with Li33 Ig2 Mab at 0.4 mg/ml (A), 7 mg/ml (B) and 27
mg/ml (C) are shown. Top panels show raw absorbance data as a
function of time. Bottom panels show relative concentrations as a
function of sedimentation coefficient.
[0043] FIG. 15: Image showing protein-protein interactions in the
Li33 Fab structure.
[0044] FIG. 16: Image depicting methods of generating PEGylated
Fabs through direct expression.
[0045] FIG. 17: Image displaying an SDS-PAGE gel under non-reducing
conditions showing the results of PEGylated Fab direct expression
studies using the methods shown in FIG. 16. Arrowhead indicates
PEGylated Fab.
[0046] FIG. 18: Image displaying an SDS-PAGE gel under non-reducing
conditions showing the results of PEGylated Fab enzymatic digestion
studies. Lane 1 shows molecular mass markers. Lane 2 shows Li33 Ig1
Mab. Lane 3 shows Li33 Ig1 Fab2. Lane 4 shows Li33 Ig1 Fab2 treated
with TCEP, and lane 5 shows Li33 Ig1 Fab2 treated with TCEP and
then with PEG. Arrow indicates PEGylated Li33 Fab' product.
[0047] FIG. 19: Graph depicting the results of a FACS assay to
assess binding of PEGylated LINGO-1 antibodies to LINGO-1.
[0048] FIG. 20: Graph depicting the results of Li81 binding
measured in a direct binding ELISA assay using LINGO-1 coated
plates.
[0049] FIG. 21: Graph depicting results of an oligodendrocyte
differentiation assay using Li81. Bar height indicates the
concentration of MBP as measured by ELISA. Li81 RTP-RO8 indicates
an Li81 (agly) reference standard.
[0050] FIG. 22: Graph depicting results of remyelination assay
using Li81 antibody and antibody fragments. Bar height represents
the intensity of black gold signal.
[0051] FIG. 23: Graphs depicting results of thermal denaturation
studies of LINGO-1 antibodies and antibody fragments. Bar height
indicates TM.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0052] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, "a LINGO-1 antibody," is
understood to represent one or more LINGO-1 antibodies. As such,
the terms "a" (or "an"), "one or more," and "at least one" can be
used interchangeably herein.
[0053] As used herein, the term "polypeptide" is intended to
encompass a singular "polypeptide" as well as plural
"polypeptides," and refers to a molecule composed of monomers
(amino acids) linearly linked by amide bonds (also known as peptide
bonds). The term "polypeptide" refers to any chain or chains of two
or more amino acids, and does not refer to a specific length of the
product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"protein," "amino acid chain," or any other term used to refer to a
chain or chains of two or more amino acids, are included within the
definition of "polypeptide," and the term "polypeptide" may be used
instead of, or interchangeably with any of these terms. The term
"polypeptide" is also intended to refer to the products of
post-expression modifications of the polypeptide, including without
limitation glycosylation, acetylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, or modification by non-naturally occurring amino acids. A
polypeptide may be derived from a natural biological source or
produced by recombinant technology, but is not necessarily
translated from a designated nucleic acid sequence. It may be
generated in any manner, including by chemical synthesis.
[0054] A polypeptide of the invention may be of a size of about 3
or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more,
75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more,
or 2,000 or more amino acids. Polypeptides may have a defined
three-dimensional structure, although they do not necessarily have
such structure. Polypeptides with a defined three-dimensional
structure are referred to as folded, and polypeptides which do not
possess a defined three-dimensional structure, but rather can adopt
a large number of different conformations, and are referred to as
unfolded. As used herein, the term glycoprotein refers to a protein
coupled to at least one carbohydrate moiety that is attached to the
protein via an oxygen-containing or a nitrogen-containing side
chain of an amino acid residue, e.g., a serine residue or an
asparagine residue.
[0055] By an "isolated" polypeptide or a fragment, variant, or
derivative thereof is intended a polypeptide that is not in its
natural milieu. No particular level of purification is required.
For example, an isolated polypeptide can be removed from its native
or natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for
purposed of the invention, as are native or recombinant
polypeptides which have been separated, fractionated, or partially
or substantially purified by any suitable technique.
[0056] Also included as polypeptides of the present invention are
fragments, derivatives, analogs, or variants of the foregoing
polypeptides, and any combination thereof. The terms "fragment,"
"variant," "derivative" and "analog" when referring to LINGO-1
antibodies or antibody polypeptides of the present invention
include any polypeptides which retain at least some of the
antigen-binding properties of the corresponding native antibody or
polypeptide. Fragments of polypeptides of the present invention
include proteolytic fragments, as well as deletion fragments, in
addition to specific antibody fragments discussed elsewhere herein.
Variants of LINGO-1 antibodies and antibody polypeptides of the
present invention include fragments as described above, and also
polypeptides with altered amino acid sequences due to amino acid
substitutions, deletions, or insertions. Variants may occur
naturally or be non-naturally occurring Non-naturally occurring
variants may be produced using art-known mutagenesis techniques.
Variant polypeptides may comprise conservative or non-conservative
amino acid substitutions, deletions or additions. Derivatives of
LINGO-1 antibodies and antibody polypeptides of the present
invention, are polypeptides which have been altered so as to
exhibit additional features not found on the native polypeptide.
Examples include fusion proteins. Variant polypeptides may also be
referred to herein as "polypeptide analogs." As used herein a
"derivative" of a LINGO-1 antibody or antibody polypeptide refers
to a subject polypeptide having one or more residues chemically
derivatized by reaction of a functional side group. Also included
as "derivatives" are those peptides which contain one or more
naturally occurring amino acid derivatives of the twenty standard
amino acids. For example, 4-hydroxyproline may be substituted for
proline; 5-hydroxylysine may be substituted for lysine;
3-methylhistidine may be substituted for histidine; homoserine may
be substituted for serine; and ornithine may be substituted for
lysine.
[0057] The term "polynucleotide" is intended to encompass a
singular nucleic acid as well as plural nucleic acids, and refers
to an isolated nucleic acid molecule or construct, e.g., messenger
RNA (mRNA) or plasmid DNA (pDNA). A polynucleotide may comprise a
conventional phosphodiester bond or a non-conventional bond (e.g.,
an amide bond, such as found in peptide nucleic acids (PNA)). The
term "nucleic acid" refer to any one or more nucleic acid segments,
e.g., DNA or RNA fragments, present in a polynucleotide. By
"isolated" nucleic acid or polynucleotide is intended a nucleic
acid molecule, DNA or RNA, which has been removed from its native
environment. For example, a recombinant polynucleotide encoding a
LINGO-1 antibody contained in a vector is considered isolated for
the purposes of the present invention. Further examples of an
isolated polynucleotide include recombinant polynucleotides
maintained in heterologous host cells or purified (partially or
substantially) polynucleotides in solution. Isolated RNA molecules
include in vivo or in vitro RNA transcripts of polynucleotides of
the present invention. Isolated polynucleotides or nucleic acids
according to the present invention further include such molecules
produced synthetically. In addition, polynucleotide or a nucleic
acid may be or may include a regulatory element such as a promoter,
ribosome binding site, or a transcription terminator.
[0058] As used herein, a "coding region" is a portion of nucleic
acid which consists of codons translated into amino acids. Although
a "stop codon" (TAG, TGA, or TAA) is not translated into an amino
acid, it may be considered to be part of a coding region, but any
flanking sequences, for example promoters, ribosome binding sites,
transcriptional terminators, introns, and the like, are not part of
a coding region. Two or more coding regions of the present
invention can be present in a single polynucleotide construct,
e.g., on a single vector, or in separate polynucleotide constructs,
e.g., on separate (different) vectors. Furthermore, any vector may
contain a single coding region, or may comprise two or more coding
regions, e.g., a single vector may separately encode an
immunoglobulin heavy chain variable region and an immunoglobulin
light chain variable region. In addition, a vector, polynucleotide,
or nucleic acid of the invention may encode heterologous coding
regions, either fused or unfused to a nucleic acid encoding a
LINGO-1 antibody or fragment, variant, or derivative thereof.
Heterologous coding regions include without limitation specialized
elements or motifs, such as a secretory signal peptide or a
heterologous functional domain.
[0059] In certain embodiments, the polynucleotide or nucleic acid
is DNA. In the case of DNA, a polynucleotide comprising a nucleic
acid which encodes a polypeptide normally may include a promoter
and/or other transcription or translation control elements operably
associated with one or more coding regions. An operable association
is when a coding region for a gene product, e.g., a polypeptide, is
associated with one or more regulatory sequences in such a way as
to place expression of the gene product under the influence or
control of the regulatory sequence(s). Two DNA fragments (such as a
polypeptide coding region and a promoter associated therewith) are
"operably associated" if induction of promoter function results in
the transcription of mRNA encoding the desired gene product and if
the nature of the linkage between the two DNA fragments does not
interfere with the ability of the expression regulatory sequences
to direct the expression of the gene product or interfere with the
ability of the DNA template to be transcribed. Thus, a promoter
region would be operably associated with a nucleic acid encoding a
polypeptide if the promoter was capable of effecting transcription
of that nucleic acid. The promoter may be a cell-specific promoter
that directs substantial transcription of the DNA only in
predetermined cells. Other transcription control elements, besides
a promoter, for example enhancers, operators, repressors, and
transcription termination signals, can be operably associated with
the polynucleotide to direct cell-specific transcription. Suitable
promoters and other transcription control regions are disclosed
herein.
[0060] A variety of transcription control regions are known to
those skilled in the art. These include, without limitation,
transcription control regions which function in vertebrate cells,
such as, but not limited to, promoter and enhancer segments from
cytomegaloviruses (the immediate early promoter, in conjunction
with intron-A), simian virus 40 (the early promoter), and
retroviruses (such as Rous sarcoma virus). Other transcription
control regions include those derived from vertebrate genes such as
actin, heat shock protein, bovine growth hormone and rabbit
.beta.-globin, as well as other sequences capable of controlling
gene expression in eukaryotic cells. Additional suitable
transcription control regions include tissue-specific promoters and
enhancers as well as lymphokine-inducible promoters (e.g.,
promoters inducible by interferons or interleukins).
[0061] Similarly, a variety of translation control elements are
known to those of ordinary skill in the art. These include, but are
not limited to ribosome binding sites, translation initiation and
termination codons, and elements derived from picornaviruses
(particularly an internal ribosome entry site, or IRES, also
referred to as a CITE sequence).
[0062] In other embodiments, a polynucleotide of the present
invention is RNA, for example, in the form of messenger RNA
(mRNA).
[0063] Polynucleotide and nucleic acid coding regions of the
present invention may be associated with additional coding regions
which encode secretory or signal peptides, which direct the
secretion of a polypeptide encoded by a polynucleotide of the
present invention. According to the signal hypothesis, proteins
secreted by mammalian cells have a signal peptide or secretory
leader sequence which is cleaved from the mature protein once
export Of the growing protein chain across the rough endoplasmic
reticulum has been initiated. Those of ordinary skill in the art
are aware that polypeptides secreted by vertebrate cells generally
have a signal peptide fused to the N-terminus of the polypeptide,
which is cleaved from the complete or "full length" polypeptide to
produce a secreted or "mature" feint of the polypeptide. In certain
embodiments, the native signal peptide, e.g., an immunoglobulin
heavy chain or light chain signal peptide is used, or a functional
derivative of that sequence that retains the ability to direct the
secretion of the polypeptide that is operably associated with it.
Alternatively, a heterologous mammalian signal peptide, or a
functional derivative thereof, may be used. For example, the
wild-type leader sequence may be substituted with the leader
sequence of human tissue plasminogen activator (TPA) or mouse
.beta.-glucuronidase.
[0064] The present invention is directed to certain LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof. Unless specifically referring to full-sized antibodies
such as naturally-occurring antibodies, the term "LINGO-1
antibodies" encompasses full-sized antibodies as well as
antigen-binding fragments, variants, analogs, or derivatives of
such antibodies, e.g., naturally occurring antibody or
immunoglobulin molecules or engineered antibody molecules or
fragments that bind antigen in a manner similar to antibody
molecules.
[0065] The terms "antibody" and "immunoglobulin" are used
interchangeably herein. An antibody or immunoglobulin comprises at
least the variable domain of a heavy chain, and normally comprises
at least the variable domains of a heavy chain and a light chain.
Basic immunoglobulin structures in vertebrate systems are
relatively well understood. See, e.g., Harlow at al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed,
1988).
[0066] As will be discussed in more detail below, the term
"immunoglobulin" comprises various broad classes of polypeptides
that can be distinguished biochemically. Those skilled in the art
will appreciate that heavy chains are classified as gamma, mu,
alpha, delta, or epsilon, (.gamma., .mu., .alpha., .delta.,
.epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the stature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, etc. are
well characterized and are known to confer functional
specialization. Modified versions of each of these classes and
isotypes are readily discernable to the skilled artisan in view of
the instant disclosure and, accordingly, are within the scope of
the instant invention. All immunoglobulin classes are clearly
within the scope of the present invention, the following,
discussion will generally be directed to the IgG class of
immunoglobulin molecules. With regard to IgG, a standard
immunoglobulin molecule comprises two identical light chain
polypeptides of molecular weight approximately 23,000 Daltons, and
two identical heavy chain polypeptides of molecular weight
53,000-70,000. The four chains are typically joined by disulfide
bonds in a "Y" configuration wherein the light chains bracket the
heavy chains starting at the mouth of the "Y" and continuing
through the variable region.
[0067] Light chains are classified as either kappa or lambda
(.kappa., .lamda.). Each heavy chain class may be bound with either
a kappa or lambda light chain. In general, the light and heavy
chains are covalently bonded to each other, and the "tail" portions
of the two heavy chains are bonded to each other by covalent
disulfide linkages or non-covalent linkages when the
immunoglobulins are generated either by hybridomas, B cells or
genetically engineered host cells. In the heavy chain, the amino
acid sequences run from an N-terminus at the forked ends of the Y
configuration to the C-terminus at the bottom of each chain.
[0068] Both the light and heavy chains are divided into regions of
structural and functional homology. The terms "constant" and
"variable" are used functionally. In this regard, it will be
appreciated that the variable domains of both the light (V.sub.L)
and heavy (V.sub.H) chain portions determine antigen recognition
and specificity. Conversely, the constant domains of the light
chain (C.sub.L) and the heavy chain (C.sub.H1, C.sub.H2 or
C.sub.H3) confer important biological properties such as secretion,
transplacental mobility, Fc receptor binding, complement binding,
and the like. By convention the numbering of the constant region
domains increases as they become more distal from the antigen
binding site or amino-terminus of the antibody. The N-terminal
portion is a variable region and at the C-terminal portion is a
constant region; the C.sub.H3 and C.sub.L domains actually comprise
the carboxy-terminus of the heavy and light chain,
respectively.
[0069] As indicated above, the variable region allows the antibody
to selectively recognize and specifically bind epitopes on
antigens. That is, the V.sub.L domain and V.sub.H domain, or subset
of the complementarity determining regions (CDRs), of an antibody
combine to form the variable region that defines a three
dimensional antigen binding site. This quaternary antibody
structure forms the antigen binding site present at the end of each
arm of the Y. More specifically, the antigen binding site is
defined by three CDRs on each of the V.sub.H and V.sub.L chains. In
some instances, e.g., certain immunoglobulin molecules derived from
camelid species or engineered based on camelid immunoglobulins, a
complete immunoglobulin molecule may consist of heavy chains only,
with no light chains. See, e.g., Hamers-Casterman et al., Nature
363:446-448 (1993).
[0070] In naturally occurring antibodies, the six "complementarity
determining regions" or "CDRs" present in each antigen binding
domain are short, non-contiguous sequences of amino acids that are
specifically positioned to form the antigen binding domain as the
antibody assumes its three dimensional configuration in an aqueous
environment. The remainder of the amino acids in the antigen
binding domains, referred to as "framework" regions, show less
inter-molecular variability. The framework regions largely adopt a
.beta.-sheet conformation and the CDRs form loops which connect,
and in some cases form part of, the .beta.-sheet structure. Thus,
framework regions act to form a scaffold that provides for
positioning the CDRs in correct orientation by inter-chain,
non-covalent interactions. The antigen binding domain formed by the
positioned CDRs defines a surface complementary to the epitope on
the immunoreactive antigen. This complementary surface promotes the
non-covalent binding of the antibody to its cognate epitope. The
amino acids comprising the CDRs and the framework regions,
respectively, can be readily identified for any given heavy or
light chain variable region by one of ordinary skill in the art,
since they have been precisely defined (see, "Sequences of Proteins
of Immunological Interest," Kabat, E., et al., U.S. Department of
Health and Human Services, (1983); and Chothia and Lesk, J. Mol.
Biol., 196:901-917 (1987), which are incorporated herein by
reference in their entireties).
[0071] In the case where there are two or more definitions of a
term which is used and/or accepted within the art, the definition
of the term as used herein is intended to include all such meanings
unless explicitly stated to the contrary. A specific example is the
use of the term "complementarity determining region" ("CDR") to
describe the non-contiguous antigen combining sites found within
the variable region of both heavy and light chain polypeptides.
This particular region has been described by Kabat et al., U.S.
Dept. of Health and Human Services, "Sequences of Proteins of
Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol.
196:901-917 (1987), which are incorporated herein by reference,
where the definitions include overlapping or subsets of amino acid
residues when compared against each other. Nevertheless,
application of either definition to refer to a CDR of an antibody
or variants thereof is intended to be within the scope of the term
as defined and used herein. The appropriate amino acid residues
which encompass the CDRs as defined by each of the above cited
references are set forth below in Table I as a comparison. The
exact residue numbers which encompass a particular CDR will vary
depending on the sequence and size of the CDR. Those skilled in the
art can routinely determine which residues comprise a particular
CDR given the variable region amino acid sequence of the
antibody.
TABLE-US-00001 TABLE 1 CDR Definitions.sup.1 Kabat Chothia V.sub.H
CDR1 31-35 26-32 V.sub.H CDR2 50-65 52-58 V.sub.H CDR3 95-102
95-102 V.sub.L CDR1 24-34 26-32 V.sub.L CDR2 50-56 50-52 V.sub.L
CDR3 89-97 91-96 .sup.1Numbering of all CDR definitions in Table 1
is according to the numbering conventions set forth by Kabat et al.
(see below).
[0072] Kabat et al. also defined a numbering system for variable
domain sequences that is applicable to any antibody. One of
ordinary skill in the art can unambigously assign this system of
"Kabat numbering" to any variable domain sequence, without reliance
on any experimental data beyond the sequence itself. As used
herein, "Kabat numbering" refers to the numbering system set forth
by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence
of Proteins of Immunological Interest" (1983). Unless otherwise
specified, references to the numbering of specific amino acid
residue positions in a LINGO-1 antibody or antigen-binding
fragment, variant, or derivative thereof of the present invention
are according to the Kabat numbering system.
[0073] In camelid species, the heavy chain variable region,
referred to as V.sub.HH, forms the entire antigen-binding domain.
The main differences between camelid V.sub.HH variable regions and
those derived from conventional antibodies (V.sub.H) include (a)
more hydrophobic amino acids in the light chain contact surface of
V.sub.H as compared to the corresponding region in V.sub.HH, (b) a
longer CDR3 in V.sub.HH, and (c) the frequent occurrence of a
disulfide bond between CDR1 and CDR3 in V.sub.HH.
[0074] Antibodies or antigen-binding fragments, variants, or
derivatives thereof of the invention include, but are not limited
to, polyclonal, monoclonal, multispecific, human, humanized,
primatized, or chimeric antibodies, single chain antibodies,
epitope-binding fragments, e.g., Fab, Fab' and F(ab').sub.2, Fd,
Fvs, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv), fragments comprising either a V.sub.L
or V.sub.H domain, fragments produced by a Fab expression library,
and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to LINGO-1 antibodies disclosed herein). ScFv molecules
are known in the art and are described, e.g., in U.S. Pat. No.
5,892,019. Immunoglobulin or antibody molecules of the invention
can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class
(e.g., IgG 1 , IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of
immunoglobulin molecule.
[0075] Antibody fragments, including single-chain antibodies, may
comprise the variable region(s) alone or in combination with the
entirety or a portion of the following: hinge region, C.sub.H1,
C.sub.H2, and C.sub.H3 domains. Also included in the invention are
antigen-binding fragments also comprising any combination of
variable region(s) with a hinge region, C.sub.H1, C.sub.H2, and
C.sub.H3 domains. Antibodies or immunospecific fragments thereof
for use in the diagnostic and therapeutic methods disclosed herein
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, donkey, rabbit, goat,
guinea pig, camel, llama, horse, or chicken antibodies. In another
embodiment, the variable region may be condricthoid in origin
(e.g., from sharks). As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human immunoglobulin
and include antibodies isolated from human immunoglobulin libraries
or from animals transgenic for one or more human immunoglobulins
and that do not express endogenous immunoglobulins, as described
infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati
et al.
[0076] As used herein, the term "heavy chain portion" includes
amino acid sequences derived from an immunoglobulin heavy chain. A
polypeptide comprising a heavy chain portion comprises at least one
of: a C.sub.H1 domain, a hinge (e.g., upper, middle, and/or lower
hinge region) domain, a C.sub.H2 domain, a C.sub.H3 domain, or a
variant or fragment thereof. For example, a binding polypeptide for
use in the invention may comprise a polypeptide chain comprising a
C.sub.H1 domain; a polypeptide chain comprising a C.sub.H1 domain,
at least a portion of a hinge domain, and a C.sub.H2 domain; a
polypeptide chain comprising a C.sub.H1 domain and a C.sub.H3
domain; a polypeptide chain comprising a C.sub.H1 domain, at least
a portion of a hinge domain, and a C.sub.H3 domain, or a
polypeptide chain comprising a C.sub.H1 domain, at least a portion
of a hinge domain, a C.sub.H2 domain, and a C.sub.H3 domain. In
another embodiment, a polypeptide of the invention comprises a
polypeptide chain comprising a C.sub.H3 domain. Further, a binding
polypeptide for use in the invention may lack at least a portion of
a C.sub.H2 domain (e.g., all or part of a C.sub.H2 domain). As set
forth above, it will be understood by one of ordinary skill in the
art that these domains (e.g., the heavy chain portions) may be
modified such that they vary in amino acid sequence from the
naturally occurring immunoglobulin molecule.
[0077] In certain LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof disclosed herein, the heavy chain
portions of one polypeptide chain of a multimer are identical to
those on a second polypeptide chain of the multimer. Alternatively,
heavy chain portion-containing monomers of the invention are not
identical. For example, each monomer may comprise a different
target binding site, forming, for example, a bispecific
antibody.
[0078] The heavy chain portions of a binding polypeptide for use in
the diagnostic and treatment methods disclosed herein may be
derived from different immunoglobulin molecules. For example, a
heavy chain portion of a polypeptide may comprise a C.sub.H1 domain
derived from an IgG1 molecule and a hinge region derived from an
IgG3 molecule. In another example, a heavy chain portion can
comprise a hinge region derived, in part, from an IgG1 molecule
and, in part, from an IgG3 molecule. In another example, a heavy
chain portion can comprise a chimeric hinge derived, in part, from
an IgG1 molecule and, in part, from an IgG4 molecule.
[0079] As used herein, the term "light chain portion" includes
amino acid sequences derived from an immunoglobulin light chain.
Preferably, the light chain portion comprises at least one of a
V.sub.L or C.sub.L domain.
[0080] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof disclosed herein may be described or
specified in terms of the epitope(s) or portion(s) of an antigen,
e.g., a target polypeptide (LINGO-1) that they recognize or
specifically bind. The portion of a target polypeptide which
specifically interacts with the antigen binding domain of an
antibody is an "epitope," or an "antigenic determinant." A target
polypeptide may comprise a single epitope, but typically comprises
at least two epitopes, and can include any number of epitopes,
depending on the size, conformation, and type of antigen.
Furthermore, it should be noted that an "epitope" on a target
polypeptide may be or include non-polypeptide elements, e.g., an
"epitope may include a carbohydrate side chain.
[0081] The minimum size of a peptide or polypeptide epitope for an
antibody is thought to be about four to five amino acids. Peptide
or polypeptide epitopes preferably contain at least seven, more
preferably at least nine and most preferably between at least about
15 to about 30 amino acids. Since a CDR can recognize an antigenic
peptide or polypeptide in its tertiary form, the amino acids
comprising an epitope need not be contiguous, and in some cases,
may not even be on the same peptide chain. In the present
invention, peptide or polypeptide epitope recognized by LINGO-1
antibodies of the present invention contains a sequence of at least
4, at least 5, at least 6, at least 7, more preferably at least 8,
at least 9, at least 10, at least 15, at least 20, at least 25, or
between about 15 to about 30 contiguous or non-contiguous amino
acids of LINGO-1.
[0082] By "specifically binds," it is generally meant that an
antibody binds to an epitope via its antigen binding domain, and
that the binding entails some complementarity between the antigen
binding domain and the epitope. According to this definition, an
antibody is said to "specifically bind" to an epitope when it binds
to that epitope, via its antigen binding domain more readily than
it would bind to a random, unrelated epitope. The term
"specificity" is used herein to qualify the relative affinity by
which a certain antibody binds to a certain epitope. For example,
antibody "A" may be deemed to have a higher specificity for a given
epitope than antibody "B," or antibody "A" may be said to bind to
epitope "C" with a higher specificity than it has for related
epitope "D."
[0083] By "preferentially binds," it is meant that the antibody
specifically binds to an epitope more readily than it would bind to
a related, similar, homologous, or analogous epitope. Thus, an
antibody which "preferentially binds" to a given epitope would more
likely bind to that epitope than to a related epitope, even though
such an antibody may cross-react with the related epitope.
[0084] By way of non-limiting example, an antibody may be
considered to bind a first epitope preferentially if it binds said
first epitope with a dissociation constant (K.sub.D) that is less
than the antibody's K.sub.D for the second epitope. In another
non-limiting example, an antibody may be considered to bind a first
antigen preferentially if it binds the first epitope with an
affinity that is at least one order of magnitude less than the
antibody's K.sub.D for the second epitope. In another non-limiting
example, an antibody may be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that
is at least two orders of magnitude less than the antibody's
K.sub.D for the second epitope.
[0085] In another non-limiting example, an antibody may be
considered to bind a first epitope preferentially if it binds the
first epitope with an off rate (k(off)) that is less than the
antibody's k(off) for the second epitope. In another non-limiting
example, an antibody may be considered to bind a first epitope
preferentially if it binds the first epitope with an affinity that
is at least one order of magnitude less than the antibody's k(off)
for the second epitope. In another non-limiting example, an
antibody may be considered to bind a first epitope preferentially
if it binds the first epitope with an affinity that is at least two
orders of magnitude less than the antibody's k(off) for the second
epitope.
[0086] An antibody or or antigen-binding fragment, variant, or
derivative disclosed herein may be said to bind a target
polypeptide disclosed herein or a fragment or variant thereof with
an off rate (k(off)) of less than or equal to 5.times.10.sup.-2
sec.sup.-1, 10.sup.-2 sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1 or
10.sup.-3 sec.sup.-1. More preferably, an antibody of the invention
may be said to bind a target polypeptide disclosed herein or a
fragment or variant thereof with an off rate (k(off)) less than or
equal to 5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1.
[0087] An antibody or or antigen-binding fragment, variant, or
derivative disclosed herein may be said to bind a target
polypeptide disclosed herein or a fragment or variant thereof with
an on rate (k(on)) of greater than or equal to 10.sup.3 M.sup.-1
sec.sup.-1, 5.times.10.sup.3 M.sup.-1 sec.sup.-1, 10.sup.4 M.sup.-1
sec.sup.-1 or 5.times.10.sup.4 M.sup.-1 sec.sup.-1. More
preferably, an antibody of the invention may be said to bind a
target polypeptide disclosed herein or a fragment or variant
thereof with an on rate (k(on)) greater than or equal to 10.sup.5
M.sup.-1 sec.sup.-1, 5.times.10.sup.5 M.sup.-1 sec.sup.-1, 10.sup.6
M.sup.-1 sec.sup.-1, or 5.times.10.sup.6 M.sup.-1 sec.sup.-1 or
10.sup.7 M.sup.-1 sec.sup.-1.
[0088] An antibody is said to competitively inhibit binding of a
reference antibody to a given epitope if it preferentially binds to
that epitope to the extent that it blocks, to some degree, binding
of the reference antibody to the epitope. Competitive inhibition
may be determined by any method known in the art, for example,
competition ELISA assays. An antibody may be said to competitively
inhibit binding of the reference antibody to a given epitope by at
least 90%, at least 80%, at least 70%, at least 60%, or at least
50%.
[0089] As used herein, the term "affinity" refers to a measure of
the strength of the binding of an individual epitope with the CDR
of an immunoglobulin molecule. See, e.g., Harlow et al.,
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed. 1988) at pages 27-28. As used herein, the term
"avidity" refers to the overall stability of the complex between a
population of immunoglobulins and an antigen, that is, the
functional combining strength of an immunoglobulin mixture with the
antigen. See, e.g., Harlow at pages 29-34. Avidity is related to
both the affinity of individual immunoglobulin molecules in the
population with specific epitopes, and also the valencies of the
immunoglobulins and the antigen. For example, the interaction
between a bivalent monoclonal antibody and an antigen with a highly
repeating epitope structure, such as a polymer, would be one of
high avidity.
[0090] LINGO-1 antibodies or antigen-binding fragments, variants or
derivatives thereof of the invention may also be described or
specified in terms of their cross-reactivity. As used herein, the
term "cross-reactivity" refers to the ability of an antibody,
specific for one antigen, to react with a second antigen; a measure
of relatedness between two different antigenic substances. Thus, an
antibody is cross reactive if it binds to an epitope other than the
one that induced its formation. The cross reactive epitope
generally contains many of the same complementary structural
features as the inducing epitope, and in some cases, may actually
fit better than the original.
[0091] For example, certain antibodies have some degree of
cross-reactivity, in that they bind related, but non-identical
epitopes, e.g., epitopes with at least 95%, at least 90%, at least
85%, at least 80%, at least 75%, at least 70%, at least 65%, at
least 60%, at least 55%, and at least 50% identity (as calculated
using methods known in the art and described herein) to a reference
epitope. An antibody may be said to have little or no
cross-reactivity if it does not bind epitopes with less than 95%,
less than 90%, less than 85%, less than 80%, less than 75%, less
than 70%, less than 65%, less than 60%, less than 55%, and less
than 50% identity (as calculated using methods known in the art and
described herein) to a reference epitope. An antibody may be deemed
"highly specific" for a certain epitope, if it does not bind any
other analog, ortholog, or homolog of that epitope.
[0092] LINGO-1 antibodies or antigen-binding fragments, variants or
derivatives thereof of the invention may also be described or
specified in terms of their binding affmity to a polypeptide of the
invention. Preferred binding affinities include those with a
dissociation constant or Kd less than 5.times.10.sup.-2M,
10.sup.-2M, 5.times.10.sup.-3M, 10.sup.-3M, 5.times.10.sup.-4 M,
10.sup.-4 M, 5.times.10.sup.-5 M, 10.sup.-5 M, 5.times.10.sup.-6 M,
10.sup.-6 M, 5.times.10.sup.-7 M, 10.sup.-7 M, 5.times.10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0093] LINGO-1 antibodies or antigen-binding fragments, variants or
derivatives thereof of the invention may be "multispecific," e.g.,
bispecific, trispecific or of greater multispecificity, meaning
that it recognizes and binds to two or more different epitopes
present on one or more different antigens (e.g., proteins) at the
same time. Thus, whether a LINGO-1 antibody is "monospecfic" or
"multispecific," e.g., "bispecific," refers to the number of
different epitopes with which a binding polypeptide reacts.
Multispecific antibodies may be specific for different epitopes of
a target polypeptide described herein or may be specific for a
target polypeptide as well as for a heterologous epitope, such as a
heterologous polypeptide or solid support material.
[0094] As used herein the term "valency" refers to the number of
potential binding domains, e.g., antigen binding domains, present
in a LINGO-1 antibody, binding polypeptide or antibody. Each
binding domain specifically binds one epitope. When a LINGO-1
antibody, binding polypeptide or antibody comprises more than one
binding domain, each binding domain may specifically bind the same
epitope, for an antibody with two binding domains, termed "bivalent
monospecific," or to different epitopes, for an antibody with two
binding domains, termed "bivalent bispecific." An antibody may also
be bispecific and bivalent for each specificity (termed "bispecific
tetravalent antibodies"). In another embodiment, tetravalent
minibodies or domain deleted antibodies can be made.
[0095] Bispecific bivalent antibodies, and methods of making them,
are described, for instance in U.S. Pat. Nos. 5,731,168; 5,807,706;
5,821,333; and U.S. Appl. Publ. Nos. 2003/020734 and 2002/0155537,
the disclosures of all of which are incorporated by reference
herein. Bispecific tetravalent antibodies, and methods of making
them are described, for instance, in WO 02/096948 and WO 00/44788,
the disclosures of both of which are incorporated by reference
herein. See generally, PCT publications WO 93/17715; WO 92/08802;
WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147:60-69
(1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920;
5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
[0096] As previously indicated, the subunit structures and three
dimensional configuration of the constant regions of the various
immunoglobulin classes are well known. As used herein, the term
"V.sub.H domain" includes the amino terminal variable domain of an
immunoglobulin heavy chain and the term "C.sub.H1 domain" includes
the first (most amino terminal) constant region domain of an
immunoglobulin heavy chain. The C.sub.H1 domain is adjacent to the
V.sub.H domain and is amino terminal to the hinge region of an
immunoglobulin heavy chain molecule.
[0097] As used herein the term "C.sub.H2 domain" includes the
portion of a heavy chain molecule that extends, e.g., from about
residue 244 to residue 360 of an antibody using conventional
numbering schemes (residues 244 to 360, Kabat numbering system; and
residues 231-340, EU numbering system; see Kabat EA et al. op. cit.
The C.sub.H2 domain is unique in that it is not closely paired with
another domain. Rather, two N-linked branched carbohydrate chains
are interposed between the two C.sub.H2 domains of an intact native
IgG molecule. It is also well documented that the C.sub.H3 domain
extends from the C.sub.H2 domain to the C-terminal of the IgG
molecule and comprises approximately 108 residues.
[0098] As used herein, the term "hinge region" includes the portion
of a heavy chain molecule that joins the C.sub.H1 domain to the
C.sub.H2 domain. This hinge region comprises approximately 25
residues and is flexible, thus allowing the two N-terminal antigen
binding regions to move independently. Hinge regions can be
subdivided into three distinct domains: upper, middle, and lower
hinge domains (Roux et al., J. Immunol. 161:4083 (1998)).
[0099] As used herein the term "disulfide bond" includes the
covalent bond formed between two sulfur atoms. The amino acid
cysteine comprises a thiol group that can form a disulfide bond or
bridge with a second thiol group. In most naturally occurring IgG
molecules, the C.sub.H1 and C.sub.L regions are linked by a
disulfide bond and the two heavy chains are linked by two disulfide
bonds at positions corresponding to 239 and 242 using the Kabat
numbering system (position 226 or 229, EU numbering system).
[0100] As used herein, the term "chimeric antibody" will be held to
mean any antibody wherein the immunoreactive region or site is
obtained or derived from a first species and the constant region
(which may be intact, partial or modified in accordance with the
instant invention) is obtained from a second species. In preferred
embodiments the target binding region or site will be from a
non-human source (e.g. mouse or primate) and the constant region is
human.
[0101] As used herein, the term "engineered antibody" refers to an
antibody in which the variable domain in either the heavy and light
chain or both is altered by at least partial replacement of one or
more CDRs from an antibody of known specificity and, if necessary,
by partial framework region replacement and sequence changing.
Although the CDRs may be derived from an antibody of the same class
or even subclass as the antibody from which the framework regions
are derived, it is envisaged that the CDRs will be derived from an
antibody of different class and preferably from an antibody from a
different species. An engineered antibody in which one or more
"donor" CDRs from a non-human antibody of known specificity is
grafted into a human heavy or light chain framework region is
referred to herein as a "humanized antibody." It may not be
necessary to replace all of the CDRs with the complete CDRs from
the donor variable region to transfer the antigen binding capacity
of one variable domain to another. Rather, it may only be necessary
to transfer those residues that are necessary to maintain the
activity of the target binding site. Given the explanations set
forth in, e.g., U. S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762,
and 6,180,370, it will be well within the competence of those
skilled in the art, either by carrying out routine experimentation
or by trial and error testing to obtain a functional engineered or
humanized antibody.
[0102] As used herein the term "properly folded polypeptide"
includes polypeptides (e.g., LINGO-1 antibodies) in which all of
the functional domains comprising the polypeptide are distinctly
active. As used herein, the term "improperly folded polypeptide"
includes polypeptides in which at least one of the functional
domains of the polypeptide is not active. In one embodiment, a
properly folded polypeptide comprises polypeptide chains linked by
at least one disulfide bond and, conversely, an improperly folded
polypeptide comprises polypeptide chains not linked by at least one
disulfide bond.
[0103] As used herein the term "engineered" includes manipulation
of nucleic acid or polypeptide molecules by synthetic means (e.g.
by recombinant techniques, in vitro peptide synthesis, by enzymatic
or chemical coupling of peptides or some combination of these
techniques).
[0104] As used herein, the terms "linked," "fused" or "fusion" are
used interchangeably. These terms refer to the joining together of
two more elements or components, by whatever means including
chemical conjugation or recombinant means. An "in-frame fusion"
refers to the joining of two or more polynucleotide open reading
frames (ORFs) to form a continuous longer ORF, in a manner that
maintains the correct translational reading frame of the original
ORFs. Thus, a recombinant fusion protein is a single protein
containing two ore more segments that correspond to polypeptides
encoded by the original ORFs (which segments are not normally so
joined in nature.) Although the reading frame is thus made
continuous throughout the fused segments, the segments may be
physically or spatially separated by, for example, in-frame linker
sequence. For example, polynucleotides encoding the CDRs of an
immunoglobulin variable region may be fused, in-frame, but be
separated by a polynucleotide encoding at least one immunoglobulin
framework region or additional CDR regions, as long as the "fused"
CDRs are co-translated as part of a continuous polypeptide.
[0105] In the context of polypeptides, a "linear sequence" or a
"sequence" is an order of amino acids in a polypeptide in an amino
to carboxyl terminal direction in which residues that neighbor each
other in the sequence are contiguous in the primary structure of
the polypeptide.
[0106] The term "expression" as used herein refers to a process by
which a gene produces a biochemical, for example, an RNA or
polypeptide. The process includes any manifestation of the
functional presence of the gene within the cell including, without
limitation, gene knockdown as well as both transient expression and
stable expression. It includes without limitation transcription of
the gene into messenger RNA (mRNA), transfer RNA (tRNA), small
hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA
product, and the translation of such mRNA into polypeptide(s). If
the final desired product is a biochemical, expression includes the
creation of that biochemical and any precursors. Expression of a
gene produces a "gene product." As used herein, a gene product can
be either a nucleic acid, e.g., a messenger RNA produced by
transcription of a gene, or a polypeptide which is translated from
a transcript. Gene products described herein further include
nucleic acids with post transcriptional modifications, e.g.,
polyadenylation, or polypeptides with post translational
modifications, e.g., methylation, glycosylation, the addition of
lipids, association with other protein subunits, proteolytic
cleavage, and the like.
[0107] As used herein, the terms "treat" or "treatment" refer to
both therapeutic treatment and prophylactic or preventative
measures, wherein the object is to prevent or slow down (lessen) an
undesired physiological change or disorder, such as the progression
of multiple sclerosis. Beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0108] By "subject" or "individual" or "animal" or "patient" or
"mammal," is meant any subject, particularly a mammalian subject,
for whom diagnosis, prognosis, or therapy is desired. Mammalian
subjects include humans, domestic animals, farm animals, and zoo,
sports, or pet animals such as dogs, cats, guinea pigs, rabbits,
rats, mice, horses, cattle, cows, and so on.
[0109] As used herein, phrases such as "a subject that would
benefit from administration of a LINGO-1 antibody" and "an animal
in need of treatment" includes subjects, such as mammalian
subjects, that would benefit from administration of a LINGO-1
antibody used, e.g., for detection of a LINGO-1 polypeptide (e.g.,
for a diagnostic procedure) and/or from treatment, i.e., palliation
or prevention of a disease such as MS, with a LINGO-1 antibody. As
described in more detail herein, the LINGO-1 antibody can be used
in unconjugated form or can be conjugated, e.g., to a drug,
prodrug, or an isotope.
II. LINGO-1
[0110] Naturally occurring humanLINGO-1 (LINGO-1) is a glycosylated
central nervous system-specific protein which is predicted to have
614 amino acids (SEQ ID NO: 51), including a 33 amino acid signal
sequence. As used herein, the term "LINGO-1" is used
interchangeably with the term "Sp35" as described in International
Applications PCT/US2006/026271, filed Jul. 7, 2006,
PCT/US2004/008323, filed Mar. 17, 2004, PCT/US2005/022881, filed
Jun. 24, 2005 and PCT/US2008/000316, filed Jan. 9, 2008, each of
which is incorporated herein by reference in its entirety. LINGO-1
is also known in the art by the names LRRN6, LRRN6A, FLJ14594,
LERN1, MGC17422 and UNQ201. The human, full-length wild-type
LINGO-1 polypeptide contains an LRR domain consisting of 14
leucine-rich repeats (including N- and C-terminal caps), an Ig
domain, a transmembrane region, and a cytoplasmic domain. The
cytoplasmic domain contains a canonical tyrosine phosphorylation
site. In addition, the naturally occurring LINGO-1 protein contains
a signal sequence, a short basic region between the LRRCT and Ig
domain, and a transmembrane region between the Ig domain and the
cytoplasmic domain. The human LINGO-1 gene (SEQ ID NO:52) contains
alternative translation start codons, so that six additional amino
acids, i.e., MQVSKR (SEQ ID NO:87) may or may not be present at the
N-terminus of the LINGO-1 signal sequence. Table 2 lists the
LINGO-1 domains and other regions, according to amino acid residue
number, based on the LINGO-1 amino acid sequence presented herein
as SEQ ID NO: 51. The LINGO-1 polypeptide is characterized in more
detail in PCT Publication No. WO 2004/085648, which is incorporated
herein by reference in its entirety.
TABLE-US-00002 TABLE 2 LINGO-1 Domains Domain or Region Beginning
Residue Ending Residue Signal Sequence 1 33 or 35 LRRNT 34 or 36 64
LRR 66 89 LRR 90 113 LRR 114 137 LRR 138 161 LRR 162 185 LRR 186
209 LRR 210 233 LRR 234 257 LRR 258 281 LRR 282 305 LRR 306 329 LRR
330 353 LRRCT 363 414 or 416 Basic 415 or 417 424 Ig 419 493
Connecting sequence 494 551 Transmembrane 552 576 Cytoplasmic 577
614
[0111] Tissue distribution and developmental expression of LINGO-1
has been studied in humans and rats. LINGO-1 biology has been
studied in an experimental animal (rat) model. Expression of rat
LINGO-1 is localized to neurons and oligodendrocytes, as determined
by northern blot and immuno-histochemical staining. Rat LINGO-1
mRNA expression level is regulated developmentally, peaking shortly
after birth, i.e., ca. postnatal day one. In a rat spinal cord
transection injury model, LINGO-1 is up-regulated at the injury
site, as determined by RT-PCR. See Mi et al. Nature Neurosci.
7:221-228 (2004).
[0112] In the context of the amino acids comprising the various
structural and functional domains of a LINGO-1 polypeptide, the
term "about" includes the particularly recited value and values
larger or smaller by several (e.g., 10, 9, 8, 7, 6, 5, 4, 3, 2, or
1) amino acids. Since the location of these domains as listed in
Table 2 have been predicted by computer graphics, one of ordinary
skill would appreciate that the amino acid residues constituting
the domains may vary slightly (e.g., by about 1 to 15 residues)
depending on the criteria used to define the domain.
[0113] The inventors have discovered that full-length, wild-type
LINGO-1 binds to NgR1. See PCT Publication No. WO 2004/085648. The
inventors have also discovered that LINGO-1 is expressed in
oligodendrocytes and that the LINGO-1 protein is involved in the
regulation of oligodendrocyte-mediated myelination of axons. See
U.S Patent Publication No. 2006/0009388 A1, which is incorporated
herein by reference in its entirety.
[0114] The nucleotide sequence for the full-length LINGO-1 molecule
is as follows:
TABLE-US-00003 (SEQ ID NO: 52)
ATGCTGGCGGGGGGCGTGAGGAGCATGCCCAGCCCCCTCCTGGCCTGCTG
GCAGCCCATCCTCCTGCTGGTGCTGGGCTCAGTGCTGTCAGGCTCGGCCA
CGGGCTGCCCGCCCCGCTGCGAGTGCTCCGCCCAGGACCGCGCTGTGCTG
TGCCACCGCAAGCGCTTTGTGGCAGTCCCCGAGGGCATCCCCACCGAGAC
GCGCCTGCTGGACCTAGGCAAGAACCGCATCAAAACGCTCAACCAGGACG
AGTTCGCCAGCTTCCCGCACCTGGAGGAGCTGGAGCTCAACGAGAACATC
GTGAGCGCCGTGGAGCCCGGCGCCTTCAACAACCTCTTCAACCTCCGGAC
GCTGGGTCTCCGCAGCAACCGCCTGAAGCTCATCCCGCTAGGCGTCTTCA
CTGGCCTCAGCAACCTGACCAAGCTGGACATCAGCGAGAACAAGATTGTT
ATCCTGCTGGACTACATGTTTCAGGACCTGTACAACCTCAAGTCACTGGA
GGTTGGCGACAATGACCTCGTCTACATCTCTCACCGCGCCTTCACCGGCC
TCAACAGCCTGGAGCAGCTGACGCTGGAGAAATGCAACCTGACCTCCATC
CCCACCGAGGCGCTGTCCCACCTGCACGGCCTCATCGTCCTGAGGCTCCG
GCACCTCAACATCAATGCCATCCGGGACTACTCCTTCAAGAGGCTCTACC
GACTCAAGGTCTTGGAGATCTCCCACTGGCCCTACTTGGACACCATGACA
CCCAACTGCCTCTACGGCCTCAACCTGACGTCCCTGTCCATCACACACTG
CAATCTGACCGCTGTGCCCTACCTGGCCGTCCGCCACCTAGTCTATCTCC
GCTTCCTCAACCTCTCCTACAACCCCATCAGCACCATTGAGGGCTCCATG
TTGCATGAGCTGCTCCGGCTGCAGGAGATCCAGCTGGTGGGCGGGCAGCT
GGCCGTGGTGGAGCCCTATGCCTTCCGCGGCCTCAACTACCTGCGCGTGC
TCAATGTCTCTGGCAACCAGCTGACCACACTGGAGGAATCAGTCTTCCAC
TCGGTGGGCAACCTGGAGACACTCATCCTGGACTCCAACCCGCTGGCCTG
CGACTGTCGGCTCCTGTGGGTGTTCCGGCGCCGCTGGCGGCTCAACTTCA
ACCGGCAGCAGCCCACGTGCGCCACGCCCGAGTTTGTCCAGGGCAAGGAG
TTCAAGGACTTCCCTGATGTGCTACTGCCCAACTACTTCACCTGCCGCCG
CGCCCGCATCCGGGACCGCAAGGCCCAGCAGGTGTTTGTGGACGAGGGCC
ACACGGTGCAGTTTGTGTGCCGGGCCGATGGCGACCCGCCGCCCGCCATC
CTCTGGCTCTCACCCCGAAAGCACCTGGTCTCAGCCAAGAGCAATGGGCG
GCTCACAGTCTTCCCTGATGGCACGCTGGAGGTGCGCTACGCCCAGGTAC
AGGACAACGGCACGTACCTGTGCATCGCGGCCAACGCGGGCGGCAACGAC
TCCATGCCCGCCCACCTGCATGTGCGCAGCTACTCGCCCGACTGGCCCCA
TCAGCCCAACAAGACCTTCGCTTTCATCTCCAACCAGCCGGGCGAGGGAG
AGGCCAACAGCACCCGCGCCACTGTGCCTTTCCCCTTCGACATCAAGACC
CTCATCATCGCCACCACCATGGGCTTCATCTCTTTCCTGGGCGTCGTCCT
CTTCTGCCTGGTGCTGCTGTTTCTCTGGAGCCGGGGCAAGGGCAACACAA
AGCACAACATCGAGATCGAGTATGTGCCCCGAAAGTCGGACGCAGGCATC
AGCTCCGCCGACGCGCCCCGCAAGTTCAACATGAAGATGATATGA.
[0115] The polypeptide sequence for the full-length LINGO-1
polypeptide is as follows:
TABLE-US-00004 (SEQ ID NO: 51)
MLAGGVRSMPSPLLACWQPILLLVLGSVLSGSATGCPPRCECSAQDRAVL
CHRKRFVAVPEGIPTETRLLDLGKNRIKTLNQDEFASFPHLEELELNENI
VSAVEPGAFNNLFNLRTLGLRSNRLKLIPLGVFTGLSNLTKLDISENKIV
ILLDYMFQDLYNLKSLEVGDNDLVYISHRAFSGLNSLEQLTLEKCNLTSI
PTEALSHLHGLIVLRLRHLNINAIRDYSFKRLYRLKVLEISHWPYLDTMT
PNCLYGLNLTSLSITHCNLTAVPYLAVRHLVYLRFLNLSYNPISTIEGSM
LHELLRLQEIQLVGGQLAVVEPYAFRGLNYLRVLNVSGNQLTTLEESVFH
SVGNLETLILDSNPLACDCRLLWVFRRRWRLNFNRQQPTCATPEFVQGKE
FKDFPDVLLPNYFTCRRARIRDRKAQQVFVDEGHTVQFVCRADGDPPPAI
LWLSPRKHLVSAKSNGRLTVFPDGTLEVRYAQVQDNGTYLCIAANAGGND
SMPAHLHVRSYSPDWPHQPNKTFAFISNQPGEGEANSTRATVPFPFDIKT
LIIATTMGFISFLGVVLFCLVLLFLWSRGKGNTKHNIEIEYVPRKSDAGI
SSADAPRKFNMKMI.
III. LINGO-1 Antibodies
[0116] In one embodiment, the present invention is directed to
LINGO-1 antibodies, or antigen-binding fragments, variants, or
derivatives thereof. For example, the present invention includes at
least the antigen-binding domains of Li62, Li81 and fragments,
variants, and derivatives thereof.
[0117] As used herein, the term "antigen binding domain" includes a
site that specifically binds an epitope on an antigen (e.g., an
epitope of LINGO-1). The antigen binding domain of an antibody
typically includes at least a portion of an immunoglobulin heavy
chain variable region and at least a portion of an immunoglobulin
light chain variable region. The binding site formed by these
variable regions determines the specificity of the antibody.
[0118] The present invention is more specifically directed to a
LINGO-1 antibody, or antigen-binding fragment, variant or
derivatives thereof, where the LINGO-1 antibody binds to the same
epitope as Li62 or Li81.
[0119] The invention is further drawn to a LINGO-1 antibody, or
antigen-binding fragment, variant or derivatives thereof, where the
LINGO-1 antibody competitively inhibits Li62 or Li81 from binding
to LINGO-1.
[0120] The invention is also drawn to a LINGO-1 antibody, or
antigen-binding fragment, variant or derivatives thereof, where the
LINGO-1 antibody comprises at least the antigen binding region of
Li62 or Li81.
[0121] In certain embodiments, the present invention is directed to
an antibody, or antigen-binding fragment, variant, or derivative
thereof which specifically or preferentially binds to a particular
LINGO -1 polypeptide fragment or domain. Such LINGO-1 polypeptide
fragments include, but are not limited to, a LINGO-1 polypeptide
comprising, consisting essentially of; or consisting of amino acids
34 to 532; 34 to 417; 34 to 425; 34 to 493; 66 to 532; 66 to 417;
66 to 426; 66 to 493; 66 to 532; 417 to 532; 417 to 425 (the
LINGO-1 basic region); 417 to 493; 417 to 532; 419 to 493 (the
LINGO-1 Ig region); or 425 to 532 of SEQ ID NO:51; or a LINGO-1
variant polypeptide at least 70%, 75%, 80%, 85%, 90%, or 95%
identical to amino acids 34 to 532; 34 to 417; 34 to 425; 34 to
493; 66 to 532; 66 to 417; 66 to 426; 66 to 493; 66 to 532; 417 to
532; 417 to 425 (the LINGO-1 basic region); 417 to 493; 417 to 532;
419 to 493 (the LINGO-1 Ig region); or 425 to 532 of SEQ ID
NO:51.
[0122] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, varaots, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of one or more leucine-rich-repeats (LRR) of LINGO-1.
Such fragments, include, for example, fragments comprising,
consisting essentially of, or consisting of amino acids 66 to 89;
66 to 113; 66 to 137; 90 to 113; 114 to 137; 138 to 161; 162 to
185; 186 to 209; 210 to 233; 234 to 257; 258 to 281; 282 to 305;
306 to 329; or 330 to 353 of SEQ ID NO:51. Corresponding fragments
of a variant LINGO-1 polypeptide at least 70%, 75%, 80%, 85%, 90%,
or 95% identical to amino acids 66 to 89; 66 to 113; 90 to 113; 114
to 137; 138 to 161; 162 to 185; 186 to 209; 210 to 233; 234 to 257;
258 to 281; 282 to 305; 306 to 329; or 330 to 353 of SEQ ID NO:51
are also contemplated.
[0123] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of one or more cysteine rich regions flanking the LRR of
LINGO-1. Such fragments, include, for example, a fragment
comprising, consisting essentially of, or consisting of amino acids
34 to 64 of SEQ ID NO:51 (the N-terminal LRR flanking region
(LRRNT)), or a fragment comprising, consisting essentially of, or
consisting of amino acids 363 to 416 of SEQ ID NO:51 (the
C-terminal LRR flanking region (LRRCT)), amino acids Corresponding
fragments of a variant LINGO-1 polypeptide at least 70%, 75%, 80%,
85%, 90%, or 95% identical to amino acids 34 to 64 and 363 to 416
of SEQ ID NO:51 are also contemplated.
[0124] As known in the art, "sequence identity" between two
polypeptides is determined by comparing the amino acid sequence of
one polypeptide to the sequence of a second polypeptide. When
discussed herein, whether any particular polypeptide is at least
about 70%, 75%, 80%, 85%, 90% or 95% identical to another
polypeptide can be determined using methods and computer
programs/software known in the art such as, but not limited to, the
BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for
Unix, Genetics Computer Group, University Research Park, 575
Science Drive, Madison, Wis. 53711). BESTFIT uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981), to find the best segment of homology
between two sequences. When using BESTFIT or any other sequence
alignment program to determine whether a particular sequence is,
for example, 95% identical to a reference sequence according to the
present invention, the parameters are set, of course, such that the
percentage of identity is calculated over the full length of the
reference polypeptide sequence and that gaps in homology of up to
5% of the total number of amino acids in the reference sequence are
allowed.
[0125] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 41 to 525 of SEQ ID NO:51; 40 to 526 of
SEQ ID NO:51; 39 to 527 of SEQ ID NO:51; 38 to 528 of SEQ ID NO:51;
37 to 529 of SEQ ID NO:51; 36 to 530 of SEQ ID NO:51; 35 to 531 of
SEQ ID NO:51; 34 to 531 of SEQ ID NO:51; 46 to 520 of SEQ ID NO:51;
45 to 521 of SEQ ID NO:51; 44 to 522 of SEQ ID NO:51; 43 to 523 of
SEQ ID NO:51; and 42 to 524 of SEQ ID NO:51.
[0126] Still additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 1 to 33 of SEQ ID NO:51; 1 to 35 of SEQ
ID NO:51; 34 to 64 of SEQ ID NO:51; 36 to 64 of SEQ ID NO:51; 66 to
89 of SEQ ID NO:51; 90 to 113 of SEQ ID NO:51; 114 to 137 of SEQ ID
NO:51; 138 to 161 of SEQ ID NO:51; 162 to 185 of SEQ ID NO:51; 186
to 209 of SEQ ID NO:51; 210 to 233 of SEQ ID NO:51; 234 to 257 of
SEQ ID NO:51; 258 to 281 of SEQ ID NO:51; 282 to 305 of SEQ ID
NO:51; 306 to 329 of SEQ ID NO:51; 330 to 353 of SEQ ID NO:51; 363
to 416 of SEQ ID NO:51; 417 to 424 of SEQ ID NO:51; 419 to 493 of
SEQ ID NO:51; and 494 to 551 of SEQ ID NO:51.
[0127] Further still, LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 1 to 33 of SEQ ID NO:51; 1 to 35 of SEQ
ID NO:51; 1 to 64 of SEQ ID NO:51; 1 to 89 of SEQ ID NO:51; 1 to
113 of SEQ ID NO:51; 1 to 137 of SEQ ID NO:51; 1 to 161 of SEQ ID
NO:51; 1 to 185 of SEQ ID NO:51; 1 to 209 of SEQ ID NO:51; 1 to 233
of SEQ ID NO:51; 1 to 257 of SEQ ID NO:51; 1 to 281 of SEQ ID
NO:51; 1 to 305 of SEQ ID NO:51; 1 to 329 of SEQ ID NO:51; 1 to 353
of SEQ ID NO:51; 1 to 416 of SEQ ID NO:51; 1 to 424 of SEQ ID
NO:51; 1 to 493 of SEQ ID NO:51; 1 to 551 of SEQ ID NO:51; 1 to 531
of SEQ ID NO:51 and 1 to 532 of SEQ ID NO:51.
[0128] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 34 to 64 of SEQ ID NO:51; 34 to 89 of SEQ
ID NO:51; 34 to 113 of SEQ ID NO:51; 34 to 137 of SEQ ID NO:51; 34
to 161 of SEQ ID NO:51; 34 to 185 of SEQ ID NO:51; 34 to 209 of SEQ
ID NO:51; 34 to 233 of SEQ ID NO;51; 34 to 257 of SEQ ID NO:51; 34
to 281 of SEQ ID NO:51; 34 to 305 of SEQ ID NO:51; 34 to 329 of SEQ
ID NO:51; 34 to 353 of SEQ ID NO:51; 34 to 416 of SEQ ID NO:51; 34
to 424 of SEQ ID NO:51; 34 to 493 of SEQ ID NO:51; and 34 to 551 of
SEQ ID NO:51.
[0129] More additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 34 to 530 of SEQ ID NO:51; 34 to 531 of
SEQ ID NO:51; 34 to 532 of SEQ ID NO:51; 34 to 533 of SEQ ID NO:51;
34 to 534 of SEQ ID NO: 51; 34 to 535 of SEQ ID NO:51; 34 to 536 of
SEQ ID NO:51; 34 to 537 of SEQ ID NO:51; 34 to 538 of SEQ ID NO:51;
34 to 539 of SEQ ID NO:51; 30 to 532 of SEQ ID NO:51; 31 to 532 of
SEQ ID NO:51; 32 to 532 of SEQ ID NO:51; 33 to 532 of SEQ ID NO:51;
34 to 532 of SEQ ID NO:51; 35 to 532 of SEQ ID NO:51; 36 to 532 of
SEQ ID NO:51; 30 to 531 of SEQ ID NO:51; 31 to 531 of SEQ ID NO:51;
32 to 531 of SEQ ID NO:51; 33 to 531 of SEQ ID NO:51; 34 to 531 of
SEQ ID NO:51; 35 to 531 of SEQ ID NO:51; and 36 to 531 of SEQ ID
NO:51.
[0130] Further still, LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 36 to 64 of SEQ ID NO:51; 36 to 89 of SEQ
ID NO:51; 36 to 113 of SEQ ID NO:51; 36 to 137 of SEQ ID NO:51; 36
to 161 of SEQ ID NO:51; 36 to 185 of SEQ ID NO:51; 36 to 209 of SEQ
ID NO:51; 36 to 233 of SEQ ID NO:51; 36 to 257 of SEQ ID NO:51; 36
to 281 of SEQ ID NO:51; 36 to 305 of SEQ ID NO:51; 36 to 329 of SEQ
ID NO:51; 36 to 353 of SEQ ID NO:51; 36 to 416 of SEQ ID NO:51; 36
to 424 of SEQ ID NO:51; 36 to 493 of SEQ ID NO:51; and 36 to 551 of
SEQ ID NO:51.
[0131] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, but are not limited
to those fragments comprising, consisting essentially of, or
consisting of amino acids 36 to 530 of SEQ ID NO:51; 36 to 531 of
SEQ ID NO:51; 36 to 532 of SEQ ID NO:51; 36 to 533 of SEQ ID NO:51;
36 to 534 of SEQ ID NO:51; 36 to 535 of SEQ ID NO:51; 36 to 536 of
SEQ ID NO:51; 36 to 537 of SEQ ID NO:51; 36 to 538 of SEQ ID NO:51;
and 36 to 539 of SEQ ID NO:51.
[0132] More LINGO-1 peptide fragments to which certain antibodies,
or antigen-binding fragments, variants, or derivatives thereof of
the present invention bind include, but are not limited to those
fragments comprising, consisting essentially of, or consisting of
amino acids 417 to 493 of SEQ ID NO:51; 417 to 494 of SEQ ID NO:51;
417 to 495 of SEQ ID NO:51; 417 to 496 of SEQ ID NO:51; 417 to 497
of SEQ ID NO:51; 417 to 498 of SEQ ID NO:51; 417 to 499 of SEQ ID
NO:51; 417 to 500 of SEQ ID NO:51; 417 to 492 of SEQ ID NO:51; 417
to 491 of SEQ ID NO:51; 412 to 493 of SEQ ID NO:51; 413 to 493 of
SEQ ID NO:51; 414 to 493 of SEQ ID NO:51; 415 to 493 of SEQ ID
NO:51; 416 to 493 of SEQ ID NO:51; 411 to 493 of SEQ ID NO:51; 410
to 493 of SEQ ID NO:51; 410 to 494 of SEQ ID NO:51; 411 to 494 of
SEQ ID NO:51; 412 to 494 of SEQ ID NO:51; 413 to 494 of SEQ ID
NO:51; 414 to 494 of SEQ ID NO:51; 415 to 494 of SEQ ID NO:51; 416
to 494 of SEQ ID NO:51; 417 to 494 of SEQ ID NO:51; and 418 to 494
of SEQ ID NO:51.
[0133] In an additional embodiment LINGO-1 peptide fragments to
which certain antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the present invention bind include, a
LINGO-1 polypeptide comprising, consisting essentially of, or
consisting of peptides of the Ig domain of LINGO-1 or fragments,
variants, or derivatives of such polypeptides. Specifically,
polypeptides comprising, consisting essentially of, or consisting
of the following polypeptide sequences: ITX.sub.1X.sub.2X.sub.3
(SEQ ID NO:88), ACX.sub.1X.sub.2X.sub.3 (SEQ ID NO:89),
VCX.sub.1X.sub.2X.sub.3 (SEQ ID NO:90) and SPX.sub.1X.sub.2X.sub.3
(SEQ ID NO:91) where X.sub.1 is lysine, arginine, histidine,
glutamine, or asparagine, X.sub.2 is lysine, arginine, histidine,
glutamine, or asparagine and X.sub.3 is lysine, arginine,
histidine, glutamine, or asparagine. For example, LINGO-1 peptide
fragments to which certain antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the present
invention bind include, those fragments comprising, consisting
essentially of, or consisting of the following polypeptide
sequences: SPRKH (SEQ ID NO:92), SPRKK (SEQ ID NO:93), SPRKR (SEQ
ID NO:94), SPKKH (SEQ ID NO:95), SPHKH (SEQ ID NO:96), SPRRH (SEQ
ID NO:97), SPRHH (SEQ ID NO:98), SPRRR (SEQ ID NO:99), SPHHH (SEQ
ID NO:100) SPKKK (SEQ ID NO:101), LSPRKH (SEQ ID NO:102), LSPRKK
(SEQ ID NO:103), LSPRKR (SEQ ID NO:104), LSPKKH (SEQ ID NO:105),
LSPHKH (SEQ ID NO:106), LSPRRH (SEQ ID NO:107), LSPRHH (SEQ ID
NO:108), LSPRRR (SEQ ID NO:109), LSPHHH (SEQ ID NO:110) LSPKKK (SEQ
ID NO:111), WLSPRKH (SEQ ID NO:112), WLSPRKK (SEQ ID NO:113),
WLSPRKR (SEQ ID NO:114), WLSPKKH (SEQ ID NO:115), WLSPHKH (SEQ ID
NO:116), WLSPRRH (SEQ ID NO:117), WLSPRHH (SEQ ID NO:118), WLSPRRR
(SEQ ID NO:119), WLSPHHH (SEQ ID NO:120) WLSPKKK (SEQ ID NO:121).
These LINGO-1 polypeptides include the basic "RKH loop"
(Arginine-Lysine-Histidine amino acids 456-458) in the Ig domain of
LINGO-1. Additional LINGO-1 peptides which include a basic
tripeptide are ITPKRR (SEQ ID NO:122), ACHHK (SEQ ID NO:123) and
VCHHK (SEQ ID NO:124).
[0134] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, a LINGO-1
polypeptide comprising, consisting essentially of, or consisting of
peptides of the Ig domain of LINGO-1 or fragments, variants, or
derivatives of such polypeptides. Specifically, peptides
comprising, consisting essentially of, or consisting of the
following polypeptide sequences: X.sub.4X.sub.5RKH (SEQ ID NO:125),
X.sub.4X.sub.5RRR (SEQ ID NO:126), X.sub.4X.sub.5KKK (SEQ ID
NO:127), X.sub.4X.sub.5HHH (SEQ ID NO:128), X.sub.4X.sub.5RKK (SEQ
ID NO:129), X.sub.4X.sub.5RKR (SEQ ID NO:130), X.sub.4X.sub.5KKH
(SEQ ID NO:131), X.sub.4X.sub.5HKH (SEQ ID NO:132),
X.sub.4X.sub.5RRH (SEQ ID NO:133) and X.sub.4X.sub.5RHH (SEQ ID
NO:134) where X.sub.4 is any amino acid and X.sub.5 is any amino
acid.
[0135] In other embodiments LINGO-1 peptide fragments to which
certain antibodies, or antigen-binding fragments, variants, or
derivatives thereof of the present invention bind include, a
LINGO-1 polypeptide comprising, consisting essentially of, or
consisting of peptides of the Ig domain of LINGO-1 or fragments,
variants, or derivatives of such polypeptides. Specifically,
polypeptides comprising, consisting essentially of, or consisting
of the following polypeptide sequences: ITX.sub.6X.sub.7X.sub.8
(SEQ ID NO:135), ACX.sub.6X.sub.7X.sub.8 (SEQ ID NO:136),
VCX.sub.6X.sub.7X.sub.8 (SEQ ID NO:137) and SPX.sub.6X.sub.7X.sub.8
(SEQ ID NO:138) where X.sub.6 is lysine, arginine, histidine,
glutamine, or asparagine, X.sub.7 is any amino acid and X.sub.8 is
lysine, arginine, histidine, glutamine, or asparagine. For example,
a polypeptide comprising, consisting essentially of, or consisting
of the following polypeptide sequence: SPRLH (SEQ ID NO:139).
[0136] LINGO-1 peptide fragments to which certain antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
present invention bind include, a LINGO-1 polypeptide comprising,
consisting essentially of, or consisting of peptides which contain
amino acids 452-458 in the Ig domain of LINGO-1, or derivatives
thereof, wherein amino acid 452 is a tryptophan or phenylalanine
residue.
[0137] Additional LINGO-1 peptide fragments to which certain
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the present invention bind include, a LINGO-1
polypeptide comprising, consisting essentially of, or consisting of
peptides of the basic domain of LINGO-1. Specifically, peptides
comprising, consisting essentially of, or consisting of the
following polypeptide sequences: RRARIRDRK (SEQ ID NO:140),
KKVKVKEKR (SEQ ID NO:141), RRLRLRDRK (SEQ ID NO:142), RRGRGRDRK
(SEQ ID NO:143) and RRIRARDRK (SEQ ID NO:144).
[0138] Additional exemplary soluble LINGO-1 polypeptides and
methods and materials for obtaining these molecules for producing
antibodies or antibody fragments of the present invention may be
found, e.g., in International Patent Application No.
PCT/US2004/008323, incorporated herein by reference in its
entirety.
[0139] Methods of making antibodies are well known in the art and
described herein. Once antibodies to various fragments of, or to
the full-length LINGO-1 without the signal sequence, have been
produced, determining which amino acids, or epitope, of LINGO-1 to
which the antibody or antigen binding fragment binds can be
determined by eptiope mapping protocols as described herein as well
as methods known in the art (e.g. double antibody-sandwich ELISA as
described in "Chapter 11--Immunology," Current Protocols in
Molecular Biology, Ed. Ausubel et al., v.2, John Wiley & Sons,
Inc. (1996)). Additional epitope mapping protocols may be found in
Morris, G. Epitope Mapping Protocols, New Jersey: Humana Press
(1996), which are both incorporated herein by reference in their
entireties. Epitope mapping can also be performed by commercially
available means (i.e. ProtoPROBE, Inc. (Milwaukee, Wis.)).
[0140] Additionally, antibodies produced which bind to any portion
of LINGO-1 can then be screened for their ability to act as an
antagonist of LINGO-1 and thus promote neurite outgrowth, neuronal
and oligodendrocyte survival, proliferation and differentiation as
well as promote myelination. Antibodies can be screened for
oligodendrocyte/neuronal survival for example by using the methods
described herein such as in Examples 11 or 12 or as described in
PCT/US2008/000316, filed Jan. 9, 2008, and PCT/US2006/026271, filed
Jul. 7, 2006, which are incorporated herein by reference in their
entireties. Additionally, antibodies can be screened for example by
their ability to promote myelination by using the methods described
herein such as in Examples 2, 6, 9, 10, 11 or 13 or as described in
PCT/US2008/000316 and/or PCT/US2006/026271. Finally, antibodies can
be screened for their ability to promote oligodendrocyte
proliferation and differentiation, as well as neurite outgrowth for
example by using the methods described herein such as in Examples 4
or 5 or as described in PCT/US2008/000316 and/or PCT/US2006/026271.
Other antagonist functions of antibodies of the present invention
can be tested using other assays as described in the Examples
herein.
[0141] In other embodiments, the present invention includes an
antibody, or antigen-binding fragment, variant, or derivative
thereof which specifically or preferentially binds to at least one
epitope of LINGO-1, where the epitope comprises, consists
essentially of, or consists of at least about four to five amino
acids of SEQ ID NO:51, at least seven, at least nine, or between at
least about 15 to about 30 amino acids of SEQ ID NO:51. The amino
acids of a given epitope of SEQ ID NO:51 as described may be, but
need not be contiguous or linear. In certain embodiments, the at
least one epitope of LINGO-1 comprises, consists essentially of, or
consists of a non-linear epitope formed by the extracellular domain
of LINGO-1 as expressed on the surface of a cell or as a soluble
fragment, e.g., fused to an IgG Fc region. Thus, in certain
embodiments the at least one epitope of LINGO-1 comprises, consists
essentially of, or consists of at least 4, at least 5, at least 6,
at least 7, at least 8, at least 9, at least 10, at least 15, at
least 20, at least 25, between about 15 to about 30, or at least
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, or 100 contiguous or non-contiguous amino acids of SEQ ID
NO:51, where the non-contiguous amino acids form an epitope through
protein folding.
[0142] In other embodiments, the present invention includes an
antibody, or antigen-binding fragment, variant, or derivative
thereof which specifically or preferentially binds to at least one
epitope of LINGO-1, where the epitope comprises, consists
essentially of, or consists of, in addition to one, two, three,
four, five, six or more contiguous or non-contiguous amino acids of
SEQ ID NO:51 as described above, and an additional moiety which
modifies the protein, e.g., a carbohydrate moiety may be included
such that the LINGO-1 antibody binds with higher affinity to
modified target protein than it does to an unmodified version of
the protein. Alternatively, the LINGO-1 antibody does not bind the
unmodified version of the target protein at all.
[0143] In certain aspects, the present invention is directed to an
antibody, or antigen-binding fragment, variant, or derivative
thereof which specifically binds to a LINGO-1 polypeptide or
fragment thereof, or a LINGO-1 variant polypeptide, with an
affinity characterized by a dissociation constant (K.sub.D) which
is less than the K.sub.D for said reference monoclonal
antibody.
[0144] In certain embodiments, an antibody, or antigen-binding
fragment, variant, or derivative thereof of the invention binds
specifically to at least one epitope of LINGO-1 or fragment or
variant described above, i.e., binds to such an epitope more
readily than it would bind to an unrelated, or random epitope;
binds preferentially to at least one epitope of LINGO-1 or fragment
or variant described above, i.e., binds to such an epitope more
readily than it would bind to a related, similar, homologous, or
analogous epitope; competitively inhibits binding of a reference
antibody which itself binds specifically or preferentially to a
certain epitope of LINGO-1 or fragment or variant described above;
or binds to at least one epitope of LINGO-1 or fragment or variant
described above with an affinity characterized by a dissociation
constant K.sub.D of less than about 5.times.10.sup.-2 M, about
10.sup.-2 M, about 5.times.10.sup.-3 M, about 10.sup.-3 M, about
5.times.10.sup.-4 M, about 10.sup.-4 M, about 5.times.10.sup.-5 M,
about 10.sup.-5 M, about 5.times.10.sup.-6 M, about 10.sup.-6 M,
about 5.times.10.sup.-7 M, about 10.sup.-7 M, about
5.times.10.sup.-8 M, about 10.sup.-8 M, about 5.times.10.sup.-9 M,
about 10.sup.-9 M, about 5.times.10.sup.-10 M, about 10.sup.-10 M,
about 5.times.10.sup.-11 M, about 10.sup.-11 M, about
5.times.10.sup.-12 M, about 10.sup.-12 M, about 5.times.10.sup.-13
M, about 10.sup.-13 M, about 5.times.10.sup.-14 M, about 10.sup.-14
M, about 5.times.10.sup.-15 M, or about 10.sup.-15 M. In a
particular aspect, the antibody or fragment thereof preferentially
binds to a human LINGO-1 polypeptide or fragment thereof, relative
to a murine LINGO-1 polypeptide or fragment thereof.
[0145] As used in the context of antibody binding dissociation
constants, the term "about" allows for the degree of variation
inherent in the methods utilized for measuring antibody affinity.
For example, depending on the level of precision of the
instrumentation used, standard error based on the number of samples
measured, and rounding error, the term "about 10.sup.-2 M" might
include, for example, from 0.05 M to 0.005 M.
[0146] In specific embodiments, an antibody, or antigen-binding
fragment, variant, or derivative thereof of the invention binds
LINGO-1 polypeptides or fragments or variants thereof with an off
rate (k(off)) of less than or equal to 5.times.10.sup.-2
sec.sup.-1, 10.sup.-2 sec.sup.-1, 5.times.10.sup.-3 sec.sup.-1 or
10.sup.-3 sec.sup.-1. Alternatively, an antibody, or
antigen-binding fragment, variant, or derivative thereof of the
invention binds LINGO-1 polypeptides or fragments or variants
thereof with an off rate (k(off)) of less than or equal to
5.times.10.sup.-4 sec.sup.-1, 10.sup.-4 sec.sup.-1,
5.times.10.sup.-5 sec.sup.-1, or 10.sup.-5 sec.sup.-1
5.times.10.sup.-6 sec.sup.-1, 10.sup.-6 sec.sup.-1,
5.times.10.sup.-7 sec.sup.-1 or 10.sup.-7 sec.sup.-1.
[0147] In other embodiments, an antibody, or antigen-binding
fragment, variant, or derivative thereof of the invention binds
LINGO-1 polypeptides or fragments or variants thereof with an on
rate (k(on)) of greater than or equal to 10.sup.3 M.sup.-1
sec.sup.-1, 5.times.10.sup.3 M.sup.-1 sec.sup.-1, 10.sup.4 M.sup.-1
sec.sup.-1 or 5.times.10.sup.4 M.sup.-1 sec.sup.-1. Alternatively,
an antibody, or antigen-binding fragment, variant, or derivative
thereof of the invention binds LINGO-1 polypeptides or fragments or
variants thereof with an on rate (k(on)) greater than or equal to
10.sup.5 M.sup.-1 sec.sup.-1, 5.times.10.sup.5 M.sup.-1 sec.sup.-1,
10.sup.6 M.sup.-1 sec.sup.-1, or 5.times.106 M.sup.-1 sec.sup.-1 or
10.sup.7 M.sup.-1 sec.sup.-1.
[0148] In various embodiments, a LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof as
described herein is an antagonist of LINGO-1 activity. In certain
embodiments, for example, binding of an antagonist LINGO-1 antibody
to LINGO-1, as expressed on neurons, blocks myelin-associated
neurite outgrowth inhibition or neuronal cell death. In other
embodiments, binding of the LINGO-1 antibody to LINGO-1, as
expressed on oligodendrocytes, blocks inhibition of oligodendrocyte
growth or differentiation, or blocks demyelination or
dysmyelination of CNS neurons.
[0149] Unless it is specifically noted, as used herein a "fragment
thereof" in reference to an antibody refers to an antigen-binding
fragment, i.e., a portion of the antibody which specifically binds
to the antigen. In one embodiment, a LINGO-1 antibody, e.g., an
antibody of the invention is a bispecific LINGO-1 antibody, binding
polypeptide, or antibody, e.g., a bispecific antibody, minibody,
domain deleted antibody, or fusion protein having binding
specificity for more than one epitope, e.g., more than one antigen
or more than one epitope on the same antigen. In one embodiment, a
bispecific LINGO-1 antibody, binding polypeptide, or antibody has
at least one binding domain specific for at least one epitope on a
target polypeptide disclosed herein, e.g., LINGO-1. In another
embodiment, a bispecific LINGO-1 antibody, binding polypeptide, or
antibody has at least one binding domain specific for an epitope on
a target polypeptide and at least one target binding domain
specific for a drug or toxin. In yet another embodiment, a
bispecific LINGO-1 antibody, binding polypeptide, or antibody has
at least one binding domain specific for an epitope on a target
polypeptide disclosed herein, and at least one binding domain
specific for a prodrug. A bispecific LINGO-1 antibody, binding
polypeptide, or antibody may be a tetravalent antibody that has two
target binding domains specific for an epitope of a target
polypeptide disclosed herein and two target binding domains
specific for a second target. Thus, a tetravalent bispecific
LINGO-1 antibody, binding polypeptide, or antibody may be bivalent
for each specificity.
[0150] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention, as known by those of
ordinary skill in the art, can comprise a constant region which
mediates one or more effector functions. For example, binding of
the C1 component of complement to an antibody constant region may
activate the complement system. Activation of complement is
important in the opsonisation and lysis of cell pathogens. The
activation of complement also stimulates the inflammatory response
and may also be involved in autoimmune hypersensitivity. Further,
antibodies bind to receptors on various cells via the Fc region,
with a Fc receptor binding site on the antibody Fc region binding
to a Fc receptor (FcR) on a cell. There are a number of Fc
receptors which are specific for different classes of antibody,
including IgG (gamma receptors), IgE (epsilon receptors), IgA
(alpha receptors) and IgM (mu receptors). Binding of antibody to Fc
receptors on cell surfaces triggers a number of important and
diverse biological responses including engulfment and destruction
of antibody-coated particles, clearance of immune complexes, lysis
of antibody-coated target cells by killer cells (called
antibody-dependent cell-mediated cytotoxicity, or ADCC), release of
inflammatory mediators, placental transfer and control of
immunoglobulin production.
[0151] Accordingly, certain embodiments of the invention include a
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof, in which at least a fraction of one or more of
the constant region domains has been deleted or otherwise altered
so as to provide desired biochemical characteristics such as
reduced effector functions, the ability to non-covalently dimerize,
increased ability to localize at the site of a tumor, reduced serum
half-life, or increased serum half-life when compared with a whole,
unaltered antibody of approximately the same inummogenicity. For
example, certain antibodies for use in the diagnostic and treatment
methods described herein are domain deleted antibodies which
comprise a polypeptide chain similar to an immunoglobulin heavy
chain, but which lack a least a portion of one or more heavy chain
domains. For instance, in certain antibodies, one entire domain of
the constant region of the modified antibody will be deleted, for
example, all or part of the C.sub.H2 domain will be deleted.
[0152] In certain LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof described herein, the Fc portion
may be mutated to decrease effector function using, techniques
known in the art. For example, the deletion or inactivation
(through point mutations or other means) of a constant region
domain may reduce Fc receptor binding of the circulating modified
antibody thereby increasing tumor localization. In other cases it
may be that constant region modifications consistent with the
instant invention moderate complement binding and thus reduce the
serum half life and nonspecific association of a conjugated
cytotoxin. Yet other modifications of the constant region may be
used to modify disulfide linkages or oligosaccharide moieties that
allow for enhanced localization due to increased antigen
specificity or antibody flexibility. The resulting physiological
profile, bioavailability and other biochemical effects of the
modifications, such as tumor localization, biodistribution and
serum half-life, may easily be measured and quantified using well
know immunological techniques without undue experimentation.
[0153] Modified forms of LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention can be
made from whole precursor or parent antibodies using techniques
known in the art. Exemplary techniques are discussed in more detail
herein.
[0154] In certain embodiments both the variable and constant
regions of LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof are fully human. Fully human
antibodies can be made using techniques that are known in the art
and as described herein. For example, fully human antibodies
against a specific antigen can be prepared by administering the
antigen to a transgenic animal which has been modified to produce
such antibodies in response to antigenic challenge, but whose
endogenous loci have been disabled. Exemplary techniques that can
be used to make such antibodies are described in U.S. Pat. Nos.
6,150,584; 6,458,592; 6,420,140 which are incorporated by reference
in their entireties. Other techniques are known in the art. Fully
human antibodies can likewise be produced by various display
technologies, e.g., phage display or other viral display systems,
as described in more detail elsewhere herein.
[0155] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention can be made or manufactured
using techniques that are known in the art. In certain embodiments,
antibody molecules or fragments thereof are "recombinantly
produced," i.e., are produced using recombinant DNA technology.
Exemplary techniques for making antibody molecules or fragments
thereof are discussed in more detail elsewhere herein.
[0156] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention also include derivatives
that are modified, e.g., by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody from specifically binding to its cognate
epitope. For example, but not by way of limitation, the antibody
derivatives include antibodies that have been modified, e.g., by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or
more non-classical amino acids.
[0157] In certain embodiments, LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention will not elicit a deleterious immune response in the
animal to be treated, e.g., in a human. In one embodiment, LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the invention are modified to reduce their
immunogenicity using art-recognized techniques. For example,
antibodies can be humanized, primatized, deimmunized, or chimeric
antibodies can be made. These types of antibodies are derived from
a non-human antibody, typically a murine or primate antibody, that
retains or substantially retains the antigen-binding properties of
the parent antibody, but which is less immunogenic in humans. This
may be achieved by various methods, including (a) grafting the
entire non-human variable domains onto human constant regions to
generate chimeric antibodies; (b) grafting at least a part of one
or more of the non-human complementarity determining regions (CDRs)
into a human framework and constant regions with or without
retention of critical framework residues; or (c) transplanting the
entire non-human variable domains, but "cloaking" them with a
human-like section by replacement of surface residues. Such methods
are disclosed in Morrison et al., Proc. Natl. Acad. Sci.
81:6851-6855 (1984); Morrison at al., Adv. Immunol. 44:65-92
(1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan,
Molec. Immun. 28:489-498 (1991); Padlan, Molec. Immun. 31:169-217
(1994), and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, and
6,190,370, all of which are hereby incorporated by reference in
their entirety.
[0158] De-immunization can also be used to decrease the
immunogenicity of an antibody. As used herein, the term
"de-immunization" includes alteration of an antibody to modify T
cell epitopes (see, e.g., WO9852976A1, WO0034317A2). For example,
V.sub.H and V.sub.L sequences from the starting antibody are
analyzed and a human T cell epitope "map" from each V region
showing the location of epitopes in relation to
complementarity-determining regions (CDRs) and other key residues
within the sequence. Individual T cell epitopes from the T cell
epitope map are analyzed in order to identify alternative amino
acid substitutions with a low risk of altering activity of the
final antibody. A range of alternative V.sub.H and V.sub.L
sequences are designed comprising combinations of amino acid
substitutions and these sequences are subsequently incorporated
into a range of binding polypeptides, e.g., LINGO-1-specific
antibodies or immunospecific fragments thereof for use in the
diagnostic and treatment methods disclosed herein, which are then
tested for function. Typically, between 12 and 24 variant
antibodies are generated and tested. Complete heavy and light chain
genes comprising modified V and human C regions are then cloned
into expression vectors and the subsequent plasmids introduced into
cell lines for the production of whole antibody. The antibodies are
then compared in appropriate biochemical and biological assays, and
the optimal variant is identified.
[0159] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention may be generated by any
suitable method known in the art. Polyclonal antibodies to an
antigen of interest can be produced by various procedures well know
in the art. For example, a LINGO-1 antibody, e.g., a binding
polypeptide, e.g., a LINGO-1-specific antibody or immunospecific
fragment thereof can be administered to various host animals
including, but not limited to, rabbits, mice, rats, chickens,
hamsters, goats, donkeys, etc., to induce the production of sera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Such adjuvants are also well known in the art.
[0160] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, 2nd ed. (1988); Hammerling et
al., in: Monoclonal Antibodies and T-Cell Hybridomas Elsevier,
N.Y., 563-681 (1981) (said references incorporated by reference in
their entireties). The term "monoclonal antibody" as used herein is
not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced. Thus,
the term "monoclonal antibody" is not limited to antibodies
produced through hybridoma technology. Monoclonal antibodies can be
prepared using LINGO-1 knockout mice to increase the regions of
epitope recognition. Monoclonal antibodies can be prepared using a
wide variety of techniques known in the art including the use of
hybridoma and recombinant and phage display technology as described
elsewhere herein.
[0161] Using art recognized protocols, in one example, antibodies
are raised in mammals by multiple subcutaneous or intraperitoneal
injections of the relevant antigen (e.g., purified tumor associated
antigens such as LINGO-1 or cells or cellular extracts comprising
such antigens) and an adjuvant. This immunization typically elicits
an immune response that comprises production of antigen-reactive
antibodies from activated splenocytes or lymphocytes. While the
resulting antibodies may be harvested from the serum of the animal
to provide polyclonal preparations, it is often desirable to
isolate individual lymphocytes from the spleen, lymph nodes or
peripheral blood to provide homogenous preparations of monoclonal
antibodies (MAbs). Preferably, the lymphocytes are obtained from
the spleen.
[0162] In this well known process (Kohler et al., Nature 256:495
(1975)) the relatively short-lived, or mortal, lymphocytes from a
mammal which has been injected with antigen are fused with an
immortal tumor cell line (e.g. a myeloma cell line), thus,
producing hybrid cells or "hybridomas" which are both immortal and
capable of producing the genetically coded antibody of the B cell.
The resulting hybrids are segregated into single genetic strains by
selection, dilution, and regrowth with each individual strain
comprising specific genes for the formation of a single antibody.
They produce antibodies which are homogeneous against a desired
antigen and, in reference to their pure genetic parentage, are
termed "monoclonal."
[0163] Hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. Those skilled in the art will appreciate
that reagents, cell lines and media for the formation, selection
and growth of hybridomas are commercially available from a number
of sources and standardized protocols are well established.
Generally, culture medium in which the hybridoma cells are growing
is assayed for production of monoclonal antibodies against the
desired antigen. Preferably, the binding specificity of the
monoclonal antibodies produced by hybridoma cells is determined by
in vitro assays such as immunoprecipitation, radioimmunoassay (RIA)
or enzyme-linked immunoabsorbent assay (ELISA). After hybridoma
cells are identified that produce antibodies of the desired
specificity, affinity and/or activity, the clones may be subcloned
by limiting dilution procedures and grown by standard methods
(Goding, Monoclonal Antibodies: Principles and Practice, Academic
Press, pp 59-103 (1986)). It will further be appreciated that the
monoclonal antibodies secreted by the subclones may be separated
from culture medium, ascites fluid or serum by conventional
purification procedures such as, for example, protein-A,
hydroxylapatite chromatography, gel electrophoresis, dialysis or
affinity chromatography.
[0164] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the variable region, the light chain constant
region and the C.sub.H1 domain of the heavy chain.
[0165] Those skilled in the art will also appreciate that DNA
encoding antibodies or antibody fragments (e.g., antigen binding
sites) may also be derived from antibody libraries, such as phage
display libraries. In a particular, such phage can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 binding domains expressed from phage with Fab,
Fv OE DAB (individual Fv region from light or heavy chains) or
disulfide stabilized Fv antibody domains recombinantly fused to
either the phage gene III or gene VIII protein. Exemplary methods
are set forth, for example, in EP 368 684 B1; U.S. Pat. No.
5,969,108, Hoogenboom, H. R. and Chames, Immunol. Today 21:371
(2000); Nagy et al. Nat. Med. 8:801 (2002); Huie et al., Proc.
Natl. Acad Sci. USA 98:2682 (2001); Lui et al., J. Mol. Biol.
315:1063 (2002), each of which is incorporated herein by reference.
Several publications (e.g., Marks et al., Bio/Technology 10:779-783
(1992)) have described the production of high affinity human
antibodies by chain shuffling, as well as combinatorial infection
and in vivo recombination as a strategy for constructing large
phage libraries. In another embodiment, Ribosomal display can be
used to replace bacteriophage as the display platform (see, e.g.,
Hanes et al., Nat. Biotechnol. 18:1287 (2000); Wilson et al., Proc.
Natl. Acad Sci. USA 98:3750 (2001); or Irving et al., J. Immunol
Methods 248:31 (2001)). In yet another embodiment, cell surface
libraries can be screened for antibodies (Boder et al., Proc. Natl.
Acad. Sci. USA 97:10701 (2000); Daugherty et al., J. Immunol.
Methods 243:211 (2000)). Such procedures provide alternatives to
traditional hybridoma techniques for the isolation and subsequent
cloning of monoclonal antibodies.
[0166] In phage display methods, functional antibody domains are
displayed on the surface of phage particles which carry the
polynucleotide sequences encoding them. For example, DNA sequences
encoding V.sub.H and V.sub.L regions are amplified from animal cDNA
libraries (e.g., human or murine cDNA libraries of lymphoid
tissues) or synthetic cDNA libraries. In certain embodiments, the
DNA encoding the V.sub.H and V.sub.L regions are joined together by
an scFv linker by PCR and cloned into a phagemid vector (e.g., p
CANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli
and the E. coli is infected with helper phage. Phage used in these
methods are typically filamentous phage including fd and M13 and
the V.sub.H or V.sub.L regions are usually recombinantly fused to
either the phage gene III or gene VIII. Phage expressing an antigen
binding domain that binds to an antigen of interest (i.e., a
LINGO-1 polypeptide or a fragment thereof) can be selected or
identified with antigen, e.g., using labeled antigen or antigen
bound or captured to a solid surface or bead.
[0167] Additional examples of phage display methods that can be
used to make the antibodies include those disclosed in Brinkman et
al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol.
Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
24:952-958 (1994); Persic et al., Gene 187:9-18 (1997); Burton et
al., Advances in Immunology 57:191-280 (1994); PCT Application No.
PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO
92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and
U.S. Pat. Nos. 5,698,426; 5,223,40; 5,403,484; 5,580,717;
5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637;
5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is
incorporated herein by reference in its entirety.
[0168] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab').sub.2 fragments can also be employed
using methods known in the art such as those disclosed in PCT
publication WO 92/22324; Mullinax et al., BioTechniques
12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34 (1995); and
Better et al., Science 240:1041-1043 (1988) (said references
incorporated by reference in their entireties).
[0169] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in
Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993);
and Skerra et al., Science 240:1038-1040 (1988). For some uses,
including in vivo use of antibodies in humans and in vitro
detection assays, it may be preferable to use chimeric, humanized,
or human antibodies. A chimeric antibody is a molecule in which
different portions of the antibody are derived from different
animal species, such as antibodies having a variable region derived
from a murine monoclonal antibody and a human immunoglobulin
constant region. Methods for producing chimeric antibodies are
known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi
et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol.
Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567;
and 4,816397, which are incorporated herein by reference in their
entireties. Humanized antibodies are antibody molecules derived
from a non-human species antibody that bind the desired antigen
having one or more complementarity determining regions (CDRs) from
the non-human species and framework regions from a human
immunoglobulin molecule. Often, framework residues in the human
framework regions will be substituted with the corresponding
residue from the CDR donor antibody to alter, preferably improve,
antigen binding. These framework substitutions are identified by
methods well known in the art, e.g., by modeling of the
interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089;
Riechmann et al., Nature 332:323 (1988), which are incorporated
herein by reference in their entireties.) Antibodies can be
humanized using a variety of techniques known in the art including,
for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967;
U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or
resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology
28(4/5):489-498 (1991); Studnicka et al., Protein Engineering
7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and
chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by
reference in its entirety).
[0170] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See also, U.S. Pat. Nos.
4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO
98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and
WO 91/10741; each of which is incorporated herein by reference in
its entirety.
[0171] Human antibodies can also be produced using transgenic mice
which are incapable of expressing functional endogenous
immunoglobulins, but which can express human immunoglobulin genes.
For example, the human heavy and light chain immunoglobulin gene
complexes may be introduced randomly or by homologous recombination
into mouse embryonic stem cells. Alternatively, the human variable
region, constant region, and diversity region may be introduced
into mouse embryonic stem cells in addition to the human heavy and
light chain genes. The mouse heavy and light chain immunoglobulin
genes may be rendered non-functional separately or simultaneously
with the introduction of human immunoglobulin loci by homologous
recombination. In particular, homozygous deletion of the JH region
prevents endogenous antibody production. The modified embryonic
stem cells are expanded and microinjected into blastocysts to
produce chimeric mice. The chimeric mice are then bred to produce
homozygous offspring that express human antibodies. The transgenic
mice are immunized in the normal fashion with a selected antigen,
e.g., all or a portion of a desired target polypeptide. Monoclonal
antibodies directed against the antigen can be obtained from the
immunized, transgenic mice using conventional hybridoma technology.
The human immunoglobulin transgenes harbored by the transgenic mice
rearrange during B-cell differentiation, and subsequently undergo
class switching and somatic mutation. Thus, using such a technique,
it is possible to produce therapeutically useful IgG, IgA, IgM and
IgE antibodies. For an overview of this technology for producing
human antibodies, see Lonberg and Huszar Int. Rev. Immunol.
13:65-93 (1995). For a detailed discussion of this technology for
producing human antibodies and human monoclonal antibodies and
protocols for producing such antibodies, see, e.g., PCT
publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.
5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;
5,814,318; and 5,939,598, which are incorporated by reference
herein in their entirety. In addition, companies such as Abgenix,
Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
[0172] Completely human antibodies which recognize a selected
epitope can be generated using a technique referred to as "guided
selection." In this approach a selected non-human monoclonal
antibody, e.g., a mouse antibody, is used to guide the selection of
a completely human antibody recognizing the same epitope. (Jespers
et al., Bio/Technology 12:899-903 (1988). See also, U.S. Pat. No.
5,565,332, which is incorporated by reference in its entirety.)
[0173] Further, antibodies to target polypeptides of the invention
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" target polypeptides using techniques well known to those
skilled in the art. (See, e.g., Greenspan & Bona, FASEB J.
7(5):437-444 (1989) and Nissinoff, J. Immunol. 147(8):2429-2438
(1991)). For example, antibodies which bind to and competitively
inhibit polypeptide multimerization and/or binding of a polypeptide
of the invention to a ligand can be used to generate anti-idiotypes
that "mimic" the polypeptide multimerization and/or binding domain
and, as a consequence, bind to and neutralize polypeptide and/or
its ligand. Such neutralizing anti-idiotypes or Fab fragments of
such anti-idiotypes can be used in therapeutic regimens to
neutralize polypeptide ligand. For example, such anti-idiotypic
antibodies can be used to bind a desired target polypeptide and/or
to bind its ligands/receptors, and thereby block its biological
activity.
[0174] In another embodiment, DNA encoding desired monoclonal
antibodies may be readily isolated and sequenced using conventional
procedures (e.g., by using oligonucleotide probes that are capable
of binding specifically to genes encoding the heavy and light
chains of murine antibodies). The isolated and subcloned hybridoma
cells serve as a preferred source of such DNA. Once isolated, the
DNA may be placed into expression vectors, which are then
transfected into prokaryotic or eukaryotic host cells such as E.
coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells or
myeloma cells that do not otherwise produce immunoglobulins. More
particularly, the isolated DNA (which may be synthetic as described
herein) may be used to clone constant and variable region sequences
for the manufacture antibodies as described in Newman et al., U.S.
Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated by
reference herein. Essentially, this entails extraction of RNA from
the selected cells, conversion to cDNA, and amplification by PCR
using Ig specific primers. Suitable primers for this purpose are
also described in U.S. Pat. No. 5,658,570. As will be discussed in
more detail below, transformed cells expressing the desired
antibody may be grown up in relatively large quantities to provide
clinical and commercial supplies of the immunoglobulin.
[0175] In one embodiment, a LINGO-1 antibody of the invention
comprises at least one heavy or light chain CDR of an antibody
molecule. In another embodiment, a LINGO-1 antibody of the
invention comprises at least two CDRs from one or more antibody
molecules. In another embodiment, a LINGO-1 antibody of the
invention comprises at least three CDRs from one or more antibody
molecules. In another embodiment, a LINGO-1 antibody of the
invention comprises at least four CDRs from one or more antibody
molecules. In another embodiment, a LINGO-1 antibody of the
invention comprises at least five CDRs from one or more antibody
molecules. In another embodiment, a LINGO-1 antibody of the
invention comprises at least six CDRs from one or more antibody
molecules. Exemplary antibody molecules comprising at least one CDR
that can be included in the subject LINGO-1 antibodies are
described herein.
[0176] In a specific embodiment, the amino acid sequence of the
heavy and/or light chain variable domains may be inspected to
identify the sequences of the complementarity determining regions
(CDRs) by methods that are well know in the art, e.g., by
comparison to known amino acid sequences of other heavy and light
chain variable regions to determine the regions of sequence
hypervariability. Using routine recombinant DNA techniques, one or
more of the CDRs may be inserted within framework regions, e.g.,
into human framework regions to humanize a non-human antibody. The
framework regions may be naturally occurring or consensus framework
regions, and preferably human framework regions (see, e.g., Chothia
et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human
framework regions). Preferably, the polynucleotide generated by the
combination of the framework regions and CDRs encodes an antibody
that specifically binds to at least one epitope of a desired
polypeptide, e.g., LINGO-1. Preferably, one or more amino acid
substitutions may be made within the framework regions, and,
preferably, the amino acid substitutions improve binding of the
antibody to its antigen. Additionally, such methods may be used to
make amino acid substitutions or deletions of one or more variable
region cysteine residues participating in an intrachain disulfide
bond to generate antibody molecules lacking one or more intrachain
disulfide bonds. Other alterations to the polynucleotide are
encompassed by the present invention and within the skill of the
art.
[0177] In addition, techniques developed for the production of
"chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci.
81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984);
Takeda et al., Nature 314:452-454 (1985)) by splicing genes from a
mouse antibody molecule of appropriate antigen specificity together
with genes from a human antibody molecule of appropriate biological
activity can be used. As used herein, a chimeric antibody is a
molecule in which different portions are derived from different
animal species, such as those having a variable region derived from
a murine monoclonal antibody and a human immunoglobulin constant
region, e.g., humanized antibodies.
[0178] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,694,778; Bird, Science
242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989))
can be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain antibody. Techniques for the assembly of functional Fv
fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[0179] Yet other embodiments of the present invention comprise the
generation of human or substantially human antibodies in transgenic
animals (e.g., mice) that are incapable of endogenous
immunoglobulin production (see e.g., U.S. Pat. Nos. 6,075,181,
5,939,598, 5,591,669 and 5,589,369 each of which is incorporated
herein by reference). For example, it has been described that the
homozygous deletion of the antibody heavy-chain joining region in
chimeric and germ-line mutant mice results in complete inhibition
of endogenous antibody production. Transfer of a human
immunoglobulin gene array to such germ line mutant mice will result
in the production of human antibodies upon antigen challenge.
Another preferred means of generating human antibodies using SCID
mice is disclosed in U.S. Pat. No. 5,811,524 which is incorporated
herein by reference. It will be appreciated that the genetic
material associated with these human antibodies may also be
isolated and manipulated as described herein.
[0180] Yet another highly efficient means for generating
recombinant antibodies is disclosed by Newman, Biotechnology 10:
1455-1460 (1992). Specifically, this technique results in the
generation of primatized antibodies that contain monkey variable
domains and human constant sequences. This reference is
incorporated by reference in its entirety herein. Moreover, this
technique is also described in commonly assigned U.S. Pat. Nos.
5,658,570, 5,693,780 and 5,756,096 each of which is incorporated
herein by reference.
[0181] In another embodiment, lymphocytes can be selected by
micromanipulation and the variable genes isolated. For example,
peripheral blood mononuclear cells can be isolated from an
immunized mammal and cultured for about 7 days in vitro. The
cultures can be screened for specific IgGs that meet the screening
criteria. Cells from positive wells can be isolated. Individual
Ig-producing B cells can be isolated by FACS or by identifying them
in a complement-mediated hemolytic plaque assay. Ig-producing B
cells can be micromanipulated into a tube and the V.sub.H and
V.sub.L genes can be amplified using, e.g., RT-PCR. The V .sub.H
and V.sub.L genes can be cloned into an antibody expression vector
and transfected into cells (e.g., eukaryotic or prokaryotic cells)
for expression.
[0182] Alternatively, antibody-producing cell lines may be selected
and cultured using techniques well known to the skilled artisan.
Such techniques are described in a variety of laboratory manuals
and primary publications. In this respect, techniques suitable for
use in the invention as described below are described in Current
Protocols in Immunology, Coligan et al., Eds., Green Publishing
Associates and Wiley-Interscience, John Wiley and Sons, New York
(1991) which is herein incorporated by reference in its entirety,
including supplements.
[0183] Antibodies for use in the diagnostic and therapeutic methods
disclosed herein can be produced by any method known in the art for
the synthesis of antibodies, in particular, by chemical synthesis
or preferably, by recombinant expression techniques as described
herein.
[0184] In one embodiment, a LINGO-1 antibody, or antigen-binding
fragment, variant, or derivative thereof of the invention comprises
a synthetic constant region wherein one or more domains are
partially or entirely deleted ("domain-deleted antibodies"). In
certain embodiments compatible modified antibodies will comprise
domain deleted constructs or variants wherein the entire C.sub.H2
domain has been removed (.DELTA.C.sub.H2 constructs). For other
embodiments a short connecting peptide may be substituted for the
deleted domain to provide flexibility and freedom of movement for
the variable region. Those skilled in the art will appreciate that
such constructs are particularly preferred due to the regulatory
properties of the C.sub.H2 domain on the catabolic rate of the
antibody. Domain deleted constructs can be derived using a vector
(e.g., from Biogen IDEC Incorporated) encoding an IgG.sub.1 human
constant domain (see, e.g., WO 02/060955A2 and WO02/096948A2, which
are incorporated by reference in their entireties). This exemplary
vector was engineered to delete the C.sub.H2 domain and provide a
synthetic vector expressing a domain deleted IgG.sub.1 constant
region.
[0185] In certain embodiments, LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention are minibodies. Minibodies can be made using methods
described in the art (see, e.g., see e.g., U.S. Pat. No. 5,837,821
or WO 94/09817A1, which are incorporated by reference in their
entireties).
[0186] In one embodiment, a LINGO-1 antibody, or antigen.-binding
fragment, variant, or derivative thereof of the invention comprises
an immunoglobulin heavy chain having deletion or substitution of a
few or even a single amino acid as long as it permits association
between the monomeric subunits. For example, the mutation of a sin
ale amino acid in selected areas of the C.sub.H2 domain may be
enough to substantially reduce Fc binding and thereby increase
tumor localization. Similarly, it may be desirable to simply delete
that part of one or more constant region domains that control the
effector function (e.g. complement binding) to be modulated. Such
partial deletions of the constant regions may improve selected
characteristics of the antibody (serum half-life) while leaving
other desirable functions associated with the subject constant
region domain intact. Moreover, as alluded to above, the constant
regions of the disclosed antibodies may be synthetic through the
mutation or substitution of one or more amino acids that enhances
the profile of the resulting construct. In this respect it may be
possible to disrupt the activity provided by a conserved binding
site (e.g. Fc binding) while substantially maintaining the
configuration and immunogenic profile of the modified antibody. Yet
other embodiments comprise the addition of one or more amino acids
to the constant region to enhance desirable characteristics such as
effector function or provide for more cytotoxin or carbohydrate
attachment. In such embodiments it may be desirable to insert or
replicate specific sequences derived from selected constant region
domains.
[0187] The present invention also provides antibodies that
comprise, consist essentially of, or consist of, variants
(including derivatives) of antibody molecules (e.g., the V.sub.H
regions and/or V.sub.L regions) described herein, which antibodies
or fragments thereof immunospecifically bind to a LINGO-1
polypeptide or fragment or variant thereof. Standard techniques
known to those of skill in the art can be used to introduce
mutations in the nucleotide sequence encoding a LINGO-1 antibody,
including, but not limited to, site-directed mutagenesis and
PCR-mediated mutagenesis which result in amino acid substitutions.
Preferably, the variants (including derivatives) encode less than
50 amino acid substitutions, less than 40 amino acid substitutions,
less than 30 amino acid substitutions, less than 25 amino acid
substitutions, less than 20 amino acid substitutions, less than 15
amino acid substitutions, less than 10 amino acid substitutions,
less than 5 amino acid substitutions, less than 4 amino acid
substitutions, less than 3 amino acid substitutions, or less than 2
amino acid substitutions relative to the reference V.sub.H region,
V.sub.HCDR1, V.sub.HCDR2, V.sub.HCDR3, V.sub.L region, V.sub.LCDR1,
V.sub.LCDR2, or V.sub.LCDR3. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a side chain with a similar
charge. Families of amino acid residues having side chains with
similar charges have been defined in the art. These families
include amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine).
Alternatively, mutations can be introduced randomly along all or
part of the coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for biological activity to
identify mutants that retain activity (e.g., the ability to bind a
LINGO-1 polypeptide).
[0188] For example, it is possible to introduce mutations only in
framework regions or only in CDR regions of an antibody molecule.
Introduced mutations may be silent or neutral missense mutations,
i.e., have no, or little, effect on an antibody's ability to bind
antigen. These types of mutations may be useful to optimize codon
usage, or improve a hybridoma's antibody production. Alternatively,
non-neutral missense mutations may alter an antibody's ability to
bind antigen. The location of most silent and neutral missense
mutations is likely to be in the framework regions, while the
location of most non-neutral missense mutations is likely to be in
CDR, though this is not an absolute requirement. One of skill in
the art would be able to design and test mutant molecules with
desired properties such as no alteration in antigen binding
activity or alteration in binding activity (e.g., improvements in
antigen binding activity or change in antibody specificity).
Following mutagenesis, the encoded protein may routinely be
expressed and the functional and/or biological activity of the
encoded protein, (e.g., ability to immunospecifically bind at least
one epitope of a LINGO-1 polypeptide) can be determined using
techniques described herein or by routinely modifying techniques
known in the art.
IV. Polynucleotides Encoding LINGO-1 Antibodies
[0189] The present invention also provides for nucleic acid
molecules encoding LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention.
[0190] In one embodiment, the present invention provides an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region (VH), where at least one of the CDRs of the heavy
chain variable region or at least two of the CDRs of the heavy
chain variable region are at least 80%, 85%, 90% or 95% identical
to reference heavy chain CDR1, CDR2, or CDR3 amino acid sequences
of Li62 or Li81 or variants thereof as described in Table 3.
Alternatively, the CDR1, CDR2, and CDR3 regions of the VH are at
least 80%, 85%, 90% or 95% identical to reference heavy chain CDR1,
CDR2, and CDR3 amino acid sequences of Li62 or Li81 or variants
thereof as described in Table 3. Thus, according to this embodiment
a heavy chain variable region of the invention has CDR1, CDR2, or
CDR3 polypeptide sequences related to the polypeptide sequences
shown in Table 3:
TABLE-US-00005 TABLE 3 LINGO-1 Antibody VH Sequences VH VH VH
Antibody VH SEQUENCE CDR1 CDR2 CDR3 Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGHND
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL
SKNTLYLQMNSLRAEDTATYYCAREGHNDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 1) NO: 2) (SEQ ID NO: 4) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGYYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD B06
SKNTLYLQMNSLRAEDTATYYCAREGYYDWYFDQW ID DSVKG Q (SEQ GRGTLVTVSS (SEQ
ID NO: 53) NO: 2) (SEQ ID ID NO: 3) NO: 17) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD B12
SKNTLYLQMNSLRAEDTATYYCAREGQYDWYFDVW ID DSVKG V (SEQ GRGTLVTVSS (SEQ
ID NO: 54) NO: 2) (SEQ ID ID NO: 3) NO: 18) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGDYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL F06
SKNTLYLQMNSLRAEDTATYYCAREGDYDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 55) NO: 2) (SEQ ID NO: 19) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFEL B01
SKNTLYLQMNSLRAEDTATYYCAREGQYDWYFELW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 56) NO: 2) (SEQ ID NO: 20) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EADID variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WFFDL D09
SKNTLYLQMNSLRAEDTATYYCAREADIDWFFDLWG ID DSVKG (SEQ ID RGTLVTVSS
(SEQ ID NO: 57) NO: 2) (SEQ ID NO: 21) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGHYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL D12
SKNTLYLQMNSLRAEDTATYYCAREGHYDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 58) NO: 2) (SEQ ID NO: 22) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGRYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDP F01
SKNTLYLQMNSLRAEDTATYYCAREGRYDWYFDPW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 59) NO: 2) (SEQ ID NO: 23) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGDYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFGL F02
SKNTLYLQMNSLRAEDTATYYCAREGDYDWYFGLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 60) NO: 2) (SEQ ID NO: 24) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGRYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL F06
SKNTLYLQMNSLRAEDTATYYCAREGRYDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 61) NO: 2) (SEQ ID NO: 25) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG ESHID variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA RYFDL F10
SKNTLYLQMNSLRAEDTATYYCARESHIDRYFDLWG ID DSVKG (SEQ ID RGTLVTVSS
(SEQ ID NO: 62) NO: 2) (SEQ ID NO: 26) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFD G08
SKNTLYLQMNSLRAEDTATYYCAREGQYDWYFDVW ID DSVKG V (SEQ GRGTLVTVSS (SEQ
ID NO: 63) NO: 2) (SEQ ID ID NO: 3) NO: 27) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGHYN variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA GYFDL H08
SKNTLYLQMNSLRAEDTATYYCAREGHYNGYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 64) NO: 2) (SEQ ID NO: 28) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGYYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL C10
SKNTLYLQMNSLRAEDTATYYCAREGYYDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 65) NO: 2) (SEQ ID NO: 29) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGTYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYLD C02
SKNTLYLQMNSLRAEDTATYYCAREGTYDWYLDLW ID DSVKG L (SEQ GRGTLVTVSS (SEQ
ID NO: 66) NO: 2) (SEQ ID ID NO: 3) NO: 30) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGYYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFEL D05
SKNTLYLQMNSLRAEDTATYYCAREGYYDWYFELW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 67) NO: 2) (SEQ ID NO: 31) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGLID variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WFFDQ F02
SKNTLYLQMNSLRAEDTATYYCAREGLIDWFFDWQG ID DSVKG (SEQ ID RGTLVTVSS
(SEQ ID NO: 68) NO: 2) (SEQ ID NO: 32) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGQFD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL C10
SKNTLYLQMNSLRAEDTATYYCAREGQFDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 69) NO: 2) (SEQ ID NO: 33) NO: 3) Li62
EVQLLESGGGLVQPGGSLRLSCAASGFTFSIYPMFWV IYPMF WIGPSG EGTYD variant
RQAPGKGLEWVSWIGPSGGITKYADSVKGRFTISRDN (SEQ GITKYA WYFDL H08
SKNTLYLQMNSLRAEDTATYYCAREGTYDWYFDLW ID DSVKG (SEQ ID GRGTLVTVSS
(SEQ ID NO: 70) NO: 2) (SEQ ID NO: 34) NO: 3) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDI
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDIWG ID ADSVK (SEQ ID QGTTVTVSS
(SEQ ID NO: 5) NO: 6) G (SEQ NO: 8) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGEND variant
VRQAFGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDV F09
NSKNTLYLQMNSLRAEDTAVYYCATEGENDAFDVW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 71) NO: 6) G (SEQ NO: 35) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AYDT G02
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAYDTW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 72) NO: 6) G (SEQ NO: 36) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGTND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDI H03
NSKNTLYLQMNSLRAEDTAVYYCATEGTNDAFDIWG ID ADSYK (SEQ ID QGTIVTVSS
(SEQ ID NO: 73) NO: 6) G (SEQ NO: 37) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDS A12
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDSW ID ADSYK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 74) NO: 6) G (SEQ NO: 38) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRGTISRD K (SEQ GFTFY AFDT C02
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAFDTW ID ADSYK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 75) NO: 6) G (SEQ NO: 39) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AYDR C11
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDAYDRW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 76) NO: 6) G (SEQ NO: 40) ID NO: 7) Li81
EVQLLESGGGLVQFGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY VFDS D11
NSKNTLYLQMNSLRAEDTAVYYCATEGDNDVFDSW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 77) NO: 6) G (SEQ NO: 41) ID NO: 7) Li81
EVQLLESGGGLVQFGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDDD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY VFDM E05
NSKNTLYLQMNSLRAEDTAVYYCATEGDDDVFDMW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 78) NO: 6) G (SEQ NO: 42) ID NO: 7) Li81
EVQLLESGGGLVQFGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGYND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFIFY AFDF H04
NSKNTLYLQMNSLRAEDTAVYYCATEGYNDAFDFW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 79) NO: 6) G (SEQ NO: 43) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDDD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AYDM B04
NSKNTLYLQMNSLRAEDTAVYYCATEGDDDAYDMW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 80) NO: 6) G (SEQ NO: 44) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EQDYD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY TYDL A02
NSKNTLYLQMNSLRAEDTAVYYCATEQDYDTYDLW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 81) NO: 6) G (SEQ NO: 45) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGDDD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDT B12
NSKNTLYLQMNSLRAEDTAVYYCATEGDDDAFDTW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 82) NO: 6) G (SEQ NO: 46) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EADDD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDI H06
NSKNTLYLQMNSLRAEDTAVYYCATEADDDAFDIWG ID ADSVK (SEQ ID QGTTVTVSS
(SEQ ID NO: 83) NO: 6) G (SEQ NO: 47) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGEND variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY AFDM H08
NSKNTLYLQMNSLRAEDTAVYYCATEGENDAFDMW ID ADSVK (SEQ ID GQGTTVTVSS
(SEQ ID NO: 84) NO: 6) G (SEQ NO: 48) ID NO: 7) Li81
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKW AYEM VIGPSG EGEYD variant
VRQAPGKGLEWVSVIGPSGGFTFYADSVKGRFTISRD K (SEQ GFTFY TYDI E07
NSKNTLYLQMNSLRAEDTAVYYCATEGEYDTYDIWG ID ADSVK (SEQ ID QGTTVTVSS
(SEQ ID NO: 85) NO: 6) G (SEQ NO: 49) ID NO: 7)
[0191] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
which is identical to the polypeptide of SEQ ID NO:146 except for a
replacement of one or more of the following amino acids: W50, P53,
I57 and/or W104. In some embodiments, W50 is replaced with an H, F,
L, M, G, I, or D residue. In some embodiments, P53 is replaced with
an L, S, T, W, or G residue. In some embodiments, I57 is replaced
with a G, M, N, H, L, F, W, Y, S, P, V or T residue. In some
embodiments, W104 is replaced with a V, H, S, Q, M, L, T, or I
residue.
[0192] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region which is identical to the polypeptide of SEQ ID
NO:5 except for a replacement of amino acid P53. In some
embodiments, P53 is replaced with an L, S, T, W, or G residue.
[0193] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region which is identical to the polypeptide of SEQ ID
NO:1 except for a replacement of one or more of the following amino
acids: W50, P53, I57 and/or W104. In some embodiments, W50 is
replaced with an H, F, L, M, G, I, or D residue. In some
embodiments, P53 is replaced with an L, S, T, W, or G residue. In
some embodiments, I57 is replaced with a G, M, N, H, L, F, W, Y, S,
P, V or T residue. In some embodiments, W104 is replaced with a V,
H, S, Q, M, L, T, or I residue.
[0194] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region which is identical to the polypeptide of SEQ ID
NO:66 except for a replacement of one or more of the following
amino acids: W50, P53, I57 and/or W104. in sonic embodiments, W50
is replaced with an H, F, L, M, G, I, or D residue. In some
embodiments, P53 is replaced with an L, S, T, W, or G residue. In
some embodiments, I57 is replaced with a G, M, N, H, L, F, W, Y, S,
P, V or T residue. In some embodiments, W104 is replaced with a V,
H, S, Q, M, L, T, or I residue.
[0195] In certain embodiments, the present invention provides an
isolated polynucleotide comprising, consisting essentially of or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region (VH) in which the CDR3 region has a polypeptide
sequence at least 80%, 85%, 90%, 95% or 100% identical to the CDR3
amino acid sequences selected from the p-oup consisting of SEQ ID
NOs: 4, 8 and 17-49. In some embodiments, the CDR1 and CDR2 regions
are at least 80%, 85%, 90%, 95% or 100% identical to the CDR1 and
CDR2 amino acid sequences of SEQ ID NOs: 2 and 3, respectively, and
the CDR3 region is at least 80%, 85%, 90%, 95% or 100% identical to
a CDR3 amino acid sequence selected from the group consisting of
SEQ ID NOs: 4 and 17-34. In some embodiments, the CDR1 and CDR2
regions are at least 80%, 85%, 90%, 95% or 100% identical to the
CDR1 and CDR2 amino acid sequences of SEQ ID NOs: 6 and 7,
respectively, and the CDR3 region is at least 80%, 85%, 90%, 95% or
100% identical to a CDR3 amino acid sequence selected from the
group consisting of SEQ ID NOs: 8 and 35-49.
[0196] In certain embodiments, an antibody or antigen-binding
fragment comprising the VH encoded by the polynucleotide
specifically or preferentially binds to LINGO-1.
[0197] In another embodiment, the present invention provides an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin heavy chain
variable region (VH) in which the CDR1, CDR2, and CDR3 regions have
polypeptide sequences which are identical to the CDR1, CDR2, and
CDR3 groups shown in Table 3. In certain embodiments, an antibody
or antigen-binding fragment comprising the VH encoded by the
polynucleotide specifically or preferentially binds to LINGO-1.
[0198] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a VH encoded by one or more of the polynucleotides
described above specifically or preferentially binds to the same
epitope as a Li62 or Li81 antibody, or will competitively inhibit
such a monoclonal antibody from binding to LINGO-1.
[0199] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a VH encoded by one or more of the polynucleotides
described above specifically or preferentially binds to a LINGO-1
polypeptide or fragment thereof, or a LINGO-1 variant polypeptide,
with an affinity characterized by a dissociation constant (K.sub.D)
no greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3
M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5
M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7
M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9
M,10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-13 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0200] In a further embodiment, the present invention includes an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding a VH at least 80%, 85%, 90%
or 95% identical to a reference VH polypeptide selected from the
group consisting of SEQ ID NOs: 1, 5 and 53-85. In certain
embodiments, an antibody or antigen-binding fragment comprising the
VH encoded by the polynucleotide specifically or preferentially
binds to LINGO-1.
[0201] In some embodiments, the isolated polynucleotide comprises,
consists essentially of or consists of a nucleic acid encoding an
antibody heavy chain as shown below in. SEQ ID NO:86.
TABLE-US-00006 (SEQ ID NO: 86)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKWVRQAPGKGLEWVIG
PSGGFTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEGDN
DAFDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP
EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSSLGTQTYIC
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
[0202] In other embodiments, the isolated polynucleotide comprises,
consists essentially of or consists of a nucleic acid encoding an
aglycosylated version of an antibody heavy chain. For example, an
aglycosylated version of Li81 is described in PCT/US2008/000316,
filed Jan. 9, 2008, which is incorporated herein by reference in
its entirety. An aglycosylated version of the Li81 antibody was
created by changing a single amino acid (T to A) in the Li81 heavy
chain sequence. The sequence of an aglycosylated version of Li81
heavy Chain (SEQ ID NO:50) is shown below. The single amino acid
change is marked in hold and underlined:
TABLE-US-00007 (SEQ ID NO: 50)
EVQLLESGGGLVQPGGSLRLSCAASGFTFSAYEMKWVRQAPGKGLEWVSV
IGPSGGFTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEG
DNDAFDIWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGEVHNAKTKPREEQYNSAY
RVVSVLTVLHQDWLNGKEYCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG.
[0203] Therefore, the present invention includes an isolated
polynucleotide comprising, consisting essentially of, or consisting
of a nucleic acid encoding a heavy chain at least 80%, 85%, 90% or
95% identical to a reference polypeptide comprising the amino acids
of SEQ ID NO:50 or 86. In certain embodiments, an antibody or
antigen-binding fragment comprising the heavy chain encoded by the
polynucleotide specifically or preferentially binds to LINGO-1.
[0204] In another aspect, the present invention includes an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid sequence encoding a comprising the
amino acids of SEQ ID NO: 1 or SEQ ID NO: 5. In certain
embodiments, an antibody or antigen-binding fragment comprising the
VH encoded by the polynucleotide specifically or preferentially
binds to LINGO-1. In certain embodiments, an antibody or
antigen-binding fragment thereof comprising, consisting essentially
of, or consisting of a VH encoded by one or more of the
polynucleotides described above specifically or preferentially
binds to the same epitope as Li62, Li81 or a variant thereof as
described in Table 3 or will competitively inhibit such a
monoclonal antibody from binding to LINGO-1.
[0205] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of or
consisting of a VH encoded by one or more of the polynucleotides
described above specifically or preferentially binds to a LINGO-1
polypeptide or fragment thereof, or a LINGO-1 variant polypeptide,
with an affinity characterized by a dissociation constant (K.sub.D)
no greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3
M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5
M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7
M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9
M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
VL Sequences
[0206] In another embodiment, the present invention provides an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin light chain
variable region (VL), where at least one of the CDRs of the light
chain variable region or at least two of the CDRs of the light
chain variable region are at least 80%, 85%, 90% or 95% identical
to reference light chain CDR1, CDR2, or CDR3 amino acid sequences
from monoclonal LINGO-1 antibodies disclosed herein. Alternatively,
the CDR1, CDR2, and CDR3 regions of the VL are at least 80%, 85%,
90% or 95% identical to reference light chain CDR1, CDR2, and CDR3
amino acid sequences from monoclonal LINGO-1 antibodies disclosed
herein. Thus, according to one embodiment, a light chain variable
region of the invention has CDR1, CDR2, or CDR3 polypeptide
sequences related to the polypeptide sequences shown in Table
4.
TABLE-US-00008 TABLE 4 LINGO-1 Antibody VL Sequences VL VL VL
Antibody VL SEQUENCE CDR1 CDR2 CDR3 Li62
DIQMTQSPSFLSASVGDSVAITCRASQDISRYLAWYQQ RASQD DASNL QQYDT
RPGKAPKLLIYDASNLQTGVPSRFSGSGSGTDFTFTITS ISRYL QT (SEQ LHPS
LQPEDFGTYYCQQYDTLHPSFGPGTTVDIK (SEQ ID A (SEQ ID (SEQ ID NO: 9) ID
NO: 11) NO: 12) NO: 10) Li81 DIQMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQ
RASQS DASNR QQRSN QKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTIS VSSYL AT
(SEQ WPMY SLEPEDFAVYYCQQRSNWPMYTFGQGTKLEIK (SEQ A (SEQ ID NO: T
(SEQ ID NO: 13) ID 15) ID NO: NO: 14) 16)
[0207] In certain embodiments, an antibody or antigen-binding
fragment comprising the VL encoded by the polynucleotide
specifically or preferentially binds to LINGO-1.
[0208] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin light chain
which is identical to the polypeptide of SEQ ID NO:145 except for a
replacement of amino acid W94. In some embodiments, W94 is replaced
with an A, D, L, N, G, Q, V, or S residue.
[0209] In certain embodiments, the present invention provides an
isolated polynucleotide, comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin light chain
variable region which is identical to the polypeptide of SEQ ID
NO:5 except for a replacement of amino acid W94. In some
embodiments, W94 is replaced with an A, D, L, N, G, Q, V, or S
residue.
[0210] In another embodiment, the present invention provides an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding an immunoglobulin light chain
variable region (VL) in which the CDR1, CDR2, and CDR3 regions have
polypeptide sequences which are identical to the CDR1, CDR2, and
CDR3 groups shown in Table 4. In certain embodiments, an antibody
or antigen-binding fragment comprising the VL encoded by the
polynucleotide specifically or preferentially binds to LINGO-1.
[0211] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a VL encoded by one or more of the polynucleotides
described above specifically or preferentially binds to the same
epitope as Li62 or Li81, or will competitively inhibit such an
antibody from binding to LINGO-1.
[0212] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a VL encoded by one or more of the polynucleotides
described above specifically or preferentially binds to a LINGO-1
polypeptide or fragment thereof, or a LINGO-1 variant polypeptide,
with an affinity characterized by a dissociation constant (K.sub.D)
no greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3
M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5
M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7
M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9
M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M.sup., 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0213] In a further embodiment, the present invention includes an
isolated polynucleotide comprising, consisting essentially of, or
consisting of a nucleic acid encoding a VL at least 80%, 85%, 90%
or 95% identical to a reference VL polypeptide sequence selected
from SEQ ID NO: 9 or SEQ ID NO: 13. In certain embodiments, an
antibody or antigen-binding fragment comprising the VL encoded by
the polynucleotide specifically or preferentially binds to LINGO-1.
In another aspect, the present invention includes an isolated
polynucleotide comprising, consisting essentially of, or consisting
of a nucleic acid sequence encoding a VL of the invention, selected
from SEQ ID NO: 9 or SEQ ID NO: 13. In certain embodiments, an
antibody or antigen-binding fragment comprising the VL encoded by
the polynucleotide specifically or preferentially binds to LINGO-1.
In certain embodiments, an antibody or antigen-binding fragment
thereof comprising, consisting essentially of, or consisting of a
VL encoded by one or more of the polynucleotides described above
specifically or preferentially binds to the same epitope as Li62 or
Li81, or will competitively inhibit such a monoclonal antibody from
binding to LINGO-1.
[0214] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a VL encoded by one or more of the polynucleotides
described above specifically or preferentially binds to a LINGO-1
polypeptide or fragment thereof, or a LINGO-1 variant polypeptide,
with an affinity characterized by a dissociation constant (K.sub.D)
no greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3
M, 10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5
M, 10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7
M, 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9
M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M,
5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M, 5.times.10.sup.-13 M, 10.sup.-13 M,
5.times.10.sup.-14 M, 10.sup.-14 M, 5.times.10.sup.-15 M, or
10.sup.-15 M.
[0215] Any of the polynucleotides described above may further
include additional nucleic acids, encoding, e.g., a signal peptide
to direct secretion of the encoded polypeptide, antibody constant
regions as described herein, or other heterologous polypeptides as
described herein.
[0216] Also, as described in more detail elsewhere herein, the
present invention includes compositions comprising the
polynucleotides comprising one or more of the polynucleotides
described above. In one embodiment, the invention includes
compositions comprising a first polynucleotide and second
polynucleotide wherein said first polynucleotide encodes a VH
polypeptide as described herein and wherein said second
polynucleotide encodes a VL polypeptide as described herein.
[0217] The present invention also includes fragments of the
polynucleotides of the invention, as described elsewhere.
Additionally polynucleotides which encode fusion polynucleotides,
Fab fragments, and other derivatives, as described herein, are also
contemplated by the invention.
[0218] The polynucleotides may be produced or manufactured by any
method known in the art. For example, if the nucleotide sequence of
the antibody is known, a polynucleotide encoding the antibody may
be assembled from chemically synthesized oligonucleotides (e.g., as
described in Kutmeier et al., BioTechniques 17:242 (1994)), which,
briefly, involves the synthesis of overlapping oligonucleotides
containing portions of the sequence encoding the antibody,
annealing and ligating of those oligonucleotides, and then
amplification of the ligated oligonucleotides by PCR.
[0219] Alternatively, a polynucleotide encoding a LINGO-1 antibody,
or antigen-binding fragment, variant, or derivative thereof may be
generated from nucleic acid from a suitable source. If a clone
containing a nucleic acid encoding a particular antibody is not
available, but the sequence of the antibody molecule is known, a
nucleic acid encoding the antibody may be chemically synthesized or
obtained from a suitable source (e.g., an antibody cDNA library, or
a cDNA library generated from, or nucleic acid, preferably poly
A+RNA, isolated from, any tissue or cells expressing the antibody
or other LINGO-1 antibody, such as hybridoma cells selected to
express an antibody) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody or other LINGO-1 antibody. Amplified nucleic
acids generated by PCR may then be cloned into replicable cloning
vectors using any method well known in the art.
[0220] Once the nucleotide sequence and corresponding amino acid
sequence of the LINGO-1 antibody, or antigen-binding fragment,
variant, or derivative thereof is determined, its nucleotide
sequence may be manipulated using methods well known in the art for
the manipulation of nucleotide sequences, e.g., recombinant DNA
techniques, site directed mutagenesis, PCR, etc. (see, for example,
the techniques described in Sambrook et al., Molecular Cloning, A
Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1990) and Ausubel et al., eds., Current
Protocols in Molecular Biology, John Wiley & Sons, NY (1998),
which are both incorporated by reference herein in their
entireties), to generate antibodies having a different amino acid
sequence, for example to create amino acid substitutions,
deletions, and/or insertions.
[0221] A polynucleotide encoding a LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof can be
composed of any polyribonucleotide or polydeoxribonucleotide, which
may be unmodified RNA or DNA or modified RNA or DNA. For example, a
polynucleotide encoding LINGO-1 antibody, or antigen-binding
fragment, variant, or derivative thereof can be composed of single-
and double-stranded DNA, DNA that is a mixture of single- and
double-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, a polynucleotide encoding a
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof can be composed of triple-stranded regions
comprising RNA or DNA or both RNA and DNA. A polynucleotide
encoding a LINGO-1 antibody, or antigen-binding fragment, variant,
or derivative thereof may also contain one or more modified bases
or DNA or RNA backbones modified for stability or for other
reasons. "Modified" bases include, for example, tritylated bases
and unusual bases such as inosine. A variety of modifications can
be made to DNA and RNA; thus, "polynucleotide" embraces chemically,
enzymatically, or metabolically modified forms.
[0222] An isolated polynucleotide encoding a non-natural variant of
a polypeptide derived from an immunoglobulin (e.g., an
immunoglobulin heavy chain portion or light chain portion) can be
created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of the
immunoglobulin such that one or more amino acid substitutions,
additions or deletions are introduced into the encoded protein.
Mutations may be introduced by standard techniques, such as
site-directed mutagenesis and PCR-mediated mutagenesis. Preferably,
conservative amino acid substitutions are made at one or more
non-essential amino acid residues.
V. LINGO-1 Antibody Polypeptides
[0223] The present invention is further directed to isolated
polypeptides which make up LINGO-1 antibodies, antigen binding
fragments, variants or derivatives thereof. LINGO-1 antibodies of
the present invention comprise polypeptides, e.g., amino acid
sequences encoding LINGO-1-specific antigen binding regions derived
from immunoglobulin molecules. A polypeptide or amino acid sequence
"derived from" a designated protein refers to the origin of the
polypeptide. In certain cases, the polypeptide or amino acid
sequence which is derived from a particular starting polypeptide or
amino acid sequence has an amino acid sequence that is essentially
identical to that of the starting sequence, or a portion thereof,
wherein the portion consists of at least 10-20 amino acids, at
least 20-30 amino acids, at least 30-50 amino acids, or which is
otherwise identifiable to one of ordinary skill in the art as
having its origin in the starting sequence.
[0224] In one embodiment, the present invention provides an
isolated polypeptide comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region (VH),
where at least one of CDRs of the heavy chain variable region or at
least two of the CDRs of the heavy chain variable region are at
least 80%, 85%, 90% or 95% identical to reference heavy chain CDR1,
CDR2 or CDR3 amino acid sequences from monoclonal LINGO-1
antibodies disclosed herein. Alternatively, the CDR1, CDR2 and CDR3
regions of the VH are at least 80%, 85%, 90% or 95% identical to
reference heavy chain CDR1, CDR2 and CDR3 amino acid sequences from
monoclonal LINGO-1 antibodies disclosed herein. Thus, according to
this embodiment a heavy chain variable region of the invention has
CDR1, CDR2, and CDR3 polypeptide sequences related to the groups
shown in Table 3, supra. In certain embodiments, an antibody or
antigen-binding fragment comprising the VH polypeptide specifically
or preferentially binds to LINGO-1.
[0225] In another embodiment, the present invention provides an
isolated polypeptide comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region (VH),
wherein at least the CDR3 region is at least 80%, 85%, 90% or 95%
identical to a reference CDR3 sequence selected from the group
consisting of SEQ ID NOs: 4, 8 and 17-49. In further embodiments,
the CDR3 region is identical to a reference CDR3 sequence selected
from the group consisting of SEQ ID NOs: 4, 8 and 17-49. In still
further embodiments, the invention provides an isolated polypeptide
comprising, consisting essentially of, or consisting of an
immunoglobulin heavy chain variable region (VH), wherein, the CDR1
and CDR2 regions are at least 80%, 85%, 90%, 95% or 100% identical
to the CDR1 and CDR2 amino acid sequences of SEQ ID NOs: 2 and 3,
respectively, and the CDR3 region is at least 80%, 85%, 90%, 95% or
100% identical to a CDR3 amino acid sequence selected from the
group consisting of SEQ ID NOs: 4 and 17-34. In other embodiments,
the invention provides an isolated polypeptide comprising,
consisting essentially of, or consisting of an immunoglobulin heavy
chain vairable region (VH), wherein the CDR1 and CDR2 regions are
at least 80%, 85%, 90%, 95% or 100% identical to the CDR1 and CDR2
amino acid sequences of SEQ ID NOs: 6 and 7, respectively, and the
CDR3 region is at least 80%, 85%, 90%, 95% or 100% identical to a
CDR3 amino acid sequence selected from the group consisting of SEQ
ID NOs: 8 and 35-49.
[0226] In another embodiment, the present invention provides an
isolated polypeptide comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region (VH) in
which the CDR1, CDR2, and CDR3 regions have polypeptide sequences
which are identical to the CDR1, CDR2, and CDR3 groups shown in
Table 3. In certain embodiments, an antibody or antigen-binding
fragment comprising the VH polypeptide specifically or
preferentially binds to LINGO-1.
[0227] In a further embodiment, the present invention includes an
isolated polypeptide comprising, consisting essentially of, or
consisting of a VH polypeptide at least 80%, 85%, 90% 95% or 100%
identical to a reference VH polypeptide sequence selected from SEQ
ID NOs: 1, 5 and 53-85. In one particular embodiment, the VH
polypeptide comprises a CDR3 amino acid sequence selected from the
group consisting of SEQ ID NOs: 4, 8 and 17-49.
[0228] In certain embodiments, an antibody or antigen-binding
fragment comprising the VH polypeptide specifically or
preferentially binds to LINGO-1. In another aspect, the present
invention includes an isolated polypeptide comprising, consisting
essentially of, or consisting of a VH polypeptide selected from the
group consisting of SEQ ID NOs: 1, 5 and 53-85. In certain
embodiments, an antibody or antigen-binding fragment comprising the
VH polypeptide specifically or preferentially binds to LINGO-1.
[0229] In certain embodiments, the present invention provides an
isolated polypeptide, comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain which is identical to
the polypeptide of SEQ ID NO:146 except for a replacement of one or
more of the following amino acids: W50, P53, I57 and/or W104. In
some embodiments, W50 is replaced with an H, F, L, M, G, I, or D
residue. In some embodiments, P53 is replaced with an L, S, T, W,
or G residue. In some embodiments, I57 is replaced with a G, M, N,
H, L, F, W, Y, S, P, V or T residue. In some embodiments, W104 is
replaced with a V, H, S, Q, M, L, T, or I residue.
[0230] In certain embodiments, the present invention provides an
isolated polypeptide, comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region which
is identical to the polypeptide of SEQ ID NO:5 except for a
replacement of amino acid P53. In some embodiments, P53 is replaced
with an L, S, T, W, or G residue.
[0231] In certain embodiments, the present invention provides an
isolated polypeptide, comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region which
is identical to the polypeptide of SEQ ID NO:1 except for a
replacement of one or more of the following amino acids: W50, P53,
I57 and/or W104. In some embodiments, W50 is replaced with an H, F,
L, M, G, I, or D residue. In some embodiments, P53 is replaced with
an L, S, T, W, or G residue. In some embodiments, I57 is replaced
with a G, M, N, H, L, F, W, Y, S, P, V or T residue. In some
embodiments, W104 is replaced with a V, H, S, Q, M, L, T, or I
residue.
[0232] In certain embodiments, the present invention provides an
isolated polypeptide, comprising, consisting essentially of, or
consisting of an immunoglobulin heavy chain variable region which
is identical to the polypeptide of SEQ ID NO:66 except for a
replacement of one or more of the following amino acids: W50, P53,
I57 and/or W104. In some embodiments, W50 is replaced with an H, F,
L, M, G, I, or D residue. In some embodiments, P53 is replaced with
an L, S, T, W, or G residue. In some embodiments, I57 is replaced
with a G, M, N, H, L, F, W, Y, S, P, V or T residue. In some
embodiments, W104 is replaced with a V, H, S, Q, M, L, T, or I
residue.
[0233] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a one or more of the VH polypeptides described above
specifically or preferentially binds to the same epitope as Li62,
Li81 or a variant thereof as described in Table 3, or will
competitively inhibit such an antibody from binding to LINGO-1.
[0234] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of one or more of the VH polypeptides described above
specifically or preferentially binds to a LINGO-1 polypeptide or
fragment thereof, or a LINGO-1 variant polypeptide, with an
affinity characterized by a dissociation constant (K.sub.D) no
greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3 M,
10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5 M,
10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 5.times.10.sup.-13 M,
10.sup.-13 M, 5.times.10.sup.-14 M, 10.sup.-14 M,
5.times.10.sup.-15 M, or 10.sup.-15 M.
[0235] In another embodiment, the present invention provides an
isolated polypeptide comprising, consisting essentially of, or
consisting of an immunoglobulin light chain variable region (VL),
where at least one of the CDRs of the light chain variable region
or at least two of the CDRs of the light chain variable region are
at least 80%, 85%, 90% or 95% identical to reference heavy chain
CDR1, CDR2, or CDR3 amino acid sequences from monoclonal LINGO-1
antibodies disclosed herein. Alternatively, the CDR1, CDR2 and CDR3
regions of the VL are at least 80%, 85%, 90% or 95% identical to
reference light chain CDR1, CDR2, and CDR3 amino acid sequences
from monoclonal LINGO-1 antibodies disclosed herein. Thus,
according to this embodiment a light chain variable region of the
invention has CDR1, CDR2, and CDR3 polypeptide sequences related to
the polypeptides shown in Table 4, supra. In certain embodiments,
an antibody or antigen-binding fragment comprising the VL
polypeptide specifically or preferentially binds to LINGO-1.
[0236] In another embodiment, the present invention provides an
isolated polypeptide comprising, consisting essentially of, or
consisting of an immunoglobulin light chain variable region (VL) in
which the CDR1, CDR2, and CDR3 regions have polypeptide sequences
which are identical to the CDR1, CDR2, and CDR3 groups shown in
Table 4. In certain embodiments, an antibody or antigen-binding
fragment comprising the VL polypeptide specifically or
preferentially binds to LINGO-1.
[0237] In a further embodiment, the present invention includes an
isolated polypeptide comprising, consisting essentially of, or
consisting of a VL polypeptide at least 80%, 85%, 90% or 95%
identical to a reference VL polypeptide sequence selected from SEQ
ID NO: 9 or SEQ ID NO: 13, shown in Table 4. In certain
embodiments, an antibody or antigen-binding fragment comprising the
VL polypeptide specifically or preferentially binds to LINGO-1. In
another aspect, the present invention includes an isolated
polypeptide comprising, consisting essentially of, or consisting of
a VL polypeptide selected from SEQ ID NO: 9 or SEQ ID NO: 13, shown
in Table 4. In certain embodiments, an antibody or antigen-binding
fragment comprising the VL polypeptide specifically or
preferentially binds to LINGO-1.
[0238] In certain embodiments, the present invention provides an
isolated polypeptide consisting essentially of, or consisting of an
immunoglobulin light chain which is identical to the polypeptide of
SEQ ID NO:145 except for a replacement of amino acid W94. In some
embodiments, W94 is replaced with an A, D, L, N, G, Q, V, or S
residue.
[0239] In certain embodiments, the present invention provides an
isolated polypeptide, comprising, consisting essentially of, or
consisting of an immunoglobulin light chain variable region which
is identical to the polypeptide of SEQ ID NO:5 except for a
replacement of amino acid W94. In some embodiments, W94 is replaced
with an A, D, L, N, G, Q, V, or S residue.
[0240] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, one or more
of the VL polypeptides described above specifically or
preferentially binds to the same epitope as Li62 or Li81, or will
competitively inhibit such a monoclonal antibody from binding to
LINGO-1.
[0241] In certain embodiments, an antibody or antigen-binding
fragment thereof comprising, consisting essentially of, or
consisting of a one or more of the VL polypeptides described above
specifically or preferentially binds to a LINGO-1 polypeptide or
fragment thereof, or a LINGO-1 variant polypeptide, with an
affinity characterized by a dissociation constant (K.sub.D) no
greater than 5.times.10.sup.-2 M, 10.sup.-2 M, 5.times.10.sup.-3 M,
10.sup.-3 M, 5.times.10.sup.-4 M, 10.sup.-4 M, 5.times.10.sup.-5 M,
10.sup.-5 M, 5.times.10.sup.-6 M, 10.sup.-6 M, 5.times.10.sup.-7 M,
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M,
5.times.10.sup.-13 M, 10.sup.-13 M, 5.times.10.sup.-14 M,
10.sup.-14 M, 5.times.10.sup.-15 M, or 10.sup.-15 M.
[0242] In other embodiments, an antibody or antigen-binding
fragment thereof comprises, consists essentially of or consists of
a VH polypeptide, as shown in Table 3, and a VL polypeptide, as
shown in Table 4, selected from the group consisting of: [0243] i)
SEQ ID NO: 1 or SEQ ID NOs: 53-70 and SEQ ID NO: 9; and [0244] iii)
SEQ ID NO: 5 or SEQ ID NOs: 71-85 and SEQ ID NO: 13.
[0245] Any of the polypeptides described above may further include
additional polypeptides, e.g., a signal peptide to direct secretion
of the encoded polypeptide, antibody constant regions as described
herein, or other heterologous polypeptides as described herein.
Additionally, polypeptides of the invention include polypeptide
fragments as described elsewhere. Additionally polypeptides of the
invention include fusion polypeptide, Fab fragments, and other
derivatives, as described herein.
[0246] Also, as described in more detail elsewhere herein, the
present invention includes compositions comprising the polypeptides
described above.
[0247] It will also be understood by one of ordinary skill in the
art that LINGO-1 antibody polypeptides as disclosed herein may be
modified such that they vary in amino acid sequence from the
naturally occurring binding polypeptide from which they were
derived. For example, a polypeptide or amino acid sequence derived
from a designated protein may be similar, e.g., have a certain
percent identity to the starting sequence, e.g., it may be 60%,
70%, 75%, 80%, 85%, 90%, or 95% identical to the starting
sequence.
[0248] Furthermore, nucleotide or amino acid substitutions,
deletions, or insertions leading to conservative substitutions or
changes at "non-essential" amino acid regions may be made. For
example, a polypeptide or amino acid sequence derived from a
designated protein may be identical to the starting sequence except
for one or more individual amino acid substitutions, insertions, or
deletions, e.g., one, two, three, four, five, six, seven, eight,
nine, ten, fifteen, twenty or more individual amino acid
substitutions, insertions, or deletions. In certain embodiments, a
polypeptide or amino acid sequence derived from a designated
protein has one to five, one to ten, one to fifteen, or one to
twenty individual amino acid substitutions, insertions, or
deletions relative to the starting sequence.
[0249] Certain LINGO-1 antibody polypeptides of the present
invention comprise, consist essentially of, or consist of an amino
acid sequence derived from a human amino acid sequence. However,
certain LINGO-1 antibody polypeptides comprise one or more
contiguous amino acids derived from another mammalian species. For
example, a LINGO-1 antibody of the present invention may include a
primate heavy chain portion, hinge portion, or antigen binding
region. In another example, one or more murine-derived amino acids
may be present in a non-murine antibody polypeptide, e.g., in an
antigen binding site of a LINGO-1 antibody. In certain therapeutic
applications, LINGO-1-specific antibodies, or antigen-binding
fragments, variants, or analogs thereof are designed so as to not
be immunogenic in the animal to which, the antibody is
administered.
[0250] In certain embodiments, a LINGO-1 antibody polypeptide
comprises an amino acid sequence or one or more moieties not
normally associated with an antibody. Exemplary modifications are
described in more detail below. For example, a single-chain fv
antibody fragment of the invention may comprise a flexible linker
sequence, or may be modified to add a functional moiety (e.g., PEG,
a drug, a toxin, or a label).
[0251] A LINGO-1 antibody polypeptide of the invention may
comprise, consist essentially of, or consist of a fusion protein.
Fusion proteins are chimeric molecules which comprise, for example,
an immunoglobulin antigen-binding domain with at least one target
binding site, and at least one heterologous portion, i.e., a
portion with which it is not naturally linked in nature. The amino
acid sequences may normally exist in separate proteins that are
brought together in the fusion polypeptide or they may normally
exist in the same protein but are placed in a new arrangement in
the fusion polypeptide. Fusion proteins may be created, for
example, by chemical synthesis, or by creating and translating a
polynucleotide in which the peptide regions are encoded in the
desired relationship.
[0252] The term "heterologous" as applied to a polynucleotide or a
polypeptide, means that the polynucleotide or polypeptide is
derived from a distinct entity from that of the rest of the entity
to which it is being compared. For instance, as used herein, a
"heterologous polypeptide" to be fused to a LINGO-1 antibody, or an
antigen-binding fragment, variant, or analog thereof is derived
from a non-immunoglobulin polypeptide of the same species, or an
immunoglobulin or non-immunoglobulin polypeptide of a different
species.
[0253] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art, including basic side
chains (e.g., lysine, arginine, histidine), acidic side chains
(e.g., aspartic acid, glutamic acid), uncharged polar side chains
(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a nonessential amino acid residue in an
immunoglobulin polypeptide is preferably replaced with another
amino acid residue from the same side chain family. In another
embodiment, a string of amino acids can be replaced with a
structurally similar string that differs in order and/or
composition of side chain family members.
[0254] Alternatively, in another embodiment, mutations may be
introduced randomly along all or part of the immunoglobulin coding
sequence, such as by saturation mutagenesis, and the resultant
mutants can be incorporated into LINGO-1 antibodies for use in the
diagnostic and treatment methods disclosed herein and screened for
their ability to bind to the desired antigen, e.g., LINGO-1.
VI. Fusion Proteins and Antibody Conjugates
[0255] As discussed in more detail elsewhere herein, LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the invention may further be recombinantly fused to a
heterologous polypeptide at the N- or C-terminus or chemically
conjugated (including covalent and non-covalent conjugations) to
polypeptides or other compositions. For example, LINGO-1-specific
LINGO-1 antibodies may be recombinantly fused or conjugated to
molecules useful as labels in detection assays and effector
molecules such as heterologous polypeptides, drugs, radionuclides,
or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO
89/12624; U.S. Pat. No. 5,314,995; and EP 396,387, which are
incorporated herein by reference in their entireties.
[0256] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention include derivatives that
are modified, i.e., by the covalent attachment of any type of
molecule to the antibody such that covalent attachment does not
prevent the antibody binding LINGO-1. For example, but not by way
of limitation, the antibody derivatives include antibodies that
have been modified, e.g., by glycosylation, acetylation,
pegylation, phosphylation, phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic
cleavage, linkage to a cellular ligand or other protein, etc. Any
of numerous chemical modifications may be carried out by known
techniques, including, but not limited to specific chemical
cleavage, acetylation, formylation, metabolic synthesis of
tunicamycin, etc. Additionally, the derivative may contain one or
more non-classical amino acids.
[0257] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention can be composed of amino
acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres, and may contain amino acids other
than the 20 gene-encoded amino acids. LINGO-1-specific antibodies
may be modified by natural processes, such as posttranslational
processing, or by chemical modification techniques which are well
known in the art. Such modifications are well described in basic
texts and in more detailed monographs, as well as in a voluminous
research literature. Modifications can occur anywhere in the
LINGO-1-specific antibody, including the peptide backbone, the
amino acid side-chains and the amino or carboxyl termini, or on
moieties such as carbohydrates. It will be appreciated that the
same type of modification may be present in the same or varying
degrees at several sites in a given LINGO-1-specific antibody.
Also, a given LINGO-1-specific antibody may contain many types of
modifications. LINGO-1-specific antibodies may be branched, for
example, as a result of ubiquitination, and they may be cyclic,
with or without branching. Cyclic, branched, and branched cyclic
LINGO-1-specific antibodies may result from posttranslation natural
processes or may be made by synthetic methods. Modifications
include acetylation, acylation, ADP-ribosylation, amidation,
covalent attachment of flavin, covalent attachment of a heme
moiety, covalent attachment of a nucleotide or nucleotide
derivative, covalent attachment of a lipid or lipid derivative,
covalent attachment of phosphotidylinositol, cross-linking,
cyclization, disulfide bond formation, demethylation, formation of
covalent cross-links, formation of cysteine, formation of
pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI
anchor formation, hydroxylation, iodination, methylation,
myristoylation, oxidation, pegylation, proteolytic processing,
phosphorylation, prenylation, racemization, selenoylation,
sulfation, transfer-RNA mediated addition of amino acids to
proteins such as arginylation, and ubiquitination. (See, for
instance, Proteins--Structure And Molecular Properties, T. E.
Creighton, W. H. Freeman and Company, New York 2nd Ed., (1993);
Posttranslational Covalent Modification Of Proteins, B. C. Johnson,
Ed., Academic Press, New York, pgs. 1-12 (1983); Seifter et al.,
Meth Enzymol 182:626-646 (1990); Rattan et al., Ann NY Acad Sci
663:48-62 (1992)).
[0258] The present invention also provides for fusion proteins
comprising a LINGO-1 antibody, or antigen-binding fragment,
variant, or derivative thereof, and a heterologous polypeptide. The
heterologous polypeptide to which the antibody is fused may be
useful for function or is useful to target the LINGO-1 polypeptide
expressing cells. In one embodiment, a fusion protein of the
invention comprises, consists essentially of, or consists of, a
polypeptide having the amino acid sequence of any one or more of
the V.sub.H regions of an antibody of the invention or the amino
acid sequence of any one or more of the V.sub.L regions of an
antibody of the invention or fragments or variants thereof, and a
heterologous polypeptide sequence. In another embodiment, a fusion
protein for use in the diagnostic and treatment methods disclosed
herein comprises, consists essentially of, or consists of a
polypeptide having the amino acid sequence of any one, two, three
of the V.sub.H CDRs of a LINGO-1-specific antibody, or fragments,
variants, or derivatives thereof, or the amino acid sequence of any
one, two, three of the V.sub.L CDRs of a LINGO-1-specific antibody,
or fragments, variants, or derivatives thereof, and a heterologous
polypeptide sequence. In one embodiment, the fusion protein
comprises a polypeptide having the amino acid sequence of a V.sub.H
CDR3 of a LINGO-1-specific antibody of the present invention, or
fragment, derivative, or variant thereof, and a heterologous
polypeptide sequence, which fusion protein specifically binds to at
least one epitope of LINGO-1. In another embodiment, a fusion
protein comprises a polypeptide having the amino acid sequence of
at least one V.sub.H region of a LINGO-1-specific antibody of the
invention and the amino acid sequence of at least one V.sub.L
region of a LINGO-1-specific antibody of the invention or
fragments, derivatives or variants thereof, and a heterologous
polypeptide sequence. Preferably, the V.sub.H and V.sub.L regions
of the fusion protein correspond to a single source antibody (or
scFv or Fab fragment) which specifically binds at least one epitope
of LINGO-1. In yet another embodiment, a fusion protein for use in
the diagnostic and treatment methods disclosed herein comprises a
polypeptide having the amino acid sequence of any one, two, three
or more of the V.sub.H CDRs of a LINGO-1-specific antibody and the
amino acid sequence of any one, two, three or more of the V.sub.L
CDRs of a LINGO-1-specific antibody, or fragments or variants
thereof, and a heterologous polypeptide sequence. Preferably, two,
three, four, five, six, or more of the V.sub.HCDR(s) or
V.sub.LCDR(s) correspond to single source antibody (or scFv or Fab
fragment) of the invention. Nucleic acid molecules encoding these
fusion proteins are also encompassed by the invention.
[0259] Exemplary fusion proteins reported in the literature include
fusions of the T cell receptor (Gascoigne et al., Proc. Natl. Acad.
Sci. USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature
337:525-531 (1989); Traunecker et al., Nature 339:68-70 (1989);
Zettmeissl et al., DNA Cell Biol. USA 9:347-353 (1990); and Byrn et
al., Nature 344:667-670 (1990)); L-selectin (homing receptor)
(Watson et al., J. Cell. Biol. 110:2221-2229 (1990); and Watson et
al., Nature 349:164-167 (1991)); CD44 (Aruffo et al., Cell
61:1303-1313 (1990)); CD28 and B7 (Linsley et al., J. Exp. Med.
173:721-730 (1991)); CTLA-4 (Lisley et al., J. Exp. Med.
174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144
(1991)); TNF receptor (Ashkenazi et al., Proc. Natl. Acad. Sci. USA
88:10535-10539 (1991); Lesslauer et al., Eur. J. Immunol.
27:2883-2886 (1991); and Peppel et al., J. Exp. Med. 174:1483-1489
(1991)); and IgE receptor a (Ridgway and Gorman, J. Cell. Biol.
Vol. 115, Abstract No. 1448 (1991)).
[0260] In certain embodiments, LINGO-1 antibodies, antibody
fragments, derivatives and variants thereof further comprise a
targeting moiety. Targeting moieties include a protein or a peptide
which directs localization to a certain part of the body, for
example, to the brain or compartments therein. In certain
embodiments, LINGO-1 antibodies, antibody fragments, derivatives
and variants thereof are attached or fused to a brain targeting
moiety. The brain targeting moieties are attached covalently (e.g.,
direct, translational fusion, or by chemical linkage either
directly or through a spacer molecule, which can be optionally
cleavable) or non-covalently attached (e.g., through reversible
interactions such as avidin, biotin, protein A, IgG, etc.). In
other embodiments, the LINGO-1 antibodies, antibody fragments,
derivatives and variants thereof are attached to one more brain
targeting moieties. In additional embodiments, the brain targeting
moiety is attached to a plurality of LINGO-1 antibodies, antibody
fragments, derivatives and variants thereof.
[0261] A brain targeting moiety associated with a LINGO-1 antibody,
antibody fragment, derivative or variant thereof enhances brain
delivery of such a LINGO-1 antibodies, antibody fragments,
derivatives and variants thereof. A number of polypeptides have
been described which, when fused to a protein or therapeutic agent,
delivers the protein or therapeutic agent through the blood brain
barrier (BBB). Non-limiting examples include the single domain
antibody FC5 (Abulrob et al. (2005) J. Neurochem. 95, 1201-1214);
mAB 83-14, a monoclonal antibody to the human insulin receptor
(Pardridge et al. (1995) Pharmacol. Res. 12, 807-816); the B2, B6
and B8 peptides binding to the human transferrin receptor (hTfR)
(Xia et al. (2000) J. Virol. 74, 11359-11366); the OX26 monoclonal
antibody to the transferrin receptor (Pardridge et al. (1991) J.
Pharmacol. Exp. Ther. 259, 66-70); and SEQ ID NOs: 1-18 of U.S.
Pat. No. 6,306,365. The contents of the above references are
incorporated herein by reference in their entirety.
[0262] Enhanced brain delivery of a LINGO-1 antibody, antibody
fragment, derivative or variant thereof is determined by a number
of means well established in the art. For example, administering to
an animal a radioactively, enzymatically or fluorescently labeled
LINGO-1 antibody, antibody fragment, derivative and variant thereof
linked to a brain targeting moiety; determining brain localization;
and comparing localization with an equivalent radioactively,
enzymatically or fluorescently labeled LINGO-1 antibody, antibody
fragment, derivative or variant thereof that is not associated with
a brain targeting moiety. Other means of determining enhanced
targeting are described in the above references.
[0263] As discussed elsewhere herein, LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention may be fused to heterologous polypeptides to increase the
in vivo half life of the polypeptides or for use in immunoassays
using methods known in the art. For example, in one embodiment, PEG
can be conjugated to the LINGO-1 antibodies of the invention to
increase their half-life in vivo. Leong, S. R., et al., Cytokine
16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et
al., Biochem. Soc. Transactions 30:512 (2002).
[0264] Moreover, LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof of the invention can be fused to
marker sequences, such as a peptide to facilitate their
purification or detection. In preferred embodiments, the marker
amino acid sequence is 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. Other
peptide tags useful for purification include, but are not limited
to, the "HA" tag, which corresponds to an epitope derived from the
influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))
and the "flag" tag.
[0265] Fusion proteins can be prepared using methods that are well
known in the art (see for example U.S. Pat. Nos. 5,116,964 and
5,225,538). The precise site at which the fusion is made may be
selected empirically to optimize the secretion or binding
characteristics of the fusion protein. DNA encoding the fusion
protein is then transfected into a host cell for expression.
[0266] LINGO-1 antibodies or antigen-binding fragments, variants,
or derivatives thereof of the present invention may be used in
non-conjugated form or may be conjugated to at least one of a
variety of molecules, e.g., to improve the therapeutic properties
of the molecule, to facilitate target detection, or for imaging or
therapy of the patient. LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention can be
labeled or conjugated either before or after purification, when
purification is performed.
[0267] In particular, LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention may be
conjugated to therapeutic agents, prodrugs, peptides, proteins,
enzymes, viruses, lipids, biological response modifiers,
pharmaceutical agents, or PEG.
[0268] Those skilled in the art will appreciate that conjugates may
also be assembled using a variety of techniques depending on the
selected agent to be conjugated. For example, conjugates with
biotin are prepared e.g. by reacting a binding polypeptide with an
activated ester of biotin such as the biotin N-hydroxysuccinimide
ester. Similarly, conjugates with a fluorescent marker may be
prepared in the presence of a coupling agent, e.g. those listed
herein, or by reaction with an isothiocyanate, preferably
fluorescein-isothiocyanate. Conjugates of the LINGO-1 antibodies,
or antigen-binding fragments, variants, or derivatives thereof of
the invention are prepared in an analogous manner.
[0269] The present invention further encompasses LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the invention conjugated to a diagnostic or therapeutic
agent. The LINGO-1 antibodies can be used diagnostically to, for
example, monitor the development or progression of a neurological
disease as part of a clinical testing procedure to, e.g., determine
the efficacy of a given treatment and/or prevention regimen.
Detection can be facilitated by coupling the LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, radioactive
materials, positron emitting metals using various positron emission
tomographies, and nonradioactive paramagnetic metal ions. See, for
example, U.S. Pat. No. 4,741,900 for metal ions which can be
conjugated to antibodies for use as diagnostics according to the
present invention. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples,
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin; and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.111In or .sup.99Tc.
[0270] A LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof also can be detectably labeled by coupling it to
a chemiluminescent compound. The presence of the
chemiluminescent-tagged LINGO-1 antibody is then determined by
detecting the presence of luminescence that arises during the
course of a chemical reaction. Examples of particularly useful
chemiluminescent labeling compounds are luminol, isoluminol,
theromatic acridinium ester, imidazole, acridinium salt and oxalate
ester.
[0271] One of the ways in which a LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof can be
detectably labeled is by linking the same to an enzyme and using
the linked product in an enzyme immunoassay (EIA) (Voller, A., "The
Enzyme Linked Immunosorbent Assay (ELISA)" Microbiological
Associates Quarterly Publication, Walkersville, Md., Diagnostic
Horizons 2:1-7 (1978)); Voller et al., J. Clin. Pathol. 31:507-520
(1978); Butler, J. E., Meth. Enrymol. 73:482-523 (1981); Maggio, E.
(ed.), Enzyme Immunoassay, CRC Press, Boca Raton, Fla., (1980);
Ishikawa, E. et al., (eds.), Enzyme Immunoassay, Kgaku Shoin, Tokyo
(1981). The enzyme, which is bound to the LINGO-1 antibody will
react with an appropriate substrate, preferably a chromogenic
substrate, in such a manner as to produce a chemical moiety which
can be detected, for example, by spectrophotometric, fluorimetric
or by visual means. Enzymes which can be used to detectably label
the antibody include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate, dehydrogenase, triose
phosphate isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. Additionally, the detection can be
accomplished by colorimetric methods which employ a chromogenic
substrate for the enzyme. Detection may also be accomplished by
visual comparison of the extent of enzymatic reaction of a
substrate in comparison with similarly prepared standards.
[0272] Detection may also be accomplished using any of a variety of
other immunoassays. For example, by radioactively labeling the
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof, it is possible to detect the antibody through
the use of a radioimmunoassay (RIA) (see, for example, Weintraub,
B., Principles of Radioimmunoassays, Seventh Training Course on
Radioligand Assay Techniques, The Endocrine Society, (March,
1986)), which is incorporated by reference herein). The radioactive
isotope can be detected by means including, but not limited to, a
gamma counter, a scintillation counter, or autoradiography.
[0273] A LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof can also be detectably labeled using
fluorescence emitting metals such as 152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0274] Techniques for conjugating various moieties to a LINGO-1
antibody, or antigen-binding fragment, variant, or derivative
thereof are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53
(1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody hi Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
Academic Press pp. 303-16 (1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev. 62:119-58 (1982).
VII. Expression of Antibody Polypeptides
[0275] As is well known, RNA may be isolated from the original
hybridoma cells or from other transformed cells by standard
techniques, such as guanidinium isothiocyanate extraction and
precipitation followed by centrifugation or chromatography. Where
desirable, mRNA may be isolated from total RNA by standard
techniques such as chromatography on oligo dT cellulose. Suitable
techniques are familiar in the art.
[0276] In one embodiment, cDNAs that encode the light and the heavy
chains of the antibody may be made, either simultaneously or
separately, using reverse transcriptase and DNA polymerase in
accordance with well known methods. PCR may be initiated by
consensus constant region primers or by more specific primers based
on the published heavy and light chain DNA and amino acid
sequences. As discussed above, PCR also may be used to isolate DNA
clones encoding the antibody light and heavy chains. In this case
the libraries may be screened by consensus primers or larger
homologous probes, such as mouse constant region probes.
[0277] DNA, typically plasmid DNA, may be isolated from the cells
using techniques known in the art, restriction mapped and sequenced
in accordance with standard, well known techniques set forth in
detail, e.g., in the foregoing references relating to recombinant
DNA techniques. Of course, the DNA may be synthetic according to
the present invention at any point during the isolation process or
subsequent analysis.
[0278] Following manipulation of the isolated genetic material to
provide LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention, the polynucleotides
encoding the LINGO-1 antibodies are typically inserted in an
expression vector for introduction into host cells that may be used
to produce the desired quantity of LINGO-1 antibody.
[0279] Recombinant expression of an antibody, or fragment,
derivative or analog thereof, e.g., a heavy or light chain of an
antibody which binds to a target molecule described herein, e.g.,
LINGO-1, requires construction of an expression vector containing a
polynucleotide that encodes the antibody. Once a polynucleotide
encoding an antibody molecule or a heavy or light chain of an
antibody, or portion thereof (preferably containing the heavy or
light chain variable domain), of the invention has been obtained,
the vector for the production of the antibody molecule may be
produced by recombinant DNA technology using techniques well known
in the art. Thus, methods for preparing a protein by expressing a
polynucleotide containing an antibody encoding nucleotide sequence
are described herein. Methods which are well known to those skilled
in the art can be used to construct expression vectors containing
antibody coding sequences and appropriate transcriptional and
translational control signals. These methods include, for example,
in vitro recombinant DNA techniques, synthetic techniques, and in
vivo genetic recombination. The invention, thus, provides
replicable vectors comprising a nucleotide sequence encoding an
antibody molecule of the invention, or a heavy or light chain
thereof, or a heavy or light chain variable domain, operably linked
to a promoter. Such vectors may include the nucleotide sequence
encoding the constant region of the antibody molecule (see, e.g.,
PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.
Pat. No. 5,122,464) and the variable domain of the antibody may be
cloned into such a vector for expression of the entire heavy or
light chain.
[0280] The host cell may be co-transfected with two expression
vectors of the invention, the first vector encoding a heavy chain
derived polypeptide and the second vector encoding a light chain
derived polypeptide. The two vectors may contain identical
selectable markers which enable equal expression of heavy and light
chain polypeptides. Alternatively, a single vector may be used
which encodes both heavy and light chain polypeptides. In such
situations, the light chain is advantageously placed before the
heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci.
USA 77:2197 (1980)). The coding sequences for the heavy and light
chains may comprise cDNA or genomic DNA.
[0281] The term "vector" or "expression vector" is used herein to
mean vectors used in accordance with the present invention as a
vehicle for introducing into and expressing a desired gene in a
host cell. As known to those skilled in the art, such vectors may
easily be selected from the group consisting of plasmids, phages,
viruses and retroviruses. In general, vectors compatible with the
instant invention will comprise a selection marker, appropriate
restriction sites to facilitate cloning of the desired gene and the
ability to enter and/or replicate in eukaryotic or prokaryotic
cells.
[0282] For the purposes of this invention, numerous expression
vector systems may be employed. For example, one class of vector
utilizes DNA elements which are derived from animal viruses such as
bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus,
baculovirus, retroviruses (RSV, MMTV or MOMLV) or SV40 virus.
Others involve the use of polycistronic systems with internal
ribosome binding sites. Additionally, cells which have integrated
the DNA into their chromosomes may be selected by introducing one
or more markers which allow selection of transfected host cells.
The marker may provide for prototrophy to an auxotrophic host,
biocide resistance (e.g., antibiotics) or resistance to heavy
metals such as copper. The selectable marker gene can either be
directly linked to the DNA sequences to be expressed, or introduced
into the same cell by cotransformation. Additional elements may
also be needed for optimal synthesis of mRNA. These elements may
include signal sequences, splice signals, as well as
transcriptional promoters, enhancers, and termination signals.
[0283] In particularly preferred embodiments the cloned variable
region genes are inserted into an expression vector along with the
heavy and light chain constant region genes (preferably human)
synthetic as discussed above. In one embodiment, this is effected
using a proprietary expression vector of Biogen IDEC, Inc.,
referred to as NEOSPLA (U.S. Pat. No. 6,159,730). This vector
contains the cytomegalovirus promoter/enhancer, the mouse beta
globin major promoter, the SV40 origin of replication, the bovine
growth hormone polyadenylation sequence, neomycin
phosphotransferase exon 1 and exon 2, the dihydrofolate reductase
gene and leader sequence. This vector has been found to result in
very high level expression of antibodies upon incorporation of
variable and constant region genes, transfection in CHO cells,
followed by selection in G418 containing medium and methotrexate
amplification. Of course, any expression vector which is capable of
eliciting expression in eukaryotic cells may be used in the present
invention. Examples of suitable vectors include, but are not
limited to plasmids pcDNA3, pHCMV/Zeo, pCR3.1, pEF1/His, pIND/GS,
pRc/HCMV2, pSV40/Zeo2, pTRACER-HCMV, pUB6/V5-His, pVAX1, and
pZeoSV2 (available from Invitrogen, San Diego, Calif.), and plasmid
pCI (available from Promega, Madison, Wis.). In general, screening
large numbers of transformed cells for those which express suitably
high levels if immunoglobulin heavy and light chains is routine
experimentation which can be carried out, for example, by robotic
systems. Vector systems are also taught in U.S. Pat. Nos. 5,736,137
and 5,658,570, each of which is incorporated by reference in its
entirety herein. This system provides for high expression levels,
e.g., >30 pg/cell/day. Other exemplary vector systems are
disclosed e.g., in U.S. Pat. No. 6,413,777.
[0284] In other preferred embodiments the LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention may be expressed using polycistronic constructs such as
those disclosed in United States Patent Application Publication No.
2003-0157641 A1, filed Nov. 18, 2002 and incorporated herein in its
entirety. In these novel expression systems, multiple gene products
of interest such as heavy and light chains of antibodies may be
produced from a single polycistronic construct. These systems
advantageously use an internal ribosome entry site (IRES) to
provide relatively high levels of LINGO-1 antibodies, e.g., binding
polypeptides, e.g., LINGO-1-specific antibodies or immunospecific
fragments thereof in eukaryotic host cells. Compatible IRES
sequences are disclosed in U.S. Pat. No. 6,193,980 which is also
incorporated herein. Those skilled in the art will appreciate that
such expression systems may be used to effectively produce the full
range of LINGO-1 antibodies disclosed in the instant
application.
[0285] More generally, once the vector or DNA sequence encoding a
monomeric subunit of the LINGO-1 antibody has been prepared, the
expression vector may be introduced into an appropriate host cell.
Introduction of the plasmid into the host cell can be accomplished
by various techniques well known to those of skill in the art.
These include, but are not limited to, transfection (including
electrophoresis and electroporation), protoplast fusion, calcium
phosphate precipitation, cell fusion with enveloped DNA,
microinjection, and infection with intact virus. See, Ridgway, A.
A. G. "Mammalian Expression Vectors" Vectors, Rodriguez and
Denhardt, Eds., Butterworths, Boston, Mass., Chapter 24.2, pp.
470-472 (1988). Typically, plasmid introduction into the host is
via electroporation. The host cells harboring the expression
construct are grown under conditions appropriate to the production
of the light chains and heavy chains, and assayed for heavy and/or
light chain protein synthesis. Exemplary assay techniques include
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
or fluorescence-activated cell sorter analysis (FACS),
immunohistochemistry and the like.
[0286] The expression vector is transferred to a host cell by
conventional techniques and the transfected cells are then cultured
by conventional techniques to produce an antibody for use in the
methods described herein. Thus, the invention includes host cells
containing a polynucleotide encoding an antibody of the invention,
or a heavy or light chain thereof, operably linked to a
heterologous promoter. In preferred embodiments for the expression
of double-chained antibodies, vectors encoding both the heavy and
light chains may be co-expressed in the host cell for expression of
the entire immunoglobulin molecule, as detailed below.
[0287] As used herein, "host cells" refers to cells which harbor
vectors constructed using recombinant DNA techniques and encoding
at least one heterologous gene. In descriptions of processes for
isolation of antibodies from recombinant hosts, the terms "cell"
and "cell culture" are used interchangeably to denote the source of
antibody unless it is clearly specified otherwise. In other words,
recovery of polypeptide from the "cells" may mean either from spun
down whole cells, or from the cell culture containing both the
medium and the suspended cells.
[0288] A variety of host-expression vector systems may be utilized
to express antibody molecules for use in the methods described
herein. Such host-expression systems represent vehicles by which
the coding sequences of interest may be produced and subsequently
purified, but also represent cells which may, when transformed or
transfected with the appropriate nucleotide coding, sequences,
express an antibody molecule of the invention in situ. These
include to are not limited to microorganisms such as bacteria
(e.g., E. coli, B. subtilis) transformed with recombinant
bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors
containing antibody coding sequences; yeast (e.g., Saccharomyces,
Pichia) transformed with recombinant yeast expression vectors
containing antibody coding sequences; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing antibody coding sequences; plant cell systems infected
with recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transformed with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing antibody coding sequences; or mammalian cell systems
(e.g., COS, CHO, BLK, 293, 3T3 cells) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K promoter). Preferably, bacterial cells such as
Escherichia coli, and more preferably, eukaryotic cells, especially
for the expression of whole recombinant antibody molecule, are used
for the expression of a recombinant antibody molecule. For example,
mammalian cells such as Chinese hamster ovary cells (CHO), in
conjunction with a vector such as the major intermediate early gene
promoter element from human cytomegalovirus is an effective
expression system for antibodies (Foecking et al., Gene 45:101
(1986); Cockett et al., Bio/Technology 8:2 (1990)).
[0289] The host cell line used for protein expression is often of
mammalian origin; those skilled in the art are credited with
ability to preferentially determine particular host cell lines
which are best suited for the desired gene product to be expressed
therein. Exemplary host cell lines include, but are not limited to,
CHO (Chinese Hamster Ovary), DG44 and DUXB11 (Chinese Hamster Ovary
lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey
kidney line), COS (a derivative of CVI with SV40 T antigen), VERY,
BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster
fibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney
line), SP2/O (mouse myeloma), P3x63-Ag3.653 (mouse myeloma),
BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocyte) and
293 (human kidney). CHO cells are particularly preferred. Host cell
lines are typically available from commercial services, the
American Tissue Culture Collection or from published
literature.
[0290] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins and gene products. Appropriate cell lines or host
systems can be chosen to ensure the correct modification and
processing of the foreign protein expressed. To this end,
eukaryotic host cells which possess the cellular machinery for
proper processing of the primary transcript, glycosylation, and
phosphorylation of the gene product may be used.
[0291] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the antibody molecule may be engineered.
Rather than using expression vectors which contain viral origins of
replication, host cells can be transformed with DNA controlled by
appropriate expression control elements (e.g., promoter, enhancer,
sequences, transcription terminators, polyadenylation sites, etc.),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells may be allowed to grow for 1-2 days in an
enriched media, and then are switched to a selective media. The
selectable marker in the recombinant plasmid confers resistance to
the selection and allows cells to stably integrate the plasmid into
their chromosomes and grow to form foci which in turn can be cloned
and expanded into cell lines. This method may advantageously be
used to engineer cell lines which stably express the antibody
molecule.
[0292] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223 (1977)), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl.
Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817 1980) genes can
be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); TIB
TECH 11(5):155-215 (May, 1993); and hygro, which confers resistance
to hygromycin (Santerre et al., Gene 30:147 (1984). Methods
commonly known in the art of recombinant DNA technology which can
be used are described in Ausubel et al. (eds.), Current Protocols
in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,
Gene Transfer and Expression, A Laboratory Manual, Stockton Press,
NY (1990); and in Chapters 12 and 13, Dracopoli et al. (eds),
Current Prolocols in Human Genetics, John Wiley & Sons, NY
(1994); Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which
are incorporated by reference herein in their entireties.
[0293] The expression levels of an antibody molecule can be
increased by vector amplification (for a review, see Bebbington and
Hentschel, The use of vectors based on gene amplification for the
expression of cloned genes in mammalian cells in DNA cloning,
Academic Press, New York, Vol. 3. (1987)). When a marker in the
vector system expressing antibody is amplifiable, increase in the
level of inhibitor present in culture of host cell will increase
the number of copies of the marker gene. Since the amplified region
is associated with the antibody gene, production of the antibody
will also increase (Crouse et al., Mol. Cell. Biol. 3:257
(1983)).
[0294] In vitro production allows scale-up to give large amounts of
the desired polypeptides. Techniques for mammalian cell cultivation
under tissue culture conditions are known in the art and include
homogeneous suspension culture, e.g. in an airlift reactor or in a
continuous stirrer reactor, or immobilized or entrapped cell
culture, e.g. in hollow fibers, microcapsules, on agarose
microbeads or ceramic cartridges. If necessary and/or desired, the
solutions of polypeptides can be purified by the customary
chromatography methods, for example gel filtration, ion-exchange
chromatography, chromatography over DEAE-cellulose or
(immuno-)affinity chromatography, e.g., after preferential
biosynthesis of a synthetic hinge region polypeptide or prior to or
subsequent to the HIC chromatography step described herein.
[0295] Genes encoding LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention can
also be expressed non-mammalian cells such as bacteria or yeast or
plant cells. Bacteria which readily take up nucleic acids include
members of the enterobacteriaceae, such as strains of Escherichia
coli or Salmonella; Bacillaceae, such as Bacillus subtilis;
Pneumococcus; Streptococcus, and Haemophilus influenzae. It will
further be appreciated that, when expressed in bacteria, the
heterologous polypeptides typically become part of inclusion
bodies. The heterologouspolypeptides must be isolated, purified and
then assembled into functional molecules. Where tetravalent forms
of antibodies are desired, the subunits will then self-assemble
into tetravalent antibodies (WO02/096948A2).
[0296] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
antibody molecule being expressed. For example, when a large
quantity of such a protein is to be produced, for the generation of
pharmaceutical compositions of an antibody molecule, vectors which
direct the expression of high levels of fusion protein products
that are readily purified may be desirable. Such vectors include,
but are not limited, to the E. coli expression vector pUR278
(Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody
coding sequence may be ligated individually into the vector in
frame with the lacZ coding region so that a fusion protein is
produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
24:5503-5509 (1989)); and the like. pGEX vectors may also be used
to express foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption and
binding to a matrix glutathione-agarose beads followed by elution
in the presence of free glutathione. The pGEX vectors are designed
to include thrombin or factor Xa protease cleavage sites so that
the cloned target gene product can be released from the GST
moiety.
[0297] In addition to prokaryotes, eukaryotic microbes may also be
used. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among eukaryotic microorganisms although a
number of other strains are commonly available, e.g., Pichia
pastoris.
[0298] For expression in Saccharomyces, the plasmid YRp7, for
example, (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al.,
Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is
commonly used. This plasmid already contains the TRP1 gene which
provides a selection marker for a mutant strain of yeast lacking
the ability to grow in tryptophan, for example ATCC No. 44076 or
PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trp1
lesion as a characteristic of the yeast host cell genome then
provides an effective environment for detecting transformation by
growth in the absence of tryptophan.
[0299] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is typically used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda cells. The
antibody coding sequence may be cloned individually into
non-essential regions (for example the polyhedrin gene) of the
virus and placed under control of an AcNPV promoter (for example
the polyhedrin promoter).
[0300] Once an antibody molecule of the invention has been
recombinantly expressed, it may be purified by any method known in
the art for purification of an immunoglobulin molecule, for
example, by chromatography (e.g., ion exchange, affinity,
particularly by affinity for the specific antigen after Protein A,
and sizing column chromatography), centrifugation, differential
solubility, or by any other standard technique for the purification
of proteins. Alternatively, a preferred method for increasing the
affinity of antibodies of the invention is disclosed in US 2002
0123057 A1.
VIII. Treatment Methods Using Therapeutic LINGO-1 Antibodies
[0301] As described herein, LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention can
relieve NgR1-mediated inhibition of axonal extension that normally
takes place in CNS neurons. This is beneficial in situations where
axonal extension or neurite sprouting is needed in the brain or
spinal cord. Spinal cord injury; including partial or complete
crush or severance, exemplifies a situation in which axonal
extension is needed, but is normally inhibited through operation of
the Nogo pathway. Examples of diseases or disorders in which axonal
extension and/or neurite sprouting in the brain would be beneficial
include stroke, multiple sclerosis, and other neurodegenerative
diseases or disorders such as multiple sclerosis (MS), progressive
multifocal leukoencephalopathy (PML), encephalomyelitis (EPL),
central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's
disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell
Leucodystrophy (Krabbe's disease) and Wallerian Degeneration, optic
neuritis, transverse myelitis, amylotrophic lateral sclerosis
(ALS), Huntington's disease, Alzheimer's disease, Parkinson's
disease, spinal cord injury, traumatic brain injury, post radiation
injury, neurologic complications of chemotherapy, stroke,
neuropathy, acute ischemic optic neuropathy, vitamin E deficiency,
isolated vitamin E deficiency syndrome, AR, Bassen-Kornzweig
syndrome, Marchiafava-Bignami syndrome, metachromatic
leukodystrophy, trigeminal neuralgia, Bell's palsy, spinal cord
injury and all neurological diseases related to neuronal cell
death.
[0302] The inventors have further discovered that LINGO-1 is
expressed in oligodendrocytes, and contributes to oligodendrocyte
biology. Soluble derivatives of LINGO-1, certain polynucleotides
(e.g. RNAi), as well as certain antibodies which specifically bind
to LINGO-1, as described herein act as antagonists to LINGO-1
function in oligodendrocytes, promoting proliferation,
differentiation and survival of oligodendrocytes and promoting
myelination of neurons in vitro and in vivo. This is beneficial in
for diseases, disorders or conditions involving demyelination and
dysmyelination. Examples of diseases or disorders in which
oligodendrocyte proliferation, differentiation and survival, and/or
myelination or remyelination would be beneficial include multiple
sclerosis (MS), progressive multifocal leukoencephalopathy (PML),
encephalomyelitis (EPL), central pontine myelolysis (CPM),
adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher
disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease),
Wallerian Degeneration, optic neuritis, transverse myelitis,
amylotrophic lateral sclerosis (ALS), Huntington's disease,
Alzheimer's disease, Parkinson's disease, spinal cord injury,
traumatic brain injury, post radiation injury, neurologic
complications of chemotherapy, stroke, acute ischemic optic
neuropathy, vitamin E deficiency, isolated vitamin E deficiency
syndrome, AR, Bassen-Kornzweig syndrome, Marchiafava-Bignami
syndrome, metachromatic leukodystrophy, trigeminal neuralgia, and
Bell's palsy.
[0303] Accordingly, one embodiment of the present invention
provides methods for treating spinal cord injury, diseases or
disorders associated with inhibition of neuronal growth in the CNS,
diseases or disorders associated with inhibition of oligodendrocyte
growth or differentiation, and diseases involving demyelination or
dysmyelination of CNS neurons in an animal suffering from such
injury or disease or predisposed to contract such disease, the
method comprising, consisting essentially of, or consisting of
administering to the animal an effective amount of a LINGO-1
antibody, or antigen-binding fragment, variant, or derivative
thereof.
[0304] A therapeutic LINGO-1 antibody to be used in treatment
methods disclosed herein can be prepared and used as a therapeutic
agent which promotes CNS neurite outgrowth, neuronal survival, axon
guidance and axon regeneration, which promotes oligodendrocyte
survival, growth, and/or differentiation, and which promotes
myelination or remyelination of CNS neurons. Characteristics of
suitable therapeutic LINGO-1 antibodies include: binding to LINGO-1
epitopes which result in blocking of LINGO-1 activity, binding to
LINGO-1 with sufficient affinity to elicit a therapeutic effect,
and binding to LINGO-1 preferentially to normal binding partners,
e.g., Nogo Receptor.
[0305] Therapeutic LINGO-1 antibodies may be monoclonal, chimeric
or humanized antibodies, or fragments of antibodies that bind
specifically to LINGO-1. The antibodies may be monovalent,
bivalent, polyvalent, or bifunctional antibodies. Antibody
fragments include without limitation Fab F(ab').sub.2, and Fv
fragments.
[0306] Therapeutic LINGO-1 antibodies, or antigen-binding
fragments, variants or derivatives thereof according to the
invention can be used in unlabeled or unconjugated form, or can be
coupled or linked to drugs, labels or stabilization agents which
may or may not exert additional therapeutic effects.
[0307] A specific dosage and treatment regimen for any particular
patient will depend upon a variety of factors, including the
particular LINGO-1 antibody, or antigen-binding fragment, variant
or derivative thereof used, the patient's age, body weight, general
health, sex, and diet, and the time of administration, rate of
excretion, drug combination, and the severity of the particular
disease being treated. Judgment of such factors by medical
caregivers is within the ordinary skill in the art. The amount will
also depend on the individual patient to be treated, the route of
administration, the type of formulation, the characteristics of the
compound used, the severity of the disease, and the desired effect.
The amount used can be determined by pharmacological and
pharmacokinetic principles well known in the art.
[0308] In the methods of the invention the LINGO-1 antibodies, or
antigen-binding fragments, variants or derivatives thereof may be
administered directly to the nervous system,
intracerebroventricularly, or intrathecally, e.g. into a chronic
lesion of MS, as discussed in more detail below.
[0309] In various embodiments, a LINGO-1 antibody as described
above is an antagonist of LINGO-1 activity. In certain embodiments,
for example, binding of an antagonist LINGO-1 antibody to LINGO-1,
as expressed on neurons, blocks myelin-associated neurite outgrowth
inhibition or neuronal cell death. In other embodiments, binding of
the LINGO-1 antibody to LINGO-1, as expressed on oligodendrocytes,
blocks inhibition of oligodendrocyte growth or differentiation, or
blocks demyelination or dysmyelination of CNS neurons.
[0310] In methods of the present invention, a LINGO-1 antibody, or
an antigen-binding fragment, variant, or derivative thereof, in
particular the LINGO-1 antibodies described herein, can be
administered directly as a preformed polypeptide, or indirectly
through a nucleic acid vector, to permit beneficial axonal
outgrowth, promote oligodendrocyte proliferation, differentiation,
and survival, and/or promote myelination or remyelination.
[0311] In certain embodiments, a subject may be treated with a
nucleic acid molecule encoding a LINGO-1 antibody, or
antigen-binding fragment variant, or analog thereof, e.g., in a
vector. Doses for nucleic acids encoding polypeptides range from
about 10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300
.mu.g DNA per patient. Doses for infectious viral vectors vary from
10-100, or more, virions per dose.
[0312] In some embodiments of the present invention a LINGO-1
antibody, or an antigen-binding fragment, variant, or derivative
thereof is administered in a treatment method that includes: (1)
transforming or transfecting an implantable host cell with a
nucleic acid, e.g., a vector, that expresses a LINGO-1 antibody, or
an antigen-binding fragment, variant, or derivative thereof; and
(2) implanting the transformed host cell into a mammal, at the site
of a disease, disorder or injury. For example, the transformed host
cell can be implanted at the site of a spinal cord injury or at a
site of dysmyelination. In some embodiments of the invention, the
implantable host cell is removed from a mammal, temporarily
cultured, transformed or transfected with an isolated nucleic acid
encoding a a LINGO-1 antibody, and implanted back into the same
mammal from which it was removed. The cell can be, but is not
required to be, removed from the same site at which it is
implanted. Such embodiments, sometimes known as ex vivo gene
therapy, can provide a continuous supply of the LINGO-1
polypeptide, localized at the site of site of action, for a limited
period of time.
[0313] The methods for treating spinal cord injury, diseases or
disorders associated with inhibition of neuronal growth in the CNS,
diseases or disorders associated with inhibition of oligodendrocyte
growth or differentiation, and diseases involving demyelination or
dysmyelination of CNS neurons comprising administration of a
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof of the invention are typically tested in vitro,
and then in vivo in an acceptable animal model, for the desired
therapeutic or prophylactic activity, prior to use in humans.
Suitable animal models, including transgenic animals, are will
known to those of ordinary skill in the art. For example, in vitro
assays to demonstrate the therapeutic utility of LINGO-1 antibody
described herein include the effect of a LINGO-1 antibody on a cell
line or a patient tissue sample. The effect of the LINGO-1 antibody
on the cell line and/or tissue sample can be determined utilizing
techniques known to those of skill in the art, such as the assays
disclosed elsewhere herein. In accordance with the invention, in
vitro assays which can be used to determine whether administration
of a specific LINGO-1 antibody is indicated, include in vitro cell
culture assays in which a patient tissue sample is grown in
culture, and exposed to or otherwise administered a compound, and
the effect of such compound upon the tissue sample is observed.
[0314] Supplementary active compounds also can be incorporated into
the compositions of the invention. For example, a LINGO-1 antibody,
or antigen-binding fragment, variant, or derivative thereof of the
invention may be coformulated with and/or coadministered with one
or more additional therapeutic agents.
[0315] The invention encompasses any suitable delivery method for a
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof of the invention to a selected target tissue,
including bolus injection of an aqueous solution or implantation of
a controlled-release system. Use of a controlled-release implant
reduces the need for repeat injections.
IX. Pharmaceutical Compositions and Administration Methods
[0316] Methods of preparing and administering LINGO-1 antibodies,
or antigen-binding fragments, variants, or derivatives thereof of
the invention to a subject in need thereof are well known to or are
readily determined by those skilled in the art. The route of
administration of the LINGO-1 antibody, or antigen-binding
fragment, variant, or derivative thereof may be, for example, oral,
parenteral, by inhalation or topical. The term parenteral as used
herein includes, e.g., intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, rectal or vaginal administration.
While all these forms of administration are clearly contemplated as
being within the scope of the invention, a form for administration
would be a solution for injection, in particular for intravenous or
intraarterial injection or drip. Usually, a suitable pharmaceutical
composition for injection may comprise a buffer (e.g. acetate,
phosphate or citrate buffer), a surfactant (e.g. polysorbate),
optionally a stabilizer agent (e.g. human albumin), etc. However,
in other methods compatible with the teachings herein, LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the invention can be delivered directly to the site of
the adverse cellular population thereby increasing the exposure of
the diseased tissue to the therapeutic agent.
[0317] As previously discussed, LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention may be administered in a pharmaceutically effective
amount for the in vivo treatment of mammalian spinal cord injury,
diseases or disorders associated with inhibition of neuronal growth
in the CNS, diseases or disorders associated with inhibition of
oligodendrocyte growth or differentiation, and diseases involving
demyelination or dysmyelination of CNS. In this regard, it will be
appreciated that the disclosed antibodies will be formulated so as
to facilitate administration and promote stability of the active
agent. Preferably, pharmaceutical compositions in accordance with
the present invention comprise a pharmaceutically acceptable,
non-toxic, sterile carrier such as physiological saline, non-toxic
buffers, preservatives and the like. For the purposes of the
instant application, a pharmaceutically effective amount of a
LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof, conjugated or unconjugated, shall be held to
mean an amount sufficient to achieve effective binding to a target
and to achieve a benefit, e.g., to ameliorate symptoms of a disease
or disorder or to detect a substance or a cell.
[0318] The pharmaceutical compositions used in this invention
comprise pharmaceutically acceptable carriers, including, e.g., ion
exchangers, alumina, aluminum stearate, lecithin, serum proteins,
such as human serum albumin, buffer substances such as phosphates,
glycine, sorbic acid, potassium sorbate, partial glyceride mixtures
of saturated vegetable fatty acids, water, salts or electrolytes,
such as protamine sulfate, disodium hydrogen phosphate, potassium
hydrogen phosphate, sodium chloride, zinc salts, colloidal silica,
magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based
substances, polyethylene glycol, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0319] Preparations for parenteral administration includes sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. In the subject invention,
pharmaceutically acceptable carriers include, but are not limited
to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.8% saline.
Other common parenteral vehicles include sodium phosphate
solutions, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers, such as
those based on Ringer's dextrose, and the like. Preservatives and
other additives may also be present such as for example,
antimicrobials, antioxidants, chelating agents and inert gases and
the like.
[0320] More particularly, pharmaceutical compositions suitable for
injectable use include sterile aqueous solutions (where water
soluble) or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersions. In such
cases, the composition must be sterile and should be fluid to the
extent that easy syringability exists. It should be stable under
the conditions of manufacture and storage and will preferably be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. Suitable formulations for
use in the therapeutic methods disclosed herein are described in
Remington's Pharmaceutical Sciences, Mack Publishing Co., 16th ed.
(1980).
[0321] Prevention of the action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars, polyalcohols, such as mannitol,
sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0322] In any case, sterile injectable solutions can be prepared by
incorporating an active compound (e.g., a LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof, by itself
or in combination with other active agents) in the required amount
in an appropriate solvent with one or a combination of ingredients
enumerated herein, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle, which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying, which yields a powder of an
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The preparations for
injections are processed, filled into containers such as ampoules,
bags, bottles, syringes or vials, and sealed under aseptic
conditions according to methods known in the art. Further, the
preparations may be packaged and sold in the form of a kit such as
those described in co-pending U.S. Ser. No. 09/259,337
(US-2002-0102208 A1), which is incorporated herein by reference in
its entirety. Such articles of manufacture will preferably have
labels or package inserts indicating that the associated
compositions are useful for treating a subject suffering from, or
predisposed to autoimmune or neoplastic disorders.
[0323] Parenteral formulations may be a single bolus dose, an
infusion or a loading bolus dose followed with a maintenance dose.
These compositions may be administered at specific fixed or
variable intervals, e.g., once a day, or on an "as needed"
basis.
[0324] Certain pharmaceutical compositions used in this invention
may be orally administered in an acceptable dosage form including,
e.g., capsules, tablets, aqueous suspensions or solutions. Certain
pharmaceutical compositions also may be administered by nasal
aerosol or inhalation. Such compositions may be prepared as
solutions in saline, employing benzyl alcohol or other suitable
preservatives, absorption promoters to enhance bioavailability,
and/or other conventional solubilizing or dispersing agents.
[0325] The amount of a LINGO-1 antibody, or fragment, variant, or
derivative thereof that may be combined With the carrier materials
to produce a single dosage form will vary depending upon the host
treated and the particular mode of administration. The composition
may be administered as :a single dose, multiple doses or over an
established period of time in an infusion. Dosage regimens also may
be adjusted to provide the optimum desired response (e.g., a
therapeutic or prophylactic response).
[0326] In keeping with the scope of the present disclosure, LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof of the invention may be administered to a human or other
animal in accordance with the aforementioned methods of treatment
in an amount sufficient to produce a therapeutic effect. The
LINGO-1 antibodies, or antigen-binding fragments, variants, or
derivatives thereof of the invention can be administered to such
human or other animal in a conventional dosage form prepared by
combining the antibody of the invention with a conventional
pharmaceutically acceptable carrier or diluent according to known
techniques. It will be recognized by one of skill in the art that
the form and character of the pharmaceutically acceptable carrier
or diluent is dictated by the amount of active ingredient with
which it is to be combined, the route of administration and other
well-known variables. Those skilled in the art will further
appreciate that a cocktail comprising one or more species of
LINGO-1 antibodies, or antigen-binding fragments, variants, or
derivatives thereof of the invention may prove to be particularly
effective.
[0327] Effective doses of the compositions of the present
invention, for treatment of spinal cord injury, diseases or
disorders associated with inhibition of neuronal growth in the CNS,
diseases or disorders associated with inhibition of oligodendrocyte
growth or differentiation, and diseases involving demyelination or
dysmyelination of CNS 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 non-human mammals
including transgenic mammals can also be treated. Treatment dosages
may be titrated using routine methods known to those of skill in
the art to optimize safety and efficacy.
[0328] For treatment of spinal cord injury, diseases or disorders
associated with inhibition of neuronal growth in the CNS, diseases
or disorders associated with inhibition of oligodendrocyte growth
or differentiation, and diseases involving demyelination or
dysmyelination of CNS with a LINGO-1 antibody, or antigen-binding
fragment, variant, or derivative thereof, the dosage can range,
e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5
mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1
mg/kg, 2 mg/kg, etc.), 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, preferably at least 1 mg/kg. Doses
intermediate in the above ranges are also intended to be within the
scope of the invention. Subjects can be administered such doses
daily, on alternative days, weekly or according to any other
schedule determined by empirical analysis. An exemplary treatment
entails administration in multiple dosages over a prolonged period,
for example, of at least six months. Additional exemplary treatment
regimes entail administration once per every two weeks or once a
month or once every 3 to 6 months. Exemplary dosage schedules
include 1-10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on
alternate days or 60 mg/kg weekly. In some methods, two or more
monoclonal antibodies with different binding specificities are
administered simultaneously, in which case the dosage of each
antibody administered falls within the ranges indicated.
[0329] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention can be administered on
multiple occasions. Intervals between single dosages can be daily,
weekly, monthly or yearly. Intervals can also be irregular as
indicated by measuring blood levels of target polypeptide or target
molecule in the patient. In some methods, dosage is adjusted to
achieve a plasma polypeptide concentration of 1-1000 .mu.g/ml and
in some methods 25-300 .mu.g/ml. Alternatively, LINGO-1 antibodies,
or antigen-binding fragments, variants, or derivatives thereof of
the 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
antibody in the patient. The half-life of a LINGO-1 antibody can
also be prolonged via fusion to a stable polypeptide or moeity,
e.g., albumin or PEG. In general, humanized antibodies show the
longest half-life, followed by chimeric antibodies and nonhuman
antibodies. In one embodiment, the LINGO-1 antibodies, or
antigen-binding fragments, variants, or derivatives thereof of the
invention can be administered in unconjugated form, In another
embodiment, the LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof of the invention can be
administered multiple times in conjugated form. In still another
embodiment, LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof of the invention can be
administered in unconjugated form, then in conjugated form, or vice
versa.
[0330] The compositions of the present invention may be
administered by any suitable method, e.g., parenterally,
intraventricularly, orally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques. As
described previously, LINGO-1 antibodies, or antigen-binding
fragments, variants, or derivatives thereof of the invention act in
the nervous system to promote survival, proliferation and
differentiation of oligodendrocytes and myelination of neurons and
neuronal survival, axon regeneration and axon guidance.
Accordingly, in the methods of the invention, the LINGO-1
antibodies, or antigen-binding fragments, variants, or derivatives
thereof are administered in such a way that they cross the
blood-brain barrier. This crossing can result from the
physico-chemical properties inherent in the LINGO-1 antibody
molecule itself, from other components in a pharmaceutical
formulation, or from the use of a mechanical device such as a
needle, cannula or surgical instruments to breach the blood-brain
barrier. Where the LINGO-1 antibody is a molecule that does not
inherently cross the blood-brain barrier, e.g., a fusion to a
moiety that facilitates the crossing, suitable routes of
administration are, e.g., intrathecal or intracranial, e.g.,
directly into a chronic lesion of MS. Where the LINGO-1 antibody is
a molecule that inherently crosses the blood-brain barrier, the
route of administration may be by one or more of the various routes
described below. In some methods, antibodies are administered as a
sustained release composition or device, such as a Medipad.TM.
device. Delivery across the blood brain barrier can be enhanced by
a carrying molecule, such as anti-Fc receptor, transferrin,
anti-insulin receptor or a toxin conjugate or penetration
enhancer.
[0331] The LINGO-1 antibodies, or antigen-binding fragments,
variants, or derivatives thereof used in the methods of the
invention may be directly infused into the brain. Various implants
for direct brain infusion of compounds are known and are effective
in the delivery of therapeutic compounds to human patients
suffering from neurological disorders. These include chronic
infusion into the brain using a pump, stereotactically implanted,
temporary interstitial catheters, permanent intracranial catheter
implants, and surgically implanted biodegradable implants. See,
e.g., Gill et al., "Direct brain infusion of glial cell
line-derived neurotrophic factor in Parkinson disease," Nature Med.
9: 589-95 (2003); Scharfen et al., "High Activity Iodine-125
Interstitial Implant For Gliomas," Int. J. Radiation Oncology Biol.
Phys. 24(4):583-91 (1992); Gaspar et al., "Permanent .sup.125I
Implants for Recurrent Malignant Gliomas," Int. J. Radiation
Oncology Biol. Phys. 43(5):977-82 (1999); chapter 66, pages
577-580, Bellezza et al, "Stereotactic Interstitial Brachytherapy,"
in Gildenberg et al., Textbook of Stereotactic and Functional
Neurosurgery, McGraw-Hill (1998); and Brem et al., "The Safety of
Interstitial Chemotherapy with BCNU-Loaded Polymer Followed by
Radiation Therapy in the Treatment of Newly Diagnosed Malignant
Gliomas: Phase I Trial," J. Neuro-Oncology 26:111-23 (1995).
[0332] The compositions may also comprise a LINGO-1 antibody
dispersed in a biocompatible carrier material that functions as a
suitable delivery or support system for the compounds. Suitable
examples of sustained release carriers include semipermeable
polymer matrices in the form of shaped articles such as
suppositories or capsules. Implantable or microcapsular sustained
release matrices include polylactides (U.S. Pat. No. 3,773,319; EP
58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
(Sidman et al, Biopolymers 22:547-56 (1985));
poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate (Langer
et al., J. Biomed. Mater. Res. 15:167-277 (1981); Langer, Chem.
Tech. 12:98-105 (1982)) or poly-D-(-)-3hydroxybutyric acid (EP
133,988).
[0333] In some embodiments of the invention, a LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof of the
invention is administered to a patient by direct infusion into an
appropriate region of the brain. See, e.g., Gill et al., supra.
Alternative techniques are available and may be applied to
administer a LINGO-1 antibody according to the invention. For
example, stereotactic placement of a catheter or implant can be
accomplished using the Riechert-Mundinger unit and the ZD
(Zamorano-Dujovny) multipurpose localizing unit. A
contrast-enhanced computerized tomography (CT) scan, injecting 120
ml of omnipaque, 350 mg iodine/ml, with 2 mm slice thickness can
allow three-dimensional multiplanar treatment planning (STP,
Fischer, Freiburg, Germany). This equipment permits planning on the
basis of magnetic resonance imaging studies, merging the CT and MRI
target information for clear target confirmation.
[0334] The Leksell stereotactic system (Downs Surgical, Inc.,
Decatur, Ga.) modified for use with a GE CT scanner (General
Electric Company, Milwaukee, Wis.) as well as the
Brown-Roberts-Wells (BRW) stereotactic system (Radionics,
Burlington, Mass.) can be used for this purpose. Thus, on the
morning of the implant, the annular base ring of the BRW
stereotactic frame can be attached to the patient's skull. Serial
CT sections can be obtained at 3 mm intervals though the (target
tissue) region with a graphite rod localizer frame clamped to the
base plate. A computerized treatment planning program can be run on
a VAX 11/780 computer (Digital Equipment Corporation, Maynard,
Mass.) using CT coordinates of the graphite rod images to map
between CT space and BRW space.
[0335] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention can optionally be
administered in combination with other agents that are effective in
treating the disorder or condition in need of treatment (e.g.,
prophylactic or therapeutic).
X. Diagnostics
[0336] The invention further provides a diagnostic method useful
during diagnosis of neronal disorders or injuries, which involves
measuring the expression level of LINGO-1 protein or transcript in
tissue or other cells or body fluid from an individual and
comparing the measured expression level with a standard LINGO-1
expression levels in normal tissue or body fluid, whereby an
increase in the expression level compared to the standard is
indicative of a disorder.
[0337] LINGO-1-specific antibodies can be used to assay protein
levels in a biological sample using classical immunohistological
methods known to those of skill in the art (e.g., see Jalkanen, et
al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell
Biol. 105:3087-3096 (1987)). Other antibody-based methods useful
for detecting protein expression include immunoassays, such as the
enzyme linked immunosorbent assay (ELISA), immunoprecipitation, or
western blotting. Suitable assays are described in more detail
elsewhere herein.
[0338] By "assaying the expression level of LINGO-1 polypeptide" is
intended qualitatively or quantitatively measuring or estimating
the level of LINGO-1 polypeptide in a first biological sample
either directly (e.g., by determining or estimating absolute
protein level) or relatively (e.g., by comparing to the cancer
associated polypeptide level in a second biological sample).
Preferably, LINGO-1 polypeptide expression level in the first
biological sample is measured or estimated and compared to a
standard LINGO-1 polypeptide level, the standard being taken from a
second biological sample obtained from an individual not having the
disorder or being determined by averaging levels from a population
of individuals not having the disorder. As will be appreciated in
the art, once the "standard" LINGO-1 polypeptide level is known, it
can be used repeatedly as a standard for comparison.
[0339] By "biological sample" is intended any biological sample
obtained from an individual, cell line, tissue culture, or other
source of cells potentially expressing LINGO-1. Methods for
obtaining tissue biopsies and body fluids from mammals are well
known in the art.
[0340] LINGO-1 antibodies for use in the diagnostic methods
described above include any LINGO-1 antibody which specifically
binds to a LINGO-1 gene product, as described elsewhere herein.
XI. Immunoassays
[0341] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention may be assayed for
immunospecific binding by any method known in the art. The
immunoassays which can be used include but are not limited to
competitive and non-competitive assay systems using techniques such
as western blots, radioimmunoassays, ELISA (enzyme linked
immunosorbent assay), "sandwich" immunoassays, immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays, agglutination assays, complement-fixation
assays, immunoradiometric assays, fluorescent immunoassays, protein
A immunoassays, to name but a few. Such assays are routine and well
known in the art (see, e.g., Ausubel et al., eds, Current Protocols
in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1
(1994), which is incorporated by reference herein in its entirety).
Exemplary immunoassays are described briefly below (but are not
intended by way of limitation).
[0342] Immunoprecipitation protocols generally comprise lysing a
population of cells in a lysis buffer such as RIPA buffer (1% NP-40
or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl,
0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with
protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF,
aprotinin, sodium vanadate), adding the antibody of interest to the
cell lysate, incubating for a period of time (e.g., 1-4 hours) at
4.degree. C., adding protein A and/or protein G sepharose beads to
the cell lysate, incubating for about an hour or more at 4.degree.
C., washing the beads in lysis buffer and resuspending the beads in
SDS/sample buffer. The ability of the antibody of interest to
immunoprecipitate a particular antigen can be assessed by, e.g.,
western blot analysis. One of skill in the art would be
knowledgeable as to the parameters that can be modified to increase
the binding of the antibody to an antigen and decrease the
background (e.g., pre-clearing the cell lysate with sepharose
beads). For further discussion regarding immunoprecipitation
protocols see, e.g., Ausubel et al., eds, Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1
(1994) at 10.16.1.
[0343] Western blot analysis generally comprises preparing protein
samples, electrophoresis of the protein samples in a polyacrylamide
gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the
antigen), transferring the protein sample from the polyacrylamide
gel to a membrane such as nitrocellulose, PVDF or nylon, blocking
the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat
milk), washing the membrane in washing buffer (e.g., PBS-Tween 20),
blocking the membrane with primary antibody (the antibody of
interest) diluted in blocking buffer, washing the membrane in
washing buffer, blocking the membrane with a secondary antibody
(which recognizes the primary antibody, e.g., an anti-human
antibody) conjugated to an enzymatic substrate (e.g., horseradish
peroxidase or alkaline phosphatase) or radioactive molecule (e.g.,
32p or 1251) diluted in blocking buffer, washing the membrane in
wash buffer, and detecting the presence of the antigen. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected and to reduce the
background noise. For further discussion regarding western blot
protocols see, e.g., Ausubel et al., eds, Current Protocols in
Molecular Biology, John Wiley & Sons, Inc., New York Vol. 1
(1994) at 10.8.1.
[0344] ELISAs comprise preparing antigen, coating the well of a 96
well microtiter plate with the antigen, adding the antibody of
interest conjugated to a detectable compound such as an enzymatic
substrate (e.g., horseradish peroxidase or alkaline phosphatase) to
the well and incubating for a period of time, and detecting the
presence of the antigen. In ELISAs the antibody of interest does
not have to be conjugated to a detectable compound; instead, a
second antibody (which recognizes the antibody of interest)
conjugated to a detectable compound may be added to the well.
Further, instead of coating the well with the antigen, the antibody
may be coated to the well. In this case, a second antibody
conjugated to a detectable compound may be added following the
addition of the antigen of interest to the coated well. One of
skill in the art would be knowledgeable as to the parameters that
can be modified to increase the signal detected as well as other
variations of ELISAs known in the art. For further discussion
regarding ELISAs see, e.g., Ausubel et al., eds, Current Protocols
in Molecular Biology, John Wiley & Sons, Inc., New York, Vol. 1
(1994) at 11.2.1.
[0345] The binding affinity of an antibody to an antigen and the
off-rate of an antibody-antigen interaction can be determined by
competitive binding assays. One example of a competitive binding
assay is a radioimmunoassay comprising the incubation of labeled
antigen (e.g., .sup.3H or .sup.125I) with the antibody of interest
in the presence of increasing amounts of unlabeled antigen, and the
detection of the antibody bound to the labeled antigen. The
affinity of the antibody of interest for a particular antigen and
the binding off-rates can be determined from the data by scatchard
plot analysis. Competition with a second antibody can also be
determined using radioimmunoassays. In this case, the antigen is
incubated with antibody of interest is conjugated to a labeled
compound (e.g., .sup.3H or .sup.125I) in the presence of increasing
amounts of an unlabeled second antibody.
[0346] LINGO-1 antibodies, or antigen-binding fragments, variants,
or derivatives thereof of the invention, additionally, be employed
histologically, as in immunofluorescence, immunoelectron microscopy
or non-immunological assays, for in situ detection of cancer
antigen gene products or conserved variants or peptide fragments
thereof. In situ detection may be accomplished by removing a
histological specimen from a patient, and applying thereto a
labeled LINGO-1 antibody, or antigen-binding fragment, variant, or
derivative thereof, preferably applied by overlaying the labeled
antibody (or fragment) onto a biological sample. Through the use of
such a procedure, it is possible to determine not only the presence
of LINGO-1 protein, or conserved variants or peptide fragments, but
also its distribution in the examined tissue. Using the present
invention, those of ordinary skill will readily perceive that any
of a wide variety of histological methods (such as staining
procedures) can be modified in order to achieve such in situ
detection.
[0347] Immunoassays and non-immunoassays for LINGO-1 gene products
or conserved variants or peptide fragments thereof will typically
comprise incubating a sample, such as a biological fluid, a tissue
extract, freshly harvested cells, or lysates of cells which have
been incubated in cell culture, in the presence of a detectably
labeled antibody capable of binding to LINGO-1 or conserved
variants or peptide fragments thereof, and detecting the bound
antibody by any of a number of techniques well-known in the
art.
[0348] The biological sample may be brought in contact with and
immobilized onto a solid phase support or carrier such as
nitrocellulose, or other solid support which is capable of
immobilizing cells, cell particles or soluble proteins. The support
may then be washed with suitable buffers followed by treatment with
the detectably labeled LINGO-1 antibody, or antigen-binding
fragment, variant, or derivative thereof. The solid phase support
may then be washed with the buffer a second time to remove unbound
antibody. Optionally the antibody is subsequently labeled. The
amount of bound label on solid support may then be detected by
conventional means.
[0349] By "solid phase support or carrier" is intended any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, gabbros, and magnetite. The nature of
the carrier can be either soluble to some extent or insoluble for
the purposes of the present invention. The support material may
have virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration may be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface may be flat
such as a sheet, test strip, etc. Preferred supports include
polystyrene beads. Those skilled in the art will know many other
suitable carriers for binding antibody or antigen, or will be able
to ascertain the same by use of routine experimentation.
[0350] The binding activity of a given lot of LINGO-1 antibody, or
antigen-binding fragment, variant, or derivative thereof may be
determined according to well known methods. Those skilled in the
art will be able to determine operative and optimal assay
conditions for each determination by employing routine
experimentation.
[0351] There are a variety of methods available for measuring the
affinity of an antibody-antigen interaction, but relatively few for
determining rate constants. Most of the methods rely on either
labeling antibody or antigen, which inevitably complicates routine
measurements and introduces uncertainties in the measured
quantities.
[0352] Surface plasmon reasonance (SPR) as performed on BIAcore
offers a number of advantages over conventional methods of
measuring the affinity of antibody-antigen interactions: (i) no
requirement to label either antibody or antigen; (ii) antibodies do
not need to be purified in advance, cell culture supernatant can be
used directly; (iii) real-time measurements, allowing rapid
semi-quantitative comparison of different monoclonal antibody
interactions, are enabled and are sufficient for many evaluation
purposes; (iv) biospecific surface can be regenerated so that a
series of different monoclonal antibodies can easily be compared
under identical conditions; (v) analytical procedures are fully
automated, and extensive series of measurements can be performed
without user intervention. BIAapplications Handbook, version AB
(reprinted 1998), BIACORE code No. BR-1001-86; BIAtechnology
Handbook, version AB (reprinted 1998), BIACORE code No.
BR-1001-84.
[0353] SPR based binding studies require that one member of a
binding pair be immobilized on a sensor surface. The binding
partner immobilized is referred to as the ligand. The binding
partner in solution is referred to as the analyte. In some cases,
the ligand is attached indirectly to the surface through binding to
another immobilized molecule, which is referred as the capturing
molecule. SPR response reflects a change in mass concentration at
the detector surface as analytes bind or dissociate.
[0354] Based on SPR, real-time BIAcore measurements monitor
interactions directly as they happen. The technique is well suited
to determination of kinetic parameters. Comparative affinity
ranking is extremely simple to perform, and both kinetic and
affinity constants can be derived from the sensorgram data.
[0355] When analyte is injected in a discrete pulse across a ligand
surface, the resulting sensorgram can be divided into three
essential phases: (i) Association of analyte with ligand during
sample injection; (ii) Equilibrium or steady state during sample
injection, where the rate of analyte binding is balanced by
dissociation from the complex; (iii) Dissociation of analyte from
the surface during buffer flow.
[0356] The association and dissociation phases provide information
on the kinetics of analyte-ligand interaction (k.sub.a and k.sub.d,
the rates of complex formation and dissociation,
k.sub.d/k.sub.a=K.sub.D). The equilibrium phase provides
information on the affinity of the analyte-ligand interaction
(K.sub.D).
[0357] BIAevaluation software provides comprehensive facilities for
curve fitting using both numerical integration and global fitting
algorithms. With suitable analysis of the data, separate rate and
affinity constants for interaction can be obtained from simple
BIAcore investigations. The range of affinities measurable by this
technique is very broad ranging from mM to pM.
[0358] Epitope specificity is an important characteristic of a
monoclonal antibody. Epitope mapping with BIAcore, in contrast to
conventional techniques using radioimmunoassay, ELISA or other
surface adsorption methods, does not require labeling or purified
antibodies, and allows multi-site specificity tests using a
sequence of several monoclonal antibodies. Additionally, large
numbers of analyses can be processed automatically.
[0359] Pair-wise binding experiments test the ability of two MAbs
to bind simultaneously to the same antigen. MAbs directed against
separate epitopes will bind independently, whereas MAbs directed
against identical or closely related epitopes will interfere with
each other's binding. These binding experiments with BIAcore are
straightforward to carry out.
[0360] For example, one can use a capture molecule to bind the
first Mab, followed by addition of antigen and second MAb
sequentially. The sensorgrams will reveal: 1. how much of the
antigen binds to first Mab, 2. to what extent the second MAb binds
to the surface-attached antigen, 3. if the second MAb does not
bind, whether reversing the order of the pair-wise test alters the
results.
[0361] Peptide inhibition is another technique used for epitope
mapping. This method can complement pair-wise antibody binding
studies, and can relate functional epitopes to structural features
when the primary sequence of the antigen is known. Peptides or
antigen fragments are tested for inhibition of binding of different
MAbs to immobilized antigen. Peptides which interfere with binding
of a given MAb are assumed to be structurally related to the
epitope defined by that MAb.
[0362] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of cell biology, cell
culture, molecular biology, transgenic biology, microbiology,
recombinant DNA, and immunology, which are within the skill of the
art. Such techniques are explained fully in the literature. See,
for example, Molecular Cloning A Laboratory Manual, 2nd Ed.,
Sambrook et al., ed., Cold Spring Harbor Laboratory Press: (1989);
Molecular Cloning: A Laboratory Manual, Sambrook et al., ed., Cold
Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N.
Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M.
J. Gait ed., (1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic
Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984);
Transcription And Translation, B. D. Hames & S. J. Higgins eds.
(1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss,
Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B.
Perbal, A Practical Guide To Molecular Cloning (1984); the
treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene
Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos
eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology,
Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell
And Molecular Biology, Mayer and Walker, eds., Academic Press,
London (1987); Handbook Of Experimental Immunology, Volumes I-IV,
D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the
Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1986); and in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989).
[0363] General principles of antibody engineering are set forth in
Antibody Engineering, 2nd edition, C. A. K. Borrebaeck, Ed., Oxford
Univ. Press (1995). General principles of protein engineering are
set forth in Protein Engineering, A Practical Approach, Rickwood,
D., et al., Eds., IRL Press at Oxford Univ. Press, Oxford, Eng.
(1995). General principles of antibodies and antibody-hapten
binding are set forth in: Nisonoff, A., Molecular Immunology, 2nd
ed., Sinauer Associates, Sunderland, Mass. (1984); and Steward, M.
W., Antibodies, Their Structure and Function, Chapman and Hall, New
York, N.Y. (1984). Additionally, standard methods in immunology
known in the art and not specifically described are generally
followed as in Current Protocols in Immunology, John Wiley &
Sons, New York; Stites et al. (eds), Basic and Clinical-Immunology
(8th ed.), Appleton & Lange, Norwalk, Conn. (1994) and Mishell
and Shiigi (eds), Selected Methods in Cellular Immunology, W. H.
Freeman and Co., New York (1980).
[0364] Standard reference works setting forth general principles of
immunology include Current Protocols in Immunology, John Wiley
& Sons, New York; Klein, J., Immunology: The Science of
Self-Nonself Discrimination, John Wiley & Sons, New York
(1982); Kennett, R., et al., eds., Monoclonal Antibodies,
Hybridoma: A New Dimension in Biological Analyses, Plenum Press,
New York (1980); Campbell, A., "Monoclonal Antibody Technology" in
Burden, R., et al., eds., Laboratory Techniques in Biochemistry and
Molecular Biology, Vol. 13, Elsevere, Amsterdam (1984), Kuby
Immunnology 4.sup.th ed. Ed. Richard A. Goldsby, Thomas J. Kindt
and Barbara A. Osborne, H. Freemand & Co. (2000); Roitt, I.,
Brostoff, J. and Male D., Immunology 6.sup.th ed. London: Mosby
(2001); Abbas A., Abul, A. and Lichtman, A., Cellular and Molecular
Immunology Ed. 5, Elsevier Health Sciences Division (2005);
Kontermann and Dubel, Antibody Engineering, Springer Verlan (2001);
Sambrook and Russell, Molecular Cloning: A Laboratory Manual. Cold
Spring Harbor Press (2001); Lewin, Genes VIII, Prentice Hall
(2003); Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Press (1988); Dieffenbach and Dveksler, PCR Primer
Cold Spring Harbor Press (2003).
[0365] All of the references cited above, as well as all references
cited herein, are incorporated herein by reference in their
entireties.
EXAMPLES
Example 1
Identification of Anti-LINGO-1 Antibodies by Phage Display
[0366] Li13 and Li33 were identified as Fab-phages that
specifically bound to LINGO-1 using phage display as described in
PCT/US2006/026271, filed Jul. 7, 2006, which is herein incorporated
by reference in its entirety. Li81 is derived from Li13 and Li33.
It includes the Li13 light chain and an affinity matured heavy
chain. The isolation of Li81 is described in more detail in
PCT/US2008/000316, filed Jan. 9, 2008, which is incorporated herein
by reference in its entirety. An aglycosylated fully human
monoclonal antibody was created from the Li81 Fab, Li81 (agly), and
its production is also detailed in PCT/US2008/000316. Li62 is
derived from Li33. it includes the Li33 heavy chain and a light
chain that was identified in a library screen.
[0367] The isolation of the Fab fragments is summarized briefly as
follows. Fab fragments were isolated from phage display libraries
as described in Hoet et al., Nat. Biotech. 23:344-348 (2005);
Rauchenberger, et al., J. Biol. Chem. 278:194-205 (2003); and
Knappik, et al., J. Mol. Biol. 296:57-86 (2000), all of which are
incorporated herein by reference in their entireties.
[0368] Li62 and Li81 Fabs, as well as Li62 (agly) and Li81 (agly),
have been purified and demonstrated to bind to specifically to
LINGO-1 by both ELISA and FACS. Assays were performed as described
in PCT/US2008/000316.
Example 2
Li62 and Li81 Promote Myelination In Vitro
[0369] The role of Li62 and Li81 in myelination was investigated in
vitro by treating co-cultures of dorsal root ganglion (DRG) neurons
and oligodendrocytes with Li62 (agly) and Li81 (agly). The DRG
neurons were then tested for myelination using Western blotting.
For these studies, it was necessary to first generate primary
cultures of DRG neurons and of oligodendrocytes.
[0370] Female Long Evans rat E14-E17 embryonic dorsal root ganglia
were cultured as described by Plant et al., J. Neurosci. 22:6083-91
(2002). Dissected DRGs were plated on poly-L-lysine-coated cover
slips (100 .mu.g/ml) for 2 weeks. The cells were incubated in the
presence of fluorodeoxyuridine for days 2-6 and in NLA medium
containing 1.times.B27, 100 ng/ml NGF (Gibco) for days 8-11.
[0371] Female Long Evans post-natal day 2 (P2) rat oligodendrocytes
were cultured as described by Conn, Meth. Neurosci. 2:1-4 (Academic
Press; 1990) with modifications as follows. Briefly, the forebrain
was extirpated from P2 rats and placed in cold HBSS medium (Gibco).
The tissue fragments were cut into 1 mm pieces and incubated at
37.degree. C. for 15 min in 0.01% trypsin and 10 .mu.g/ml DNase.
Dissociated cells were plated on a poly-L-lysine coated T75 tissue
culture flasks and grown in DMEM with 20% fetal bovine serum at
37.degree. C. for 10 days. A2B5-positive oligodendrocytes were
collected by shaking the flasks overnight at 200 rpm at 37.degree.
C. The A2B5 oligodendrocytes were cultured for 7 days in DMEM
(Gibco) containing 25 mM D-glucose, 4 mM L-glutamine, 1 mM sodium
pyruvate, 50 .mu.g/ml human apo-transferrin, 5 .mu.g/ml bovine
pancreatic insulin, 30 nM sodium selenate, 10 nM hydrocortisone, 10
nM D-biotin, 1 mg/ml BSA, 10 ng/ml FGF and PDGF (Peprotech). The
cells were then harvested by trypsinization. The cells then
co-cultured with the DRG neurons in the presence or absence of 1.0,
0.30, 0.10, or 0.03 .mu.g/ml of Li62 (agly) or Li81 (agly), or a
negative control antibody (h5C8 Ctrl) in NLA medium containing 2%
fetal bovine serum, 50 .mu.g/ml ascorbic acid, 100 ng/ml NGF
(Gibco). One of skill in the art would be able to determine an
effective dose using assays described herein.
[0372] The culture medium was changed and the antibodies or
antibody fragments were replenished every three days. After 3 weeks
at 37.degree. C., the co-cultured cells were lysed and subjected to
Western blot analysis to quantify the MBP and MOG (FIG. 1). Based
on Western blot analyses, co-cultured cells treated with Li62
(agly) and Li81 (agly) showed increased levels of both MBP and MOG
compared to control-antibody treated co-cultures. Similar results
were obtained using Li62 and Li81 Fabs. These data suggest that
both Li62 and Li81 can promote myelination in vitro and can promote
mature oligodendrocyte axon interactions and myelination compared
to control-antibody treated co-cultures.
Example 3
Li62 and Li81 Variants
[0373] In order to identify antibodies with improved affinity, Li62
and Li81 variants were isolated by targeted phage display. The
variants included alterations in the amino acid sequence of the VH
CDR3 sequence in each of the Fabs. Eighteen Li62 variants had
improved affinities as shown below in Table 5.
TABLE-US-00009 TABLE 5 Li62 Variants Li62 Heavy Chain Li62 Fold-
CDR3 Sequence ELISA Variant Improvement SEQ ID NO E G H N D W Y F D
L Signal Reps B06 8 17 Y Y Q 3 B12 8 18 Q Y V 3 F06 12 19 D Y 3 12
B01 8 20 Q Y 3 D09 15 21 A D I F 3 D12 8 22 Y 3 26 F01 9 23 R Y P 3
F02 8 24 D Y 3 F06 8 25 R Y 3 2 F10 5 26 S I R 3 G08 10 27 Q Y V 3
4 H08 6 28 Y N G 0.5 C10 11 29 Y Y 3 4 C02 8 30 T Y L 3 D05 10 31 Y
Y E 3 2 F02 16 32 L I F Q 3 C10 9 33 Q F 3 H08 9 34 T Y 3
[0374] Additionally, fifteen Li81 variants had improved affinities
as shown below in Table 6.
TABLE-US-00010 TABLE 6 Li81 Variants Li81 Heavy Chain Li81 Fold-
CDR3 Sequence ELISA variant Improvement SEQ ID NO E G D N D A F D I
Signal Reps F09 9 35 E V 2.5 G02 6 36 Y T 3 H03 9 37 T 3 A12 15.1
38 S 2.6 C02 6 39 T 2.8 2 C11 15.1 40 Y R 2 D11 6 41 V S 2.1 E05 15
42 D V M 2.9 H04 6 43 Y F 3 B04 8 44 D Y M 3 A02 8 45 Q Y T Y L 3
B12 6 46 D T 3 H06 6 47 A D 3 H08 6 48 E M 3 E07 6 49 E Y T Y 3
Example 4
Li81 Promotes Rat Oligodendrocyte Differentiation
[0375] The ability of Li81 to promote the differentiation of rat
A2B5+ progenitor cells into mature MBP+ myelinating
oligodendroctyes was tested. This process was studied in vitro by
plating primary rat forebrain A2B5+ cells into 24-well culture
plates, treating cultures for 72 hr with Li81 (agly), and staining
cultures for myelin basic protein (MBP) expression by Western
blotting. In Western blots, myelin oligodendrocyte glycoprotein
(MOG) expression was also used as a marker for maturation.
[0376] Treatment with Li81 (agly) resulted in more highly
differentiated, mature oligodendrocytes as evidenced by increases
in the length of cell processes and the presence of abundant myelin
sheet structures that are stained by the anti-MBP antibody. A
dose-dependent increase in number of mature oligodendrocytes was
observed. The lowest concentration of Li81 (agly) with a detectable
effect on MBP production was 0.1 .mu.g/mL. A small percentage of
less differentiated oligodendrocytes was seen in the control
antibody treated cells. By Western blotting, there was a
dose-dependent increase in MBP and MOG expression in the Li81
(agly) treated samples (FIG. 2). No expression was observed with
the isotype control antibody at any concentration. The complex
pattern of MBP bands results from alternatively spliced forms of
the MBP protein. Similar results were obtained using Li81 Fab.
These results indicate that Li81 can promote differentiation of rat
A2B5+ progenitor cells into mature MBP+ myelinating
oligodendroctyes in vitro.
Example 5
Li81 Promotes Human Oligodendrocyte Differentiation
[0377] The ability of Li81 to promote differentiation of human
oligodendrocyte precursor cells (OPC) was also evaluated. As with
rat OPCs, Li81 Fab and Li81 (agly) had a dramatic effect on the
human OPC cultures and resulted in the formation of highly
differentiated, mature oligodendrocytes as evidenced by increases
in the length of cell processes and the presence of abundant myelin
sheet structures that are stained by the anti-MBP antibody. The
number of human OPCs that were MBP+ after treatment with a control
antibody (hIgG1) or Li81 (agly) is shown in FIG. 3. Only a small
percentage of less differentiated oligodendrocytes was seen in the
control antibody (hIgG1) treated cells (FIG. 3). Similar results
were obtained using Li81 Fab.
Example 6
Li81 Promotes Remyelination in Lysolecithin-Treated Brains
[0378] The cerebellar slice culture system is an in vitro model for
analyzing mechanisms of remyelination. Coronal cerebellar slices
from P17 rats approximately 300 .mu.m thick were placed in tissue
culture medium for 4 days, then treated with lysolecithin for 24
hours to induce demyelination and incubated with medium containing
Li81 (agly) (30, 10, 3, and 1 .mu.g/ml) or an isotype-matched
control antibody (5c8) for 3 days to allow remyelination to occur.
Remyelination was visualized by black gold immunostaining, which
selectively stains myelin in brain slices. In black gold stained
sections, myelinated white matter appears dark brown and
demyelinated lesions appear as pale brown or white.
[0379] Treatment of the brain slices with lysolecithin resulted in
almost complete demyelination of the tissue as evidenced by loss of
staining in the control antibody treated culture. Li81 (agly)
treatment resulted in robust remyelination as evidenced from the
reappearance of the staining. The immunohistochemistry data were
quantified by measuring the intensity of the black gold staining as
summarized in the bar graph of FIG. 4. Treatment with Li81 (agly)
resulted in approximately a 30-fold increase in myelinated tissue
over the level seen in the control treated brain slice. The overall
level of remyelination following Li81 (agly) treatment was
approximately half of that observed without any demyelination
treatment. Similar results were obtained using Li81 Fab.
Example 7
Decreased Binding of Aglycosylated Anti-LINGO-1 Antibody to
Fc(Gamma) Receptors
[0380] Relative binding affinities of IgG for human Fc receptors
(CD16, CD32a and b, CD64) were measured using the Amplified
Luminescent Proximity Homogeneous Assay (ALPHA) technology from
Perkin Elmer. The assay was performed in a competitive format in
which serial dilutions of test antibodies were incubated with the
receptor-GST fusion proteins and anti-GST acceptor beads overnight
at 40.degree. C. in a 96-well plate. Streptavidin donor beads and
biotinylated wild-type IgG1 were also incubated overnight at
40.degree. C. in a separate tube and then added to the assay plate
the next day. The plates were incubated at room temperature for 2
hours with gentle shaking and read in an Envision plate reader
(Perkin Elmer). The data were plotted to a 4-parameter curve fit
using Graphpad Prism software to calculate the IC.sub.50 values in
order to determine the relative binding affinities. The antibodies
tested were Li81 (agly), an isotype-matched control antibody (5c8)
and an aglycosylated version of the control antibody. The data are
plotted in FIG. 5. The IC.sub.50 values of Li81 (agly) were
calculated as follows: CD32a: 365 .mu.g/mL (down 60.times. from
wt), CD32b: 350 .mu.g/mL (down 15.times. from wt), CD16: 179
.mu.g/mL (down 50.times. from wt), and CD64: >100 .mu.g/mL (down
>100.times. from wt).
[0381] The ability of Li81 (agly) to bind certain Fc(gamma)
receptors was also evaluated in a cell bridging assay. For these
studies, CHO cells expressing human LINGO-1 were plated into
96-well tissue culture plates, then incubated with serial dilutions
of test samples, and with BCECF-AM labeled U937 cells that
naturally express both CD64 (FcgR1) and CD32 (FcgRIIa). Bound U937
cells were quantified by fluorescence (ex485/em530) using a
cytofluor plate reader. Anti-LINGO-1 monoclonal antibodies Li33 and
Li13 that bind LINGO-1 with nM EC.sub.50 values and contain wild
type Ig1 frameworks showed typical sigmoidal binding curves with
EC.sub.50 values of 0.17 and 0.23 .mu.g/mL, respectively (FIG. 6).
In contrast, Li81 (agly) showed a very poor bridging response,
consistent with a reduction in the affinity of the aglycosylated
framework for CD64 and CD32. The shift in dose response is
consistent with a >10 fold drop in binding. Control huIg1 showed
no bridging activity.
Example 8
Aglycosylated Anti-LINGO-1 Antibody Does Not Promote Complement
Activation
[0382] The effect of the aglycosylated variant of IgG1 antibodies
on reducing C1q binding and activation of the complement pathway
are well documented. To verify these effects on the Li81 (agly)
antibody, the antibody was tested for C1q binding in an ELISA
format and for complement-dependent cytotoxicity (CDC) in CHO cells
expressing human LINGO-1. For the CDC assay LINGO-1 and Lt-beta
(positive control) expressing CHO cells were treated with serial
dilutions of anti-LINGO-1 antibodies or LtbetaR-Fc, low toxicity
rabbit serum complement and propidium iodide and assayed for
killing. Li81 (agly) did not elicit a cytotoxic response whereas
the LtbetaR-Fc reagent promoted a robust killing response (FIG. 7).
To date no measurable cytotoxic response in the CDC assay has been
observed with any LINGO-1 targeted reagent including Li33 or Li13
as intact Ig1 anti-LINGO-1 Mabs (shown in FIG. 7) or aggregated 1A7
Ig1.
Example 9
Anti-LINGO-1 Antibodies Promote Myelination In Vivo in Lysolecithin
Assay
[0383] The lysolecithin (LPC)-induced demyelination model is a
simple in vivo system for investigating remyelination. LPC was
injected into the dorsal column of 9 week old adult female Sprague
Dawley rats (250 g) on day 0. Demyelination occurred within a few
hours following LPC treatment. Li81 (agly) or a control antibody
was administered IP on day 3. The animals were sacrificed on day 9,
and the region of the spinal cord encompassing the lesion was
excised and sectioned.
[0384] Sections from control antibody-treated animals showed large
lesions with extensive areas of demyelination as evident from the
absence of stain in the lesion area. Smaller lesions were apparent
in Li81 (agly)-treated rats and the lesions contained lace-like
structures representative of the remyelinated axons (FIG. 8). In
subsequent studies, the model was run with Li81 (agly) at 2, 1, and
0.3 mg/kg. The 2 and 1 mg/kg doses of Li81 (agly) were highly
efficacious, while effects from the 0.3 mg/kg treated animals were
less efficient. These results demonstrate that anti-LINGO-1
antibodies promote myelination in vivo in a dose dependent
manner.
[0385] In a similar experiment, lysolecithin-treated rats are
administered 2, 1, and 0.3 mg/kg of Li62 (agly) antibody on day 3
instead of Li81 (agly). Animals are sacrificed on day 9, and the
region of the spinal cord encompassing the lesion is excised and
sectioned. Sections are analyzed to compare lesion size and
myelination in animals treated with Li62 (agly) to lesion size and
myelination in animals treated with a control antibody.
Example 10
Anti-LINGO-1 Antibodies Promote Myelination In Vivo in MOG-EAE
Assay
[0386] Myelin oligodendrocyte glycoprotein (MOG)-induced murine
experimental autoimmune encephalomyelitis (EAE) is a widely
accepted model for studying the clinical and pathological features
of multiple sclerosis and has been described in more detail in
PCT/US2008/000316, filed Jan. 9, 2008, which is incorporated by
reference herein. Li81 (agly) was tested in the EAE model to
determine if inhibition of endogenous LINGO-1 function promotes
functional recovery.
[0387] Adult 9-week-old brown Norway female rats (150 g) were
injected with 75 .mu.g recombinant rat MOG (amino acids 1-125) in
PBS. Animals developed signs of EAE at 15 days. Li81 (agly)
treatment or isotype control (3 mg/kg) was injected IP at days 15,
18, 21, 24 and 27 (10 rats per group). The EAE clinical score was
measured daily for 2 weeks. As shown in FIG. 9, Li81 (agly)
promotes functional recovery in this model by improving hind limb
and tail movement.
[0388] In a similar experiment, MOG-treated rats are injected with
Li62 (agly) or an isotype control at days 15, 18, 21, 24 and 27.
The EAE clinical score is measured daily for 2 weeks to assess hind
limb paralysis, complete tail paralysis and distal tail paralysis,
and the paralysis in animals treated with Li62 (agly) is compared
to paralysis in animals treated with the control antibody.
Example 11
Testing the Effect of LINGO-1 Antibodies and Fragments Thereof on
Oligodendrocytes in an In Vivo Cuprizone Model
[0389] In order to determine if Li62, Li81, and variants thereof
promote myelination in vivo adult mice are fed cuprizone (0.2%
milled with ground mouse chow by weight) for 6 weeks to induce
demyelination within the corpus callosum according to the method
described by Morell P et al., Mol Cell Neurosci. 12:220-7 (1998)
and in PCT/US2008/000316, filed Jan. 9, 2008, which is incorporated
herein by reference in its entirety. Briefly, an anti-LINGO-1 Li62
or Li81 monoclonal antibody, Fab, or a variant thereof, is
stereotactically injected into the demyelinating corpus callosum at
weeks 2, 2.5, and 3 weeks of cuprizone feeding. Control mice are
stereotactically injected at the same intervals with sterilized
media containing control antibody. After the 6 weeks of cuprizone
feeding is completed, the mice are returned to a normal diet for 2,
4 and 6 weeks to allow remyelination.
[0390] The animals receiving anti-LINGO-1 antibody treatment are
evaluated for mature oligodendrocyte survival (based on CC1
antibody staining) and axon myelination by IHC using anti-MBP
protein antibody or luxol fast blue. CC1 antibody-positive
oligodendrocytes are quantitated at four weeks and six weeks.
Increased CC1 and/or MBP levels indicate that the antibodies
promote mature oligodendrocyte survival and axon myelination.
Example 12
Testing the Effect of LINGO-1 Antibodies and Fragments Thereof on
Retinal Ganglion Cell Survival in the Optic Nerve Transection
Model
[0391] Anti-LINGO-1 antibodies are tested in an optic nerve
transection model, which investigates factors that affect neuronal
function. The right optic nerve of an adult rat is transected
intraorbitally 1.5 mm from the optic disc. A piece of gelfoam
soaked with 6% Fluoro-Gold (FG) is applied to the newly transected
site right behind the optic disc to label the surviving retinal
ganglion cells (RGCs). The animals are divided into three groups
which receive Li81 or Li62 monoclonal antibodies, Fabs, variants
thereof, a control antibody, or PBS, by intravitreal injection. The
volume of each intravitreal injection is 4 .mu.l while the dosage
of each injection is 2 .mu.g. The intravitreal injections are
performed immediately after the optic nerve transection.
[0392] All animals are allowed to survive for 1 week. Two days
before sacrificing the animals, the left optic nerve of each animal
is transected and 6% FG is administered as described above to label
the surviving RGCs, to serve as the internal control. Animals are
sacrificed with an overdose of Nembutal and the retinas are
dissected in 4% paraformaldehyde. Four radial cuts are made to
divide the retinas into four quadrants (superior, inferior, nasal
and temporal). The retinas are then post-fixed in the same fixative
for 1 hour before they are flat-mounted with the counting medium
(Dako). The slides are examined under a fluorescence microscope
using an ultra-violet filter (excitation wavelength=330-380 nm).
Labeled RGCs are counted along the median line of each quadrants
starting from the optic disc to the peripheral border of the retina
at 500 .mu.m intervals, under an eyepiece grid of 200.times.200
.mu.m.sup.2. The percentage of surviving RGCs resulting from each
treatment is expressed by comparing the number of surviving RGCs in
the injured eyes with their contra-lateral eyes. Effective
antibodies show increased neuronal survival when compared to
control-antibody or PBS treated animals.
Example 13
Testing LINGO-1 Antibodies for Remyelination in the Optic Nerve
Crush Model
[0393] The right optic nerve is completely crushed by #5 forceps
for 10 seconds around 1.5 mm behind the eyeball intraorbitally just
before administration of 2 .mu.l of Li62 or Li81 monoclonal
antibody, Fab, or a variant thereof, in 2 ml by intravitreal
injection.
[0394] The animals receive a second intravitreal injection of the
same treatment one week after the surgery. Two weeks after the
surgery, the animals are perfused with EM fixatives, postfixed and
processed for semithin and ultrathin sections. The longitudinal
optic nerve sections are stained and prepared for myelin
observation. The myelination of the proximal and the distal parts
of the crushed optic nerve are compared among different treatment
groups. Animals treated with Li62 or Li81 monoclonal antibody, Fab,
or variants thereof will be analyzed for remyelination in the
distal part of the optic nerve compared to the controls.
Example 14
Testing LINGO-1 Antibodies for Axon Regeneration in the Optic Nerve
Crush Model
[0395] The right optic nerve is crushed by #5 forceps for 10
seconds around 1.5-2 mm behind the eyeball intraorbitally just
before administration of 2 .mu.g of Li62 or Li81 monoclonal
antibody, Fab, or a variant thereof in PBS via intravitreal
injection. Control animals are administered a control antibody or
PBS. The animals receive a second intravitreal injection of the
same treatment one week after the surgery. Three days prior to
sacrifice of the test animals (day 11 of the experiment), 2 ml of
CTB-FITC is injected intravitreally to label, anterograde, the
regenerative optic nerve axons. On the 14th day post surgery, the
animals are perfused and postfixed. The crushed optic nerve is
processed for frozen longitudinal sections. The CTB-FITC labeled
axons, which cross the lesion site are counted as regenerative
fibers at various distances beyond the crush site. The regeneration
of axons in animals treated with Li62 or Li81 monoclonal antibody,
Fab, or a variant thereof and compared to control animals
Example 15
Identification and Characterization of Li113
[0396] Li62 variant C02, also called Li113, was elected for further
study. A LINGO-1 ELISA assay demonstrated that the Li113 Fab bound
to LINGO-1 with an EC50 of 0.09 nM. The EC50 measurements of the
Li33 Fab, the Li62 Fab, and Li81 Fab were 0.30 nM, 0.26 nM and 0.11
nM, respectively in the same experiment (FIG. 10). The Li113 Fab
was also tested in the oligodendrocyte differentiation assay. The
assay was performed essentially as described above in Example 2,
but MBP levels were measured by ELISA. The results for a control
monoclonal antibody, the Li81 monoclonal antibody, the Li62 Fab,
and the Li113 Fab are shown in FIG. 11. These data demonstrate that
Li113 can effectively bind to LINGO-1 and promote oligodendrocyte
differentiation.
Example 16
Isotype Switching to Improve Antibody Solubility
[0397] The anti-LINGO-1 Li33 Fab was converted into a full human
antibody and expressed in mammalian cells. Three different IgG
frameworks (Ig1, Ig2, and Ig4) were evaluated both in wildtype and
aglycosyl forms. For the Ig2 framework, the V234A/G237A mutation
was also evaluated as an alternative to the glycosylation site
mutation to eliminate FcRIIa binding. Native human kappa light
chain and heavy chain signal peptides were used to direct secretion
of Li33 light and heavy chains, respectively, in mammalian cell
hosts. The variable domain fragment of the light chain was
subcloned into a shuttle vector containing the intact signal
peptide and light chain kappa chain constant region. The variable
domain fragment of the heavy chain was subcloned into shuttle
vectors containing the intact signal peptide and Ig1, Ig1agly, Ig4,
Ig4agly, Ig2, Ig2agly, and Ig2 V234A/G237A heavy chain constant
region.
[0398] Each of the generated antibodies showed typical antibody
features by SDS-PAGE gel analysis under both reducing and
non-reducing conditions. In addition, the ability of each of the
isotypes to bind LINGO-1 was assessed in an ELISA format. ELISA
plates were coated with LINGO-1, treated with serial dilutions of
each antibody, and bound Li33 was detected with an alkaline
phosphatase anti-human Fab conjugate. The seven Mabs showed similar
EC50 values for binding to LINGO-1 (Table 7) with apparent
affinities of 0.12 nM for the Ig1 wt and agly, .about.0.24 nM for
Ig2 and Ig2agly, and .about.0.36 nM for Ig4 and Ig4agly.
TABLE-US-00011 TABLE 7 Impact of Li33 Ig frameworks on solubility.
LINGO-1 Solu- binding Stability bility EC50 .degree. C. SEC % Li33
Isotype (mg/mL) (nM) TM1 TM2 monomer Ig1 0.9 0.12 69 76 99 Ig1agly
0.3 0.12 60 77 >99 Ig4 >30 0.35 64 72 98 Ig4Pagly 0.3 0.37 56
73 95 Ig2 >50 0.23 69 76 96 Ig2agly 0.2 0.26 59 76 98
Ig2-V234A/G237A 5.6 0.19 69 76 95 Ig1 Fab2 0.3 0.10 -- 77 98 Ig2
Fab2 >50 0.39 -- 77 98 Ig1 Fab >50 0.68 -- 76 95 PEG-Fab
>50 1.9 -- 77 98 Ig1agly reduced >40 0.12 55 75 98 Ig1
reduced >50 0.15 63 75 98 Ig1 pH 7.0 0.9 0.08 68 77 Ig1 pH 6.5
1.7 0.10 69 77 Ig1 pH 6.0 2.4 0.10 69 78 Ig1 pH 5.5 30 0.16 66 81
Ig1 pH 5.0 >50 0.45 66 81 Ig1 pH 4.5 >50 2.1 62 82 Ig1 pH 4.0
>50 16 54 78 Ig1 pH 3.5 >50 34 46/66 74 Ig1 pH 3.0 >50 ND
34/52 72
[0399] The solubility of the isoforms was assayed as follows.
Samples were buffer exchanged using multiples cycles of
concentration and dilution in centrifugation YM30 filter devices.
Protein concentration was determined immediately after
concentration from absorbance scans and again after 5 days at
4.degree. C. following filtration through a 0.45 .mu.m filter. If
the absorbance had decreased, samples continued to be monitored
over time at 4.degree. C. When possible, samples were concentrated
to 50 mg/mL; however, some of the purified constructs were only
concentrated to the amount indicated due to small sample size.
[0400] All three of the aglycosyl antibodies had poor solubility at
pH 7.0 with extensive precipitation at concentrations greater than
0.3 mg/mL Mab. The solubility of the Ig1wt Mab was slightly
improved (0.9 mg/mL) while the Ig2 and Ig4 Mabs were soluble at the
highest concentration tested. The solubility of the Ig2 Mab was
>50 mg/mL, representing a >150 fold increase over the
aglycosyl version of the same construct. The Ig2 V234A/G237A
variant was intermediate in terms of its solubility. Below the
solubility limits, the antibodies were stable to prolonged storage
at 4.degree. C. and to freeze-thaw.
[0401] Since the solubility of a protein can be significantly
reduced at pH near its isoelectric point (pI), pI values for the
Li33 Mabs were determined by isoelectric focusing. Samples were
subjected to isoelectric focusing on a pH 3-10 IEF minigel
(Invitrogen). Elecrophoresis was carried out at 100V for 1 hour,
200V for 1 hour and 500V for 30 minutes. The gel was fixed, stained
with Coomassie brilliant blue R-250, and destained. All of the
antibodies had basic isoelectric points with pI values >pH 8.2.
The pI values for both the Ig1 and Ig1agly Li33 were .about.9.0,
for Ig4 and Ig4agly Li33 were 8.2, and for Ig2, Ig2agly, and Ig2
V234A/G237A were 8.5.
[0402] The aggregation state of the antibodies was studied by size
exclusion chromatography (SEC). SEC was performed on a Superdex 200
FPLC column using 20 mM sodium phosphate pH 7.2 and 150 mM NaCl as
the mobile phase. The column was run at 0.3 mL/min. The column
effluent was monitored by UV detection at 280 nm, and purity was
assessed by peak height. All constructs eluted as a single
prominent peak with an apparent molecular mass of 150 kDa with
>95% purity (Table 7). Selected profiles are shown in FIG. 12.
The soluble fraction for the Ig1agly by SEC was >99% monomer
with no evidence of soluble aggregates. In contrast, the Ig2
contained 2% dimer and 2% higher molecular mass aggregates. The
aggregation state of Li33 Ig2 was further evaluated by analytical
ultracentrifugation (FIG. 13), which revealed that the antibody
actually formed reversible dimers at high concentrations. Thus,
while the Ig2 framework prevented the transition to an insoluble
aggregate, it had not ablated all protein-protein interactions.
[0403] The stability of a protein can also impact its solubility.
The thermal stability of the constructs was measured by
differential scanning fluoremetry (DSF). Measurements were
conducted on an Mx3005p real-time PCR system (Agilent Technologies)
in a 96-well format using 10 .mu.g of protein in 50-55 .mu.L
phosphate buffer (at neutral pH) supplemented with SYPRO orange
fluorophor (Invitrogen) at a final concentration of 10.times..
Samples were heated from 25.degree. C. to 95.degree. C. at
1.degree. C./min with fluorescence intensity measured 3 times every
1.degree. C. Fluorescence intensities were plotted as a function of
temperature. Melting temperatures (Tm) were derived from these
curves by taking the negative derivative ("-R'(T)" in the Mx3005p
software) and selecting the local minima of the derivative plots.
For DSF measurements at various pH values, 10 mM sodium citrate was
used as the buffering agent.
[0404] Tm values for the Fab region (TM2) were 76-77.degree. C. for
the Ig1 and Ig2 constructs and 72-73.degree. C. for the Ig4 wt and
agly. TM1 values for the CH2 region were variable. Transitions were
8-10.degree. C. lower for each of the agly constructs. The Ig4
constructs were the least stable. The stability of Li33 Ig1 and
Li33 Ig2 was also studied using, guanidine denaturation as an
alternative to thermal denaturation to assess stability. FIG. 14.
The denaturation curve for the Ig1 was monophasic with a transition
point of 3.1 M guanidine. The denaturation curve for the Ig1 Fab
was similar to that for the intact Mab. Reduction of the Ig1 Fab
shifted the transition point to 1.8 M guanidine. Li33 Ig2 denatures
at a higher guanidine concentration than the Ig1 with a 50%
transition point of 4.1 M. The shape of the curve suggests there
may be several transitions.
[0405] To further assess features of the antibodies that were
affecting solubility a variety of conditions and fragmentation were
tested (Table 7). Fab2 fragments of Ig1 and Ig2 were generated
enzymatically with pepsin, and a Fab fragment of Ig1 was generated
with papain. The solubility of the Ig2 Fab was >50 mg/mL,
whereas the solubility of the Ig1 Fab2 was only 0.3 mg/mL. The
solubility of the Ig1 Fab was >50 mg/mL.
[0406] A pegylated version of the Fab was also generated. For
pegylation, Ig1 Fab2 at 1.2 mg/mL in 40 mM sodium borate pH 7.0 and
0.1 mM TCEP was incubated for 75 min at 37.degree. C. The reduced
sample was desalted on a G25M column that had been equilibrated in
5 mM MES pH 5.0 and 50 mM NaCl (final Fab concentration 0.5 mg/mL).
After storage overnight at 4.degree. C., over 90% of the disulfide
bond holding the heavy and light chain together had reoxidized to a
Fab' leaving the 2 hinge Cys residues tree for conjugation. 10 kDa
methoxy-polyethyleneglycol maleimide (PEGmal) (Nektar) was added to
0.4 mg/mL, and MES pH 6.0 was added to 25 mM. The sample was
incubated at room temperature for 2.5 hours and overnight at
4.degree. C. The sample was then subjected to cation exchange
chromatography on a Fractogel EMD sulfate column. The column was
washed with 2 column volumes of 10 mM sodium phosphate pH 6.0, and
the PEG-Fab was eluted with 10 mM sodium phosphate pH 6.0 and 50 mM
NaCl. The pegylated version also had excellent solubility (Table
7).
[0407] Fragmentation had little impact on stability. No TM1 signal
was observed for the Fab2 and Fab moieties as expected, since the
TM1 transition is produced from the CH2 domain. Differences in
LINGO-1 binding were consistent with the nature of the products as
the three monvalent versions had reduced binding. Reduction of the
interchain disulfides that link the heavy-heavy and heavy-light
chains also had a very dramatic affect on solubility. After
reduction, the Li33 Ig1 and Ig1agly Mabs were soluble at the
highest concentration tested (Table 7). Reduction had only a slight
effect on thermal stability and had no impact on aggregation state
or LINGO-1 binding.
[0408] Finally, the effect of pH on solubility was tested. A
dramatic transition in the solubility of Li33 Ig1 occurred between
pH 6 and pH 5.5. Below pH 5.5 the protein was very soluble, and
above pH 5.5, it had poor solubility. Thermal stability and LINGO-1
binding were reduced under the more acidic conditions that had
improved solubility.
Example 17
Disulfide Bond Mapping
[0409] The disulfide structure of Li33 Ig2 was determined by
peptide mapping. In these experiments, alkylation of Li33 Ig2 was
done under denaturing and non-reducing conditions. 5 uL of 100 mM
idoacetamide solution was added to 25 .mu.L of the solution
containing .about.22.5 .mu.g of the protein, and 25 mg of guanidine
hydrochloride was immediately added to the solution. The solution
was kept at room temperature in the dark for 30 minutes. The
alkylated proteins were recovered by precipitation in cooled
ethanol. The solution was stored at -20.degree. C. for 1 hour and
then centrifuged at 20,000 g for 12 min at 4.degree. C. The
alkylated and recovered proteins were digested with 20% (w/w) of
endo-Lys-C in 2 M urea and 0.6 M Tris-HCl pH 6.5 for 8 hours at
room temperature. Then 5% (w/w) of trypsin was added to the
solution, and the solution was kept overnight at room temperature.
Another aliquot of 5% of trypsin was added the second morning, and
the solution was kept at room temperature for an additional 4
hours. Prior to analysis of the digests, 50 .mu.L of freshly
prepared 8 M urea was added to the digest, and the solution was
split into two parts: one was analyzed after reduction, which was
done by incubating the digest with 40 mM DTT at 37.degree. C. for 1
hour; the other part was not reduced before analysis. The reduced
and non-reduced digests were analyzed on an LC-MS system comprised
of a reversed-phase HPLC (Alliance, Waters, Milford Mass.) and an
LCT mass spectrometer (Waters Corp., Milford, Mass.). The
separation was carried out on a 1.0 mm.times.15-cm Vydac C4 column
(214TP5115) using a flow rate of 0.07 mL/min. The mobile phase A
was water with 0.03% trifluoroacetic acid, and mobile phase B was
acetonitrile with 0.024% trifluoroacetic acid. The gradient was
running linearly from 0 to 15% B in 65 minutes, then to 26% B in 55
minutes, then to 39% B in 30 minutes. The ESI source voltage was
set at 3,300 V, and the cone voltage was 30 V, with a desolvation
temperature set to 200.degree. C. Peaks on the maps were identified
using MassLynx 4.1 software.
[0410] Unlike Ig1 and Ig4 Mabs, which each contain a distinct
interchain disulfide pattern, the disulfide structure on an Ig2
antibody is complex and contains a mixture of different isoforms.
The detected disulfide linked peptide clusters for Li33 Ig2 are
listed in Table 8.
TABLE-US-00012 TABLE 8 Disulfide structure of Li33 IgG2. Calc. Mass
Detected Mass RT Recovery Linkage (Da) (Da) (min) (%)* Non-hinge
Intrachain LT2(C1)1 - LT6(C2) 4204.89 4204.90 133.1 100% LT11(C3) -
LT18(C4) 3555.75 3555.73 123.1 100% HT2(C1) - HT10(C2) 3442.55
3442.54 141.5 100% HT14(C4) - HT15(C5) 8073.92* 8073.78* 142.3 90%
HT21(C10) - HT26(C11) 3986.85 3986.84 124.5 100% HT34(C12) -
HT39(C13) 3844.82 3844.77 110.7 100% Interchain LT20(C5) - HT13(C3)
1535.69 1535.66 83.5 30% Hinge Pattern HT19(C6 - 9) - HT19(C6 - 9)
5350.56 5350.52 138.9 10 A + A HT19(C6 - 9)-HT19'(C6 - 9) 5125.40
5125.37 140.5 HT19' (C6-9) - HT19'(C6 - 9) 4900.26 4900.25 142.3
Pattern 2x(HT13(C3) + LT20(C5)) + 8425.62* 8425.46* 138.2 20 B + B
2xHT19(C6 - 9) 2x(HT13(C3) + LT20(C5)) + 8200.33* 8200.02* 139.4
HT19(C6 - 9) + HT19'(C6 - 9) 2x(HT13(C3) + LT20(C5)) + 7975.04*
7975.05* 140.8 2xHT19'(C6 - 9) Pattern HT13(C3) + LT20(C5) +
6885.24 6885.47 138.8 15 A + B 2xHT19(C6 - 9) HT13(C3) + LT20(C5) +
6660.08 6660.29 140.1 HT19(C6 - 9) + HT19'(C6 - 9) HT13(C3) +
LT20(C5) + 6434.92 6434.95 141.5 2xHT19'(C6 - 9) Pattern HT13(C3) +
LT20(C5) + 4209.97 4209.88 138.2 5 C HT19(C6 - 9) HT13(C3) +
LT20(C5) + 3984.82 3984.78 141.7 HT19'(C6 - 9) *indicates average
mass
[0411] All the predicted intrachain disulfides were detected with
high recovery. The recovery percentage of the intrachain disulfide
linkage between the 3rd cysteine on the heavy chain to the 5th
cysteine on the light chain was 30%. Four different forms of
disulfide linkage were detected for the hinge region. The first
form is the classic four parallel linked disulfides in the hinge
region. The second form is the 3rd cysteines in both heavy chains
and the 5th cysteines in both light chains linked to cysteines in
the diner of the hinge peptide. The third form is a mixture of the
first and second forms, on one arm, the 3rd cysteine in the heavy
chain linked to the 5th cysteine in the light chain, whereas, one
the other arm, the 3rd cysteine in the heavy chain and the 5th
cysteine in the light chain are linked to cysteines in the dimer of
the hinge peptide. The forth form is a half antibody with the 3rd
Cys in the heavy chain and the 5th cysteine in the light chain
forming disulfide bonds with cysteines in the hinge. Further
heterogeneity in the Li33 Ig2 Mab resulted from glycosylation.
Typical for a glycosylated protein, 15 different glycan structures
were observed. Glycans were largely simple bianternary core
structures (G0 55%, G1 24%, G2 3%). Six percent of the glycan
structures were sialyated.
Example 18
Targeted Mutagenesis to Improve Solubility
[0412] The crystal structures of Li33 Ig1 Fab and Li33 Ig2 Fab2
were determined in order to identify contact points that could be
altered to improve solubility.
[0413] The crystal structure of the Li33 Ig1 Fab was solved to 3.2
.ANG.. Li33 Ig1 Fab at 5 mg/ml was mixed at a volumetric ratio of
1:1 with a reservoir solution consisting of 2 M ammonium sulfate,
0.1M sodium acetate pH 3.5, and 0.1 M TCEP. Football-shaped
crystals were grown by vapor diffusion at 20.degree. C. They were
then cryoprotected by transferring them into 2M ammonium sulfate,
0.1 M citrate pH 3.5, 20% glycerol, 10% sucrose, and 10% xylitol
for 2 minutes and freezing them by a quick transfer into liquid
nitrogen. The crystals diffracted to 3.2 .ANG. at the SGXcat
beamline at the Advanced Photon Source (Argonne, Ill.). Data
processing with the HKL program package v. 1.97 [1] revealed the
crystals to belong to a P6(5)22 space group with approximate cell
dimensions a, b=90.6 .ANG., c=215.0 .ANG., and a=b=90 o, g=120 o.
The crystal structure was solved by molecular replacement based on
another IgG1 homology model (AQC2 mutant Fab PDBID: 2B2X) in PHASER
(Otwinowski and Minor, Methods in Enzymology 276: 307-326 (1997))
with all possible arrangements of the screw access leading to a
clear solution in space group P6(5)22. Model building of the single
Fab and 4 sulfates in Coot 0.5.2 (Vagin et al., Acta Crystallogr D
Biol Crystallogr 60:2184-2195 (2004)) followed by refinement using
Refmac5 (Emsley and Cowtan, Acta Crystallogr D Biol Crystallogr
60:2126-32 (2004)) to 3.2 .ANG. resolution resulted in a final
R-factor of 19.3% and Rfree of 28.9% with reasonable geometry
(Table 1).
[0414] For crystallization of the Li33 Ig2 Fab2, the sample at 7.2
mg/ml was mixed at a volumetric ratio of 1:1 with a reservoir
solution consisting of 12% Peg3350, 0.1M phosphate citrate pH 4,
and 0.2M NaCl. Rod-shaped crystals were grown by vapor diffusion at
20.degree. C. They were then cryoprotected by transferring them
into 20% Peg3350, 0.1M phosphate citrate pH 4, 0.2M NaCl, and 15%
glycerol for 2 minutes and then freezing them by a quick transfer
into liquid nitrogen. The crystals of the Li33 Ig2 Fab2 diffracted
to 2.8 .ANG. at the SGXcat beamline at the Advanced Photon Source
(Argonne, Ill.). Data processing with the HKL program package v.
1.97 (Otwinowski and Minor, Methods in Enzymology 276: 307-326
(1997)) revealed the crystals to belong to a P1 space group with
approximate cell dimensions .alpha.=91.7, .beta.=109.5 A, c=118.4,
and .alpha.=61.4 o, .beta.=74.3 o, .gamma.=87.6 o. The crystal
structure was solved by molecular replacement based on another Ig2
homology model (3GIZ) in PHASER.
[0415] These crystal structure provided a unique opportunity to
identify contact points and use rational design to address
solubility issues. FIG. 15 shows structural interfaces from the
crystal structure with CDR-CDR and CDR-framework contact points
highlighted. Five residues were identified with intermolecular
contacts, W50, W94, W104, I57, and P54 and are highlighted in the
figure. Targeted site directed mutagenesis was performed on the key
residues within the CDR sequences that contributed to contact
points. Results from selected mutations are shown in Table 9.
TABLE-US-00013 TABLE 9 Impact of targeted mutagenesis on Li33
solubility. LINGO-1 Solu- binding Stability bility EC50 .degree. C.
SEC % Li33 Isotype (mg/mL) (nM) TM1 TM2 monomer Ig1agly 0.3 0.12 60
77 >99 Ig1aglyW94V/I57V >10 0.15 59 74/82 99 Ig1aglyW94V/I57S
>7 0.38 58 75/82 99 Ig1aglyW94V/I57P >8 0.07 58 75/83 99
Ig1aglyW94V/I57T >7 0.13 59 75/83 99 Ig2 >50 0.23 69 76 96
Ig2agly 0.3 0.26 59 76 98 Ig2-V234A/G237A 5.6 0.19 69 76 95
+94V/104Q/57S >50 2.9 67 74 95 +94V/104Q/57A >50 2.4 69 73
95
[0416] The series of Ig1agly W94VI57 mutations all improved
solubility with no impact on LINGO-1 binding, stability, and level
of aggregation detected by size exclusion chromatography. The
Ig2-PDL W104QW94VI57 mutations also improved solubility with no
impact on stability and aggregation, but the additional mutation
caused a 10-fold loss in LINGO-1 binding affinity. The triple
mutants were further characterized by analytical
ultracentrifugation where there was no evidence for dimer
formation.
Example 19
PeEGylated Li33 Fab
[0417] PEGylated Li33 Fab constructs were created both by enzymatic
digestion of the Li33 Mab and by direct expression of the Fab.
[0418] In order to directly express a Li33 Fab, a Fab construct was
genetically engineered from the Li33 Ig1 construct so that the
heavy chain terminated at P231 in the hinge, thereby deleting the
Fc moiety and providing a single, unpaired cysteine from the
natural Ig1 hinge sequence that could be targeted for PEGylation.
The light chain sequence was not altered. The amino acid sequence
for the heavy chain of Li33 Fab', as predicted from the DNA
sequence, is:
TABLE-US-00014 (SEQ ID NO: 146)
EVQLLESGGGLVQPGGSLRLSCAASGETFSIYPMEWVRQAPGKGLEWVSW
IGPSGGITKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCAREG
HNDWYEDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY
ICNVNHKPSNTKVDKKVEPKSCDKTHTCPP.
The amino acid sequence for the light chain as predicted from the
DNA sequence is:
TABLE-US-00015 (SEQ ID NO: 145)
DIQMTQSPGTLSLSPGERATLSCRASQSVSSYLAWQQKPGQAPRLLIYDA
SNRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYDKVVPLTFGG
GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV
DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG
LSSPVTKSFNRGEC.
[0419] The Li33 Fab construct was expressed in CHO cells. Cells
expressing high levels of the Li33 Fab were selected by FACS
sorting. The Li33 Fab contains 11 cysteines: 5 that form disulfides
and a single free cysteine. Of these, only the disulfide that holds
the heavy and light chains together and the free cysteine are
surface exposed and potential targets for PEGylation. To verify the
reactivity of these cysteines, the Li33 Fab was reduced with 0.1 mM
TCEP, treated with an excess of PEGmaleimide (PEGmal) (0.2 mM), and
analyzed by SDS-PAGE for rapid assessment of both the extent of
reduction and PEGylation.
[0420] Reduction (step 1 in methods 1, 2, and 3 of FIG. 16) was
performed as follows. To 1 mL of Protein A purified Fab' at 1.2
mg/mL, 40 .mu.L of 1M sodium borate, pH 8.4, and 1 .mu.L of 100 mM
TCEP (final 0.1 mM) were added, and the sample was incubated for 75
minutes at 37.degree. C. Under these conditions, greater than 90%
of the product was routinely reduced to heavy and light chain when
analyzed by non-reducing SDS-PAGE. The predominant products after
reduction were free heavy and light chain that each migrated with
an apparent mass of 25 kDa.
[0421] For PEGylation with 20 kDa methoxy-polyethyleneglycol
maleimide (PEGmal) (Nektar) (step 2 in method 1 of FIG. 16), MES pH
6.0 was added to 25 mM from a 0.5 M stock solution and PEGmal was
added to 0.4 mg/mL (2.times. molar excess) from a 20 mg/mL stock
solution that was stored at -70.degree. C. The sample was incubated
at room temperature for 2.5 hours then overnight at 4.degree. C.
and then subjected to cation exchange chromatography on a Fractogel
EMD sulfate (EM Merck) resin.
[0422] When the TCEP-reduced Fab was treated with PEGmal, a nearly
complete loss of the free heavy and light chains was observed. This
observation is consistent with modification of the three cysteines.
Three new bands were detected under non-reducing conditions that
correspond to heavy chain or light chain containing a single PEG,
heavy chain containing two PEGs, and the PEGylated Fab with a
single PEG attached (FIG. 17, method 1).
[0423] The experiment was repeated with 5 kDA, 10 kDA, and 40 kDA
PEGmal, and the same three bands were present regardless of the
size of the PEGmal that was utilized. However, their molecular
weights varied in a manner that was consistent with the size of the
attached PEG moiety. For example the mono-PEGylated heavy or light
chain had apparent molecular weights of 35, 40, 50, and 100 kDa
when the samples were treated with the 5, 10, 20, and 40 kDa PEGs,
respectively, and the di-PEGylated heavy chain migrated at 45, 80,
100, and 250 kDa under the same conditions. The PEGylated Fab bands
with a single 5, 10, 20, and 40 kDa PEG attached, migrated with
masses of 70, 80, 90, and 150 kDa, respectively.
[0424] Under reducing conditions the prominent bands corresponding
to heavy chain or light chain containing a single PEG or two PEGs
were not affected, while the PEG-Fab bands disappeared. A new
prominent band corresponding to free heavy or light chain was
present after reduction. In all cases, the percent of product that
was accounted for in the PEG-Fab band was only 20% of the starting
material indicating that the predominant product was Fab with three
PEGs attached. These results confirm that after reduction, all
three of the reactive cysteines are accessible for modification.
Subsequent studies have focused on 20 kDa PEG as the reactive
group.
[0425] Follow-up studies was performed to optimize the PEGylation
reaction in order to form the desired PEG-Fab product (methods 2
and 3 in FIG. 16). The results are shown in FIG. 17 (methods 2 and
3). In both studies the TCEP reductant was removed on a desalting
column prior to reaction of the reduced Fab' with the PEG. In
method 2, the PEG was added immediately after the removal of the
TCEP, allowing both the PEGylation reaction and oxidation of the
interchain disulfide to occur concurrently. In contrast, for method
3, oxidation of the interchain disulfide was allowed to occur prior
to the addition of the PEG. Both methods resulted in a much higher
percentage of the desired PEG-Fab product than when PEGylation was
performed in the presence of TCEP (method 1). When oxidation and
PEGylation occurred simultaneously (method 2), the major
contaminants were PEGylated light chain and di-PEGylated heavy
chain. When oxidation and PEGylation were performed sequentially
(method 3), the major contaminants were unmodified Fab and Fab2.
The PEG-Fab' was purified from the reaction mixture by cation
exchange chromatography on a Fractogel EMD sulfate resin. The
unpegylated reaction products from the later scheme can be more
easily fractionated away from the PEG-Fab based on preliminary
purification studies.
[0426] In order to create a PEGylated Li33 Fab construct by
enzymatic digestion, Fab2 fragments of Li33 were first generated
with pepsin. Samples were dialyzed overnight at 4.degree. C.
against 10 mM sodium acetate pH 3.6. Pepsin was added at an
enzyme:protein ratio of 1:100 and incubated at 37.degree. C. for 6
hours for complete conversion of the Mab to Fab2. The pH of the
digest was adjusted to 7.5 with 200 mM Hepes and the sample was
loaded onto a Protein A Sepharose column at 10 mg protein/mL resin.
The column was washed with 5 column volumes of PBS, 4 column
volumes of 25 mM sodium phosphate pH 5.5 and 100 mM NaCl, and the
Fab2 was eluted from the resin with 10 mM sodium citrate pH 3.3 and
50 mM NaCl, collecting six 0.5 column volume steps. The pH of the
samples was adjusted to 4.7 with NaOH. Peak fractions were pooled,
filtered through 0.22 .mu. units, aliquoted and stored at
-70.degree. C.
[0427] For PEGylation, Ig1 Fab2 at 1.2 mg/ml in 40 mM sodium borate
pH 7.0 and 0.1 mM TCEP was incubated for 75 minutes at 37.degree.
C. The reduced sample was desalted on a G25M column that had been
equilibrated in 5 mM MES pH 5.0 and 50 mM NaCl (final Fab
concentration 0.5 mg/ml). After storage overnight at 4.degree. C.,
over 90% of the disulfide bonds holding the heavy and light chains
together had reoxidzed to a Fab' leaving the 2 hinge Cys residue
free for conjugation. 10 kDa PEGmal (Nektar) was added to 0.4 mg/mL
and MES ph 6.0 was added to 25 mM. The sample was incubated at room
temperature for 2.5 hours, overnight at 4.degree. C., and then
subjected to cation exchange chromatography on a Fractogel EMD
sulfate column. The column was washed with 2 column volumes of 10
mM sodium phosphate pH 6.0, and the PEG-Fab was eluted with 10 mM
sodium phosphate pH 6.0 and 50 mM NaCl. The results are shown in
FIG. 18.
[0428] When evaluated for function by ELISA, the PEG-Li33 Fab'
product was fully active in its ability to bind LINGO-1.
Example 20
Pegylated Li81 and Li113
[0429] Li81 fragments were created by pepsin cleavage and subject
to both C-terminal and N-terminal PEGylation.
[0430] For N-terminal PEGylation, a Fab was generated from the Li81
antibody by enzymatic digestion with papain and repurification.
Li81 Fab at .about.2 mg/mL in 10 mM sodium citrate pH 6.0, 5 mg/mL
20 kDa methoxy-polyethyleneglycol proprionaldehyde (Nektar), and 5
mM sodium cyanoborohydride were incubated at room temperature for
24 hours. The pH was adjusted to pH 4, and the samples were
concentrated to 10 mg Fab/mL and subjected to cation exchange
chromatography at room temperature on a Fractogel EMD sulfate
column (Merck) at 10 mg Fab/mL resin. The columns were washed with
2.5 column volumes of 10 mM sodium citrate pH 4.7, and 1 column
volume of 10 mM sodium citrate pH 4.7, 15 mM NaCl. The PEG-Fab was
eluted with 10 mM sodium citrate pH 4.7, 50 mM NaCl, and 0.25
column volume fractions were collected. Fractions were analyzed by
SDS-PAGE and peak fractions containing monopegylated Fab were
pooled, filtered, aliquoted and stored at -70.degree. C. Protein
concentrations were estimated from absorbance at 280 nm using the
theoretical extinction coefficients for the Fabs.
[0431] For C-terminal PEGylation, Fab2 fragments of Li81 Ig1agly
were generated with pepsin. Samples were dialyzed overnight at
4.degree. C. against 10 mM sodium acetate pH 3.6. Pepsin was added
at an enzyme:protein ratio of 1:1000 and incubated at 37.degree. C.
for 3 hours to achieve complete conversion of the Mab to Fab2. The
pH of the digest was adjusted to 7.5 with 200 mM Hepes, and the
sample was loaded onto a Protein A Sepharose column at 10 mg
protein/mL resin. The column was washed with 5 column volumes of
PBS, 4 column volumes of 25 mM sodium phosphate pH 5.5 and 100 mM
NaCl, and the Fab2 was eluted from the resin with 10 mM sodium
citrate pH 3.3, 50 mM NaCl, collecting 6.times.0.5 column volume
steps. The pH of the samples were adjusted to 4.7 with NaOH. Peak
fractions were pooled, filtered through 0.22 .mu. units, aliquoted,
and stored at -70.degree. C.
[0432] For pegylation, Li81 Ig1agly Fab2 at 2.6 mg/mL in 20 mM
sodium borate pH 7.0, 0.2 mM TCEP was incubated for 90 minutes at
37.degree. C. The reduced sample was diluted with 2 volumes of 10
mM sodium citrate pH 4.7 and loaded onto a Fractogel EMD sulfate
column (10 mg Fab'/mL resin), pre-equilibrated in the citrate pH
4.7 buffer. The column was washed with 3 column volumes of 10 mM
sodium citrate pH 4.7, 2.5 column volumes of 10 mM sodium phosphate
pH 6.0, 50 mM NaCl, and the Fab' was eluted with 7.times.0.8 column
volume steps of 10 mM sodium phosphate pH 6.0, 200 mM NaCl. The
protein eluate was diluted with water to a final protein
concentration of 1.4 mg/mL. After storage for 48 hours at 4.degree.
C., most of the disulfide bond holding the heavy and light chain
together had reoxidized to a Fab' leaving the 2 hinge Cys residues
free for conjugation. 10 kDa methoxy-polyethyleneglycol maleimide
(PEGmal) (Nektar) was added to 1.0 mg/mL, and sodium citrate pH 6.5
was added to 10 mM. The sample was incubated at room temperature
for 2.5 hours and then overnight at 4.degree. C., concentrated and
buffer exchanged in an Amicon Ultra-15 30K centrifugal filter
device to 8 mg/mL with a final buffer concentration of 10 mM
citrate pH 4.7, 6 mM NaCl, and then subjected to cation exchange
chromatography on a Fractogel EMD sulfate column (8 mg protein/mL
resin), pre-equilibrated in the citrate pH 4.7 buffer. The column
was washed with 1 column volume of 10 mM sodium citrate pH 4.7
buffer, and the PEG-Fab' was eluted with 12 0.15 column volume
steps of 10 mM sodium citrate pH 4.7, 50 mM NaCl, 2 0.15 column
volume steps of 10 mM sodium citrate pH 4.7, 100 mM NaCl, and 7
0.15 column volume steps of 10 mM sodium citrate pH 4.7, 200 mM
NaCl. Column fractions were analyzed by SDS-PAGE. Peak fractions
were pooled and the buffer adjusted to 15 mM sodium citrate pH 6.5,
125 mM NaCl. Protein concentration was determined by absorbance at
280 nm. The sample was filtered through a 0.22 .mu. unit, aliquoted
and stored at -70.degree. C.
[0433] The N-terminally PEGylated Li81 Fab2 demonstrated an EC50 of
0.15 ng/ml, while the C-terminally PEGylated Li81 Fab' demonstrated
an EC50 of 0.054 ng/ml, but both showed equivalent activity in an
oligodendrocyte proliferaction assay (0.1 .mu.g/ml).
[0434] Li113 was also N-terminally PEGylated by following the same
protocol as described for Li81 N-terminal PEGylation. The FACS
results shown in FIG. 19 demonstrate that PEGylated Li81 and Li113
can bind to LINGO-1.
Example 21
Evaluation of Functional Properties of LINGO-1 Antibody
Variants
[0435] The efficacy of Li81 antibodies and fragments thereof was
evaluated in an ELISA assay to asses LINGO-1 binding (FIG. 20), in
an oligodendrocyte proliferation assay ("OPC assay") (FIG. 21), and
in a rat remyelination assay (FIG. 22). The results demonstrate
that each of the Li81 Mab, Fab2, Fab, N-PEG-Fab, and C-PEG Fab
binds to LINGO-1 and is functional in in vitro assays. The
biochemical and in vitro properties of the molecules tested are
summarized in Table 10.
TABLE-US-00016 TABLE 10 Biochemical and in vitro LINGO-1 antibody
properties. Property Mab Fab2 Fab N-PEG C-PEG Valency Bivalent
Bivalent Mono- Mono- Mono- valent valent valent Fc fucntion Low
effector; None None None None Wt FcRn Size SEC (kDa) 140 100 50 250
250 EC50 (nM) 0.017 0.017 0.041 0.15 0.054 OPC assay 0.1 0.1 0.1
0.1 0.1 (.mu.g/ml)
Example 22
LINGO-1 Antibody and Antibody Fragment Thermal Stability
[0436] Thermal denaturation was carried out using a UV-visible
spectrophotometer fitted with a computer-controlled,
thermoelectrically-heated cuvette holder. Solutions were
equilibrated at 25.degree. C. for 15 minutes in a 200:1
microcuvette. The temperature of the cuvette holder was then ramped
from 25.degree. C. to 90.degree. C. at a rate of 2.degree. C./min,
and the denaturation of the protein was followed by continuous
monitoring of absorbance at 280 nm. The mid-point of the
cooperative unfolding event, Tm, was obtained from the melting
curves by determining the temperature at which the measured
absorbance was mid-way between the values defined by lines
extrapolated from the linear regions on each side of the
cooperative unfolding transition. The results of denaturation
experiments are shown in FIG. 23 and demonstrate that the Tm of the
LINGO-1 antibodies and Fabs tested range from about 66.degree. C.
to about 76.degree. C.
Example 23
Li33 Variants
[0437] In order to identify other Li33 variants with improved
affinity, the variants listed in Tables 11 below, were constructed
and tested. A direct binding ELISA assay was performed using
LINGO-Fc coated plates. The half maximal concentration that gave
50% saturation of binding was measured and is reported as a ratio
of the same measurement for Li33. In addition, the OD450 for
maximal saturation value was measured and is reported as a ratio of
the same measurement for Li33.
TABLE-US-00017 TABLE 11 Li33 Variants. Chain with ELISA Plateau
Variant Variant SEQ ID NO Ratio Ratio WT Li33 NA (WT) 145 and 146
1.0 0.91 W50H Heavy 147 1.3 0.87 W50F Heavy 148 1.2 0.86 W50L Heavy
149 1.8 1.00 W50M Heavy 150 2.0 0.93 P53L Heavy 151 2.2 1.13 P53S
Heavy 152 2.5 1.43 P53T Heavy 153 2.1 0.85 P53W Heavy 154 3.9 0.55
W104V Heavy 155 2.8 0.94 W104H Heavy 156 0.1 0.91 W104S Heavy 157
2.7 0.85 W104Q Heavy 158 2.4 0.91 I57G Heavy 159 2.3 0.91 I57M
Heavy 160 3.8 1.09 I57N Heavy 161 2.9 1.10 I57H Heavy 162 3.2 1.07
I57L Heavy 163 0.7 1.00 I57F Heavy 164 2.0 0.83 W94A Light 165 1.6
0.91 W94D Light 166 1.5 0.87 W94L Light 167 0.6 0.97 W94N Light 168
0.8 1.05 W94G Light 169 0.9 1.03 W94Q Light 170 0.9 1.05 W94V Light
171 1.4 0.91 W94S Light 172 1.3 0.99 W50G Heavy 173 2041.4 21.79
W50I Heavy 174 93.5 12.44 W50D Heavy 175 4.0 0.71 W104M Heavy 176
3.8 0.88 W104L Heavy 177 2.5 0.69 W104T Heavy 178 3.5 0.33 W104I
Heavy 179 6.5 0.62 P53G Heavy 180 3.1 0.83 I57W Heavy 181 0.0 I57Y
Heavy 182 0.0 I57S Heavy 183 0.6 0.96 I57P Heavy 184 0.6 1.27 I57V
Heavy 185 1.4 1.13 I57T Heavy 186 1.1 1.01 W104Q Heavy 187 1.1
0.67
[0438] Several variants with an affinity within 2 fold of Li33 and
a plateau value of at least 85% of that of Li33 were identified:
W50F, W50L, W50M, I57L, I57F, W94A, W94L, W94N, W94G, W94Q, W94V,
W94S, I57S, I57P, I57V, and I57T.
Li62 Variants
Example 24
[0439] In order to identify other Li62 variants with improved
affinity, the variants listed in Table 12 below, are constructed
and tested. A direct binding ELISA assay is performed using
LINGO-Fc coated plates. Variants that show an affinity within 2
fold of Li62 and a plateau value of at least 85% of that of Li62
are identified and analyzed in in vitro and in viro functional
assays as described above.
TABLE-US-00018 TABLE 12 Li62 Variants Variant Chain with Variant
SEQ ID NO Li62 NA (WT) 1 and 9 W50H Heavy 188 W50F Heavy 189 W50L
Heavy 190 W50M Heavy 191 P53L Heavy 192 P53S Heavy 193 P53T Heavy
194 P53W Heavy 195 W104V Heavy 196 W104H Heavy 197 W104S Heavy 198
W104Q Heavy 199 I57G Heavy 200 I57M Heavy 201 I57N Heavy 202 I57H
Heavy 203 I57L Heavy 204 I57F Heavy 205 W50G Heavy 206 W50I Heavy
207 W50D Heavy 208 W104M Heavy 209 W104L Heavy 210 W104T Heavy 211
W104I Heavy 212 P53G Heavy 213 I57W Heavy 214 I57Y Heavy 215 I57S
Heavy 216 I57P Heavy 217 I57V Heavy 218 I57T Heavy 219 W104Q Heavy
220
Li81 Variants
Example 25
[0440] In order to identify other Li81 variants with improved
affinity, the variants listed in Table 13 below, are constructed
and tested. A direct binding ELISA assay is performed using
LINGO-Fc coated plates. Variants that show an affinity within 2
fold of Li81 and a plateau value of at least 85% of that of Li81
are identified and analyzed in in vitro and in viro functional
assays as described above.
TABLE-US-00019 TABLE 13 Li81 Variants Variant Chain with Variant
SEQ ID NO Li81 NA (WT) 5 and 13 M96L Light 221 M96I Light 222 M96Q
Light 223 M96K Light 224 M96A Light 225 M96V Light 226 M96Y Light
227 M96F Light 228 P53L Heavy 229 P53S Heavy 230 P53T Heavy 231
P53W Heavy 232 P53G Heavy 233 W94A Light 234 W94D Light 235 W94L
Light 236 W94N Light 237 W94G Light 238 W94Q Light 239 W94V Light
240 W94S Light 241
Li113 Variants
Example 26
[0441] In order to identify other Li113 variants with improved
affinity, the variants listed in Table 14 below, are constructed
and tested. A direct binding ELISA assay is performed using
LINGO-Fc coated plates. Variants that show an affinity within 2
fold of Li113 and a plateau value of at least 85% of that of Li113
are identified and analyzed in in vitro and in viro functional
assays as described above.
TABLE-US-00020 TABLE 14 Li113 Variants Variant Chain with Variant
SEQ ID NO Li113 NA (WT) 66 and 9 W50H Heavy 242 W50F Heavy 243 W50L
Heavy 244 W50M Heavy 245 P53L Heavy 246 P53S Heavy 247 P53T Heavy
248 P53W Heavy 249 W104V Heavy 250 W104H Heavy 251 W104S Heavy 252
W104Q Heavy 253 I57G Heavy 254 I57M Heavy 255 I57N Heavy 256 I57H
Heavy 257 I57L Heavy 258 I57F Heavy 259 W50G Heavy 260 W50I Heavy
261 W50D Heavy 262 W104M Heavy 263 W104L Heavy 264 W104T Heavy 265
W104I Heavy 266 P53G Heavy 267 I57W Heavy 268 I57Y Heavy 269 I57S
Heavy 270 I57P Heavy 271 I57V Heavy 272 I57T Heavy 273 W104Q Heavy
274
[0442] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and any
compositions or methods which are functionally equivalent are
within the scope of this invention. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and accompanying drawings. Such modifications are
intended to fall within the scope of the appended claims.
[0443] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
2741119PRTArtificialVH sequence of Li62 antibody 1Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30
Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr
Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 25PRTArtificialVH CDR1 sequence of Li62 antibody 2Ile Tyr Pro
Met Phe 1 5 317PRTArtificialVH CDR2 sequence of Li62 antibody 3Trp
Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val Lys 1 5 10
15 Gly 410PRTArtificialVH CDR3 sequence of Li62 antibody 4Glu Gly
His Asn Asp Trp Tyr Phe Asp Leu 1 5 10 5118PRTArtificialVH sequence
of Li81 antibody 5Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro Ser
Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100
105 110 Thr Val Thr Val Ser Ser 115 65PRTArtificialVH CDR1 sequence
of Li81 antibody 6Ala Tyr Glu Met Lys 1 5 717PRTArtificialVH CDR2
sequence of Li81 antibody 7Val Ile Gly Pro Ser Gly Gly Phe Thr Phe
Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 89PRTArtificialVH CDR3
sequence of Li81 antibody 8Glu Gly Asp Asn Asp Ala Phe Asp Ile 1 5
9107PRTArtificialVL sequence of Li62 antibody 9Asp Ile Gln Met Thr
Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val
Ala Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Arg Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Arg Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Thr Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Gly Thr Tyr Tyr Cys Gln Gln Tyr Asp
Thr Leu His Pro 85 90 95 Ser Phe Gly Pro Gly Thr Thr Val Asp Ile
Lys 100 105 1011PRTArtificialVL CDR1 sequence of Li62 antibody
10Arg Ala Ser Gln Asp Ile Ser Arg Tyr Leu Ala 1 5 10
117PRTArtificialVL CDR2 sequence of Li62 antibody 11Asp Ala Ser Asn
Leu Gln Thr 1 5 129PRTArtificialVL CDR3 sequence of Li62 antibody
12Gln Gln Tyr Asp Thr Leu His Pro Ser 1 5 13108PRTArtificialVL
sequence of Li81 antibody 13Asp Ile Gln Met Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser
Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Met 85
90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
1411PRTArtificialVL CDR1 sequence of Li81 antibody 14Arg Ala Ser
Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 157PRTArtificialVL CDR2
sequence of Li81 antibody 15Asp Ala Ser Asn Arg Ala Thr 1 5
1610PRTArtificialVL CDR3 sequence of Li81 antibody 16Gln Gln Arg
Ser Asn Trp Pro Met Tyr Thr 1 5 10 1710PRTArtificialVH CDR3
sequence of Li62 variant B06 antibody 17Glu Gly Tyr Tyr Asp Trp Tyr
Phe Asp Gln 1 5 10 1810PRTArtificialVH CDR3 sequence of Li62
variant B12 antibody 18Glu Gly Gln Tyr Asp Trp Tyr Phe Asp Val 1 5
10 1910PRTArtificialVH CDR3 sequence of Li62 variant F06 antibody
19Glu Gly Asp Tyr Asp Trp Tyr Phe Asp Leu 1 5 10
2010PRTArtificialVH CDR3 sequence of Li62 variant B01 antibody
20Glu Gly Gln Tyr Asp Trp Tyr Phe Glu Leu 1 5 10
2110PRTArtificialVH CDR3 sequence of Li62 variant D09 antibody
21Glu Ala Asp Ile Asp Trp Phe Phe Asp Leu 1 5 10
2210PRTArtificialVH CDR3 sequence of Li62 variant D12 antibody
22Glu Gly His Tyr Asp Trp Tyr Phe Asp Leu 1 5 10
2310PRTArtificialVH CDR3 sequence of Li62 variant F01 antibody
23Glu Gly Arg Tyr Asp Trp Tyr Phe Asp Pro 1 5 10
2410PRTArtificialVH CDR3 sequence of Li62 variant F02 antibody
24Glu Gly Asp Tyr Asp Trp Tyr Phe Gly Leu 1 5 10
2510PRTArtificialVH CDR3 sequence of Li62 variant F06 antibody
25Glu Gly Arg Tyr Asp Trp Tyr Phe Asp Leu 1 5 10
2610PRTArtificialVH CDR3 sequence of Li62 variant F10 antibody
26Glu Ser His Ile Asp Arg Tyr Phe Asp Leu 1 5 10
2710PRTArtificialVH CDR3 sequence of Li62 variant G08 antibody
27Glu Gly Gln Tyr Asp Trp Tyr Phe Asp Val 1 5 10
2810PRTArtificialVH CDR3 sequence of Li62 variant H08 antibody
28Glu Gly His Tyr Asn Gly Tyr Phe Asp Leu 1 5 10
2910PRTArtificialVH CDR3 sequence of Li62 variant C10 antibody
29Glu Gly Tyr Tyr Asp Trp Tyr Phe Asp Leu 1 5 10
3010PRTArtificialVH CDR3 sequence of Li62 variant C02 antibody
30Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu 1 5 10
3110PRTArtificialVH CDR3 sequence of Li62 variant D05 antibody
31Glu Gly Tyr Tyr Asp Trp Tyr Phe Glu Leu 1 5 10
3210PRTArtificialVH CDR3 sequence of Li62 variant F02 antibody
32Glu Gly Leu Ile Asp Trp Phe Phe Asp Gln 1 5 10
3310PRTArtificialVH CDR3 sequence of Li62 variant C10 antibody
33Glu Gly Gln Phe Asp Trp Tyr Phe Asp Leu 1 5 10
3410PRTArtificialVH CDR3 sequence of Li62 variant H08 antibody
34Glu Gly Thr Tyr Asp Trp Tyr Phe Asp Leu 1 5 10 359PRTArtificialVH
CDR3 sequence of Li81 variant F09 antibody 35Glu Gly Glu Asn Asp
Ala Phe Asp Val 1 5 369PRTArtificialVH CDR3 sequence of Li81
variant G02 antibody 36Glu Gly Asp Asn Asp Ala Tyr Asp Thr 1 5
379PRTArtificialVH CDR3 sequence of Li81 variant H03 antibody 37Glu
Gly Thr Asn Asp Ala Phe Asp Ile 1 5 389PRTArtificialVH CDR3
sequence of Li81 variant A12 antibody 38Glu Gly Asp Asn Asp Ala Phe
Asp Ser 1 5 399PRTArtificialVH CDR3 sequence of Li81 variant C02
antibody 39Glu Gly Asp Asn Asp Ala Phe Asp Thr 1 5
409PRTArtificialVH CDR3 sequence of Li81 variant C11 antibody 40Glu
Gly Asp Asn Asp Ala Tyr Asp Arg 1 5 419PRTArtificialVH CDR3
sequence of Li81 variant D11 antibody 41Glu Gly Asp Asn Asp Val Phe
Asp Ser 1 5 429PRTArtificialVH CDR3 sequence of Li81 variant E05
antibody 42Glu Gly Asp Asp Asp Val Phe Asp Met 1 5
439PRTArtificialVH CDR3 sequence of Li81 variant H04 antibody 43Glu
Gly Tyr Asn Asp Ala Phe Asp Phe 1 5 449PRTArtificialVH CDR3
sequence of Li81 variant B04 antibody 44Glu Gly Asp Asp Asp Ala Tyr
Asp Met 1 5 459PRTArtificialVH CDR3 sequence of Li81 variant A02
antibody 45Glu Gln Asp Tyr Asp Thr Tyr Asp Leu 1 5
469PRTArtificialVH CDR3 sequence of Li81 variant B12 antibody 46Glu
Gly Asp Asp Asp Ala Phe Asp Thr 1 5 479PRTArtificialVH CDR3
sequence of Li81 variant H06 antibody 47Glu Ala Asp Asp Asp Ala Phe
Asp Ile 1 5 489PRTArtificialVH CDR3 sequence of Li81 variant H08
antibody 48Glu Gly Glu Asn Asp Ala Phe Asp Met 1 5
499PRTArtificialVH CDR3 sequence of Li81 variant E07 antibody 49Glu
Gly Glu Tyr Asp Thr Tyr Asp Ile 1 5 50447PRTArtificialAglycosylated
Li81 heavy chain 50Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro Ser
Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95
Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100
105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225
230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser Ala Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345
350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys
355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 51614PRTHomo
sapiens 51Met Leu Ala Gly Gly Val Arg Ser Met Pro Ser Pro Leu Leu
Ala Cys 1 5 10 15 Trp Gln Pro Ile Leu Leu Leu Val Leu Gly Ser Val
Leu Ser Gly Ser 20 25 30 Ala Thr Gly Cys Pro Pro Arg Cys Glu Cys
Ser Ala Gln Asp Arg Ala 35 40 45 Val Leu Cys His Arg Lys Arg Phe
Val Ala Val Pro Glu Gly Ile Pro 50 55 60 Thr Glu Thr Arg Leu Leu
Asp Leu Gly Lys Asn Arg Ile Lys Thr Leu 65 70 75 80 Asn Gln Asp Glu
Phe Ala Ser Phe Pro His Leu Glu Glu Leu Glu Leu 85 90 95 Asn Glu
Asn Ile Val Ser Ala Val Glu Pro Gly Ala Phe Asn Asn Leu 100 105 110
Phe Asn Leu Arg Thr Leu Gly Leu Arg Ser Asn Arg Leu Lys Leu Ile 115
120 125 Pro Leu Gly Val Phe Thr Gly Leu Ser Asn Leu Thr Lys Leu Asp
Ile 130 135 140 Ser Glu Asn Lys Ile Val Ile Leu Leu Asp Tyr Met Phe
Gln Asp Leu 145 150 155 160 Tyr Asn Leu Lys Ser Leu Glu Val Gly Asp
Asn Asp Leu Val Tyr Ile 165 170 175 Ser His Arg Ala Phe Ser Gly Leu
Asn Ser Leu Glu Gln Leu Thr Leu 180 185 190 Glu Lys Cys Asn Leu Thr
Ser Ile Pro Thr Glu Ala Leu Ser His Leu 195 200 205 His Gly Leu Ile
Val Leu Arg Leu Arg His Leu Asn Ile Asn Ala Ile 210 215 220 Arg Asp
Tyr Ser Phe Lys Arg Leu Tyr Arg Leu Lys Val Leu Glu Ile 225 230 235
240 Ser His Trp Pro Tyr Leu Asp Thr Met Thr Pro Asn Cys Leu Tyr Gly
245 250 255 Leu Asn Leu Thr Ser Leu Ser Ile Thr His Cys Asn Leu Thr
Ala Val 260 265 270 Pro Tyr Leu Ala Val Arg His Leu Val Tyr Leu Arg
Phe Leu Asn Leu 275 280 285 Ser Tyr Asn Pro Ile Ser Thr Ile Glu Gly
Ser Met Leu His Glu Leu 290 295 300 Leu Arg Leu Gln Glu Ile Gln Leu
Val Gly Gly Gln Leu Ala Val Val 305 310 315 320 Glu Pro Tyr Ala Phe
Arg Gly Leu Asn Tyr Leu Arg Val Leu Asn Val 325 330 335 Ser Gly Asn
Gln Leu Thr Thr Leu Glu Glu Ser Val Phe His Ser Val 340 345 350 Gly
Asn Leu Glu Thr Leu Ile Leu Asp Ser Asn Pro Leu Ala Cys Asp 355 360
365 Cys Arg Leu Leu Trp Val Phe Arg Arg Arg Trp Arg Leu Asn Phe Asn
370 375 380 Arg Gln Gln Pro Thr Cys Ala Thr Pro Glu Phe Val Gln Gly
Lys Glu 385 390 395 400 Phe Lys Asp Phe Pro Asp Val Leu Leu Pro Asn
Tyr Phe Thr Cys Arg 405 410 415 Arg Ala Arg Ile Arg Asp Arg Lys Ala
Gln Gln Val Phe Val Asp Glu 420 425 430 Gly His Thr Val Gln Phe Val
Cys Arg Ala Asp Gly Asp Pro Pro Pro 435 440 445 Ala Ile Leu Trp Leu
Ser Pro Arg Lys His Leu Val Ser Ala Lys Ser 450 455 460 Asn Gly Arg
Leu Thr Val Phe Pro Asp Gly Thr Leu Glu Val Arg Tyr 465 470 475 480
Ala Gln Val Gln Asp Asn Gly Thr Tyr Leu Cys Ile Ala Ala Asn Ala 485
490 495 Gly Gly Asn Asp Ser Met Pro Ala His Leu His Val Arg Ser Tyr
Ser 500 505 510 Pro Asp Trp Pro His Gln Pro Asn Lys Thr Phe Ala Phe
Ile Ser Asn 515 520 525 Gln Pro Gly Glu Gly Glu Ala Asn Ser Thr Arg
Ala Thr Val Pro Phe 530 535 540 Pro Phe Asp Ile Lys Thr Leu Ile Ile
Ala Thr Thr Met Gly Phe Ile 545 550 555 560 Ser Phe Leu Gly Val Val
Leu Phe Cys Leu
Val Leu Leu Phe Leu Trp 565 570 575 Ser Arg Gly Lys Gly Asn Thr Lys
His Asn Ile Glu Ile Glu Tyr Val 580 585 590 Pro Arg Lys Ser Asp Ala
Gly Ile Ser Ser Ala Asp Ala Pro Arg Lys 595 600 605 Phe Asn Met Lys
Met Ile 610 521845DNAHomo sapiens 52atgctggcgg ggggcgtgag
gagcatgccc agccccctcc tggcctgctg gcagcccatc 60ctcctgctgg tgctgggctc
agtgctgtca ggctcggcca cgggctgccc gccccgctgc 120gagtgctccg
cccaggaccg cgctgtgctg tgccaccgca agcgctttgt ggcagtcccc
180gagggcatcc ccaccgagac gcgcctgctg gacctaggca agaaccgcat
caaaacgctc 240aaccaggacg agttcgccag cttcccgcac ctggaggagc
tggagctcaa cgagaacatc 300gtgagcgccg tggagcccgg cgccttcaac
aacctcttca acctccggac gctgggtctc 360cgcagcaacc gcctgaagct
catcccgcta ggcgtcttca ctggcctcag caacctgacc 420aagctggaca
tcagcgagaa caagattgtt atcctgctgg actacatgtt tcaggacctg
480tacaacctca agtcactgga ggttggcgac aatgacctcg tctacatctc
tcaccgcgcc 540ttcagcggcc tcaacagcct ggagcagctg acgctggaga
aatgcaacct gacctccatc 600cccaccgagg cgctgtccca cctgcacggc
ctcatcgtcc tgaggctccg gcacctcaac 660atcaatgcca tccgggacta
ctccttcaag aggctctacc gactcaaggt cttggagatc 720tcccactggc
cctacttgga caccatgaca cccaactgcc tctacggcct caacctgacg
780tccctgtcca tcacacactg caatctgacc gctgtgccct acctggccgt
ccgccaccta 840gtctatctcc gcttcctcaa cctctcctac aaccccatca
gcaccattga gggctccatg 900ttgcatgagc tgctccggct gcaggagatc
cagctggtgg gcgggcagct ggccgtggtg 960gagccctatg ccttccgcgg
cctcaactac ctgcgcgtgc tcaatgtctc tggcaaccag 1020ctgaccacac
tggaggaatc agtcttccac tcggtgggca acctggagac actcatcctg
1080gactccaacc cgctggcctg cgactgtcgg ctcctgtggg tgttccggcg
ccgctggcgg 1140ctcaacttca accggcagca gcccacgtgc gccacgcccg
agtttgtcca gggcaaggag 1200ttcaaggact tccctgatgt gctactgccc
aactacttca cctgccgccg cgcccgcatc 1260cgggaccgca aggcccagca
ggtgtttgtg gacgagggcc acacggtgca gtttgtgtgc 1320cgggccgatg
gcgacccgcc gcccgccatc ctctggctct caccccgaaa gcacctggtc
1380tcagccaaga gcaatgggcg gctcacagtc ttccctgatg gcacgctgga
ggtgcgctac 1440gcccaggtac aggacaacgg cacgtacctg tgcatcgcgg
ccaacgcggg cggcaacgac 1500tccatgcccg cccacctgca tgtgcgcagc
tactcgcccg actggcccca tcagcccaac 1560aagaccttcg ctttcatctc
caaccagccg ggcgagggag aggccaacag cacccgcgcc 1620actgtgcctt
tccccttcga catcaagacc ctcatcatcg ccaccaccat gggcttcatc
1680tctttcctgg gcgtcgtcct cttctgcctg gtgctgctgt ttctctggag
ccggggcaag 1740ggcaacacaa agcacaacat cgagatcgag tatgtgcccc
gaaagtcgga cgcaggcatc 1800agctccgccg acgcgccccg caagttcaac
atgaagatga tatga 184553119PRTArtificialVH sequence of Li62 variant
B06 antibody 53Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly
Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala
Arg Glu Gly Tyr Tyr Asp Trp Tyr Phe Asp Gln Trp Gly Arg Gly 100 105
110 Thr Leu Val Thr Val Ser Ser 115 54119PRTArtificialVH sequence
of Li62 variant B12 antibody 54Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile
Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr
Cys 85 90 95 Ala Arg Glu Gly Gln Tyr Asp Trp Tyr Phe Asp Val Trp
Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
55119PRTArtificialVH sequence of Li62 variant F06 antibody 55Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Tyr
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 56119PRTArtificialVH sequence of Li62 variant B01
antibody 56Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Gln Tyr Asp Trp Tyr Phe Glu Leu Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 57119PRTArtificialVH sequence of
Li62 variant D09 antibody 57Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly
Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Ala Asp Ile Asp Trp Phe Phe Asp Leu Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
58119PRTArtificialVH sequence of Li62 variant D12 antibody 58Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Tyr
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 59119PRTArtificialVH sequence of Li62 variant F01
antibody 59Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Arg Tyr Asp Trp Tyr Phe Asp Pro Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 60119PRTArtificialVH sequence of
Li62 variant F02 antibody 60Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly
Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Asp Tyr Asp Trp Tyr Phe Gly Leu Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
61119PRTArtificialVH sequence of Li62 variant F06 antibody 61Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Arg Tyr
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 62119PRTArtificialVH sequence of Li62 variant F10
antibody 62Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Ser His Ile Asp Arg Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 63119PRTArtificialVH sequence of
Li62 variant G08 antibody 63Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly
Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Gln Tyr Asp Trp Tyr Phe Asp Val Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
64119PRTArtificialVH sequence of Li62 variant H08 antibody 64Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Tyr
Asn Gly Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 65119PRTArtificialVH sequence of Li62 variant C10
antibody 65Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Tyr Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 66119PRTArtificialVH sequence of
Li62 variant C02 antibody 66Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly
Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
67119PRTArtificialVH sequence of Li62 variant D05 antibody 67Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Tyr Tyr
Asp Trp Tyr Phe Glu Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 68119PRTArtificialVH sequence of Li62 variant F02
antibody 68Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile
Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70
75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr
Cys 85 90 95 Ala Arg Glu Gly Leu Ile Asp Trp Phe Phe Asp Gln Trp
Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
69119PRTArtificialVH sequence of Li62 variant C10 antibody 69Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Gln Phe
Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 70119PRTArtificialVH sequence of Li62 variant H08
antibody 70Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly Thr Tyr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser 115 71118PRTArtificialVH sequence of
Li81 variant F09 antibody 71Glu Val Gln Leu Leu Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly
Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Thr Glu Gly Glu Asn Asp Ala Phe Asp Val Trp Gly Gln Gly
Thr 100 105 110 Thr Val Thr Val Ser Ser 115 72118PRTArtificialVH
sequence of Li81 variant G02 antibody 72Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Tyr Asp Thr Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
73118PRTArtificialVH sequence of Li81 variant H03 antibody 73Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Thr Asn
Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val
Ser Ser 115 74118PRTArtificialVH sequence of Li81 variant A12
antibody 74Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly
Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr
Glu Gly Asp Asn Asp Ala Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110
Thr Val Thr Val Ser Ser 115 75118PRTArtificialVH sequence of Li81
variant C02 antibody 75Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro
Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Thr Trp Gly Gln Gly Thr
100 105 110 Thr Val Thr Val Ser Ser 115 76118PRTArtificialVH
sequence of Li81 variant C11 antibody 76Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Tyr Asp Arg Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
77118PRTArtificialVH sequence of Li81 variant D11 antibody 77Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn
Asp Val Phe Asp Ser Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val
Ser Ser 115 78118PRTArtificialVH sequence of Li81 variant E05
antibody 78Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly
Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr
Glu Gly Asp Asp Asp Val Phe Asp Met Trp Gly Gln Gly Thr 100 105 110
Thr Val Thr Val Ser Ser 115 79118PRTArtificialVH sequence of Li81
variant H04 antibody 79Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro
Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Glu Gly Tyr Asn Asp Ala Phe Asp Phe Trp Gly Gln Gly Thr
100 105 110 Thr Val Thr Val Ser Ser 115 80118PRTArtificialVH
sequence of Li81 variant B04 antibody 80Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asp Asp Ala Tyr Asp Met Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
81118PRTArtificialVH sequence of Li81 variant A02 antibody 81Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gln Asp Tyr
Asp Thr Tyr Asp Leu Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val
Ser Ser 115 82118PRTArtificialVH sequence of Li81 variant B12
antibody 82Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly
Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr
Glu Gly Asp Asp Asp Ala Phe Asp Thr Trp Gly Gln Gly Thr 100 105 110
Thr Val Thr Val Ser Ser 115 83118PRTArtificialVH sequence of Li81
variant H06 antibody 83Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Gly Pro
Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Glu Ala Asp Asp Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr
100 105 110 Thr Val Thr Val Ser Ser 115 84118PRTArtificialVH
sequence of Li81 variant H08 antibody 84Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Glu Asn Asp Ala Phe Asp Met Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
85118PRTArtificialVH sequence of Li81 variant E07 antibody 85Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr
20 25 30 Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Glu Tyr
Asp Thr Tyr Asp Ile Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val
Ser Ser 115 86447PRTArtificialLi81 heavy chain 86Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30
Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Val Ile Gly Pro Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser
Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185
190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser
195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300 Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310
315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445 876PRTArtificialN-terminus of LINGO-1 polypeptide 87Met Gln Val
Ser Lys Arg 1 5 885PRTArtificialLINGO-1 polypeptide 88Ile Thr Xaa
Xaa Xaa 1 5 895PRTArtificialLINGO-1 polypeptide 89Ala Cys Xaa Xaa
Xaa 1 5 905PRTArtificialLINGO-1 polypeptide 90Val Cys Xaa Xaa Xaa 1
5 915PRTArtificialLINGO-1 polypeptide 91Ser Pro Xaa Xaa Xaa 1 5
925PRTArtificialLINGO-1 polypeptide 92Ser Pro Arg Lys His 1 5
935PRTArtificialLINGO-1 polypeptide 93Ser Pro Arg Lys Lys 1 5
945PRTArtificialLINGO-1 polypeptide 94Ser Pro Arg Lys Arg 1 5
955PRTArtificialLINGO-1 polypeptide 95Ser Pro Lys Lys His 1 5
965PRTArtificialLINGO-1 polypeptide 96Ser Pro His Lys His 1 5
975PRTArtificialLINGO-1 polypeptide 97Ser Pro Arg Arg His 1 5
985PRTArtificialLINGO-1 polypeptide 98Ser Pro Arg His His 1 5
995PRTArtificialLINGO-1 polypeptide 99Ser Pro Arg Arg Arg 1 5
1005PRTArtificialLINGO-1 polypeptide 100Ser Pro His His His 1 5
1015PRTArtificialLINGO-1 polypeptide 101Ser Pro Lys Lys Lys 1 5
1026PRTArtificialLINGO-1 polypeptide 102Leu Ser Pro Arg Lys His 1 5
1036PRTArtificialLINGO-1 polypeptide 103Leu Ser Pro Arg Lys Lys 1 5
1046PRTArtificialLINGO-1 polypeptide 104Leu Ser Pro Arg Lys Arg 1 5
1056PRTArtificialLINGO-1 polypeptide 105Leu Ser Pro Lys Lys His 1 5
1066PRTArtificialLINGO-1 polypeptide 106Leu Ser Pro His Lys His 1 5
1076PRTArtificialLINGO-1 polypeptide 107Leu Ser Pro Arg Arg His 1 5
1086PRTArtificialLINGO-1 polypeptide 108Leu Ser Pro Arg His His 1 5
1096PRTArtificialLINGO-1 polypeptide 109Leu Ser Pro Arg Arg Arg 1 5
1106PRTArtificialLINGO-1 polypeptide 110Leu Ser Pro His His His 1 5
1116PRTArtificialLINGO-1 polypeptide 111Leu Ser Pro Lys Lys Lys 1 5
1127PRTArtificialLINGO-1 polypeptide 112Trp Leu Ser Pro Arg Lys His
1 5 1137PRTArtificialLINGO-1 polypeptide 113Trp Leu Ser Pro Arg Lys
Lys 1 5 1147PRTArtificialLINGO-1 polypeptide 114Trp Leu Ser Pro Arg
Lys Arg 1 5 1157PRTArtificialLINGO-1 polypeptide 115Trp Leu Ser Pro
Lys Lys His 1 5 1167PRTArtificialLINGO-1 polypeptide 116Trp Leu Ser
Pro His Lys His 1 5 1177PRTArtificialLINGO-1 polypeptide 117Trp Leu
Ser Pro Arg Arg His 1 5 1187PRTArtificialLINGO-1 polypeptide 118Trp
Leu Ser Pro Arg His His 1 5 1197PRTArtificialLINGO-1 polypeptide
119Trp Leu Ser Pro Arg Arg Arg 1 5 1207PRTArtificialLINGO-1
polypeptide 120Trp Leu Ser Pro His His His 1 5
1217PRTArtificialLINGO-1 polypeptide 121Trp Leu Ser Pro Lys Lys Lys
1 5 1226PRTArtificialLINGO-1 polypeptide 122Ile Thr Pro Lys Arg Arg
1 5 1235PRTArtificialLINGO-1 polypeptide 123Ala Cys His His Lys 1 5
1245PRTArtificialLINGO-1 polypeptide 124Val Cys His His Lys 1 5
1255PRTArtificialLINGO-1 polypeptide 125Xaa Xaa Arg Lys His 1 5
1265PRTArtificialLINGO-1 polypeptide 126Xaa Xaa Arg Arg Arg 1 5
1275PRTArtificialLINGO-1 polypeptide 127Xaa Xaa Lys Lys Lys 1 5
1285PRTArtificialLINGO-1 polypeptide 128Xaa Xaa His His His 1 5
1295PRTArtificialLINGO-1 polypeptide 129Xaa Xaa Arg Lys Lys 1 5
1305PRTArtificialLINGO-1 polypeptide 130Xaa Xaa Arg Lys Arg 1 5
1315PRTArtificialLINGO-1 polypeptide 131Xaa Xaa Lys Lys His 1 5
1325PRTArtificialLINGO-1 polypeptide 132Xaa Xaa His Lys His 1 5
1335PRTArtificialLINGO-1 polypeptide 133Xaa Xaa Arg Arg His 1 5
1345PRTArtificialLINGO-1 polypeptide 134Xaa Xaa Arg His His 1 5
1355PRTArtificialLINGO-1 polypeptide 135Ile Thr Xaa Xaa Xaa 1 5
1365PRTArtificialLINGO-1 polypeptide 136Ala Cys Xaa Xaa Xaa 1 5
1375PRTArtificialLINGO-1 polypeptide 137Val Cys Xaa Xaa Xaa 1 5
1385PRTArtificialLINGO-1 polypeptide 138Ser Pro Xaa Xaa Xaa 1 5
1395PRTArtificialLINGO-1 polypeptide 139Ser Pro Arg Leu His 1 5
1409PRTArtificialLINGO-1 polypeptide 140Arg Arg Ala Arg Ile Arg Asp
Arg Lys 1 5 1419PRTArtificialLINGO-1 polypeptide 141Lys Lys Val Lys
Val Lys Glu Lys Arg 1 5 1429PRTArtificialLINGO-1 polypeptide 142Arg
Arg Leu Arg Leu Arg Asp Arg Lys 1 5 1439PRTArtificialLINGO-1
polypeptide 143Arg Arg Gly Arg Gly Arg Asp Arg Lys 1 5
1449PRTArtificialLINGO-1 polypeptide 144Arg Arg Ile Arg Ala Arg Asp
Arg Lys 1 5 145214PRTArtificialLi33 Fab' light chain 145Asp Ile Gln
Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Asp Lys Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val
Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val
Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155
160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
146230PRTArtificialLi33 Fab' heavy chain 146Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met
Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp
Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
147230PRTArtificialLi33 Heavy Chain Variant 147Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser His Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
148230PRTArtificialLi33 Heavy Chain Variant 148Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Phe Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
149230PRTArtificialLi33 Heavy Chain Variant 149Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Leu Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
150230PRTArtificialLi33 Heavy Chain Variant 150Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Met Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85
90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210
215 220 Thr His Thr Cys Pro Pro 225 230 151230PRTArtificialLi33
Heavy Chain Variant 151Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Leu
Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90
95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly
100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro 225 230 152230PRTArtificialLi33 Heavy
Chain Variant 152Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Ser Ser
Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95
Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100
105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly
Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220
Thr His Thr Cys Pro Pro 225 230 153230PRTArtificialLi33 Heavy Chain
Variant 153Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro
Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Thr Ser Gly Gly
Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg
Glu Gly His Asn Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro 225 230 154230PRTArtificialLi33 Heavy Chain Variant
154Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Trp Ser Gly Gly Ile Thr
Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
His Asn Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125
Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130
135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr
Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys
Pro Pro 225 230 155230PRTArtificialLi33 Heavy Chain Variant 155Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn
Asp Val Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145
150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro
225 230 156230PRTArtificialLi33 Heavy Chain Variant 156Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25
30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp His
Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155
160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu
165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
157230PRTArtificialLi33 Heavy Chain Variant 157Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Ser Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
158230PRTArtificialLi33 Heavy Chain Variant 158Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Gln Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
159230PRTArtificialLi33 Heavy Chain Variant 159Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Gly Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
160230PRTArtificialLi33 Heavy Chain Variant 160Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Met Thr Lys Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp
Trp Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala
Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150
155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225
230 161230PRTArtificialLi33 Heavy Chain Variant 161Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30
Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Trp Ile Gly Pro Ser Gly Gly Asn Thr Lys Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr
Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
162230PRTArtificialLi33 Heavy Chain Variant 162Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
163230PRTArtificialLi33 Heavy Chain Variant 163Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
164230PRTArtificialLi33 Heavy Chain Variant 164Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Phe Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
165214PRTArtificialLi33 Light Chain Variant 165Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Lys Ala Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
166214PRTArtificialLi33 Light Chain Variant 166Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Lys Asp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
167214PRTArtificialLi33 Light Chain Variant 167Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Lys Leu Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
168214PRTArtificialLi33 Light Chain Variant 168Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Lys Asn Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
169214PRTArtificialLi33 Light Chain Variant 169Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp
Lys Gly Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp
Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170
175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
170214PRTArtificialLi33 Light Chain Variant 170Asp Ile Gln Met Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65
70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Lys Gln
Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 171214PRTArtificialLi33
Light Chain Variant 171Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asp Lys Val Pro Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 172214PRTArtificialLi33 Light Chain Variant
172Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr
Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Tyr Asp Lys Ser Pro Leu 85 90 95 Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130
135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr
Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly
Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys
210 173230PRTArtificialLi33 Heavy Chain Variant 173Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30
Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Gly Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr
Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165
170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
174230PRTArtificialLi33 Heavy Chain Variant 174Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ile Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
175230PRTArtificialLi33 Heavy Chain Variant 175Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Asp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
176230PRTArtificialLi33 Heavy Chain Variant 176Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Met Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
177230PRTArtificialLi33 Heavy Chain Variant 177Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Leu Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
178230PRTArtificialLi33 Heavy Chain Variant 178Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Thr Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
179230PRTArtificialLi33 Heavy Chain Variant 179Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Ile Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
180230PRTArtificialLi33 Heavy Chain Variant 180Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Gly Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp
Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys
Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180
185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
181230PRTArtificialLi33 Heavy Chain Variant 181Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Trp Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
182230PRTArtificialLi33 Heavy Chain Variant 182Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Tyr Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
183230PRTArtificialLi33 Heavy Chain Variant 183Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
184230PRTArtificialLi33 Heavy Chain Variant 184Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Pro Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
185230PRTArtificialLi33 Heavy Chain Variant 185Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Val Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
186230PRTArtificialLi33 Heavy Chain Variant 186Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Thr Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
187230PRTArtificialLi33 Heavy Chain Variant 187Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Gln Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro 225 230
188119PRTArtificialLi62 VH Variant 188Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
His Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
189119PRTArtificialLi62 VH Variant 189Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Phe Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
190119PRTArtificialLi62 VH Variant 190Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Leu Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
191119PRTArtificialLi62 VH Variant 191Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Met Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg
Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
192119PRTArtificialLi62 VH Variant 192Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Leu Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
193119PRTArtificialLi62 VH Variant 193Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Ser Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
194119PRTArtificialLi62 VH Variant 194Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Thr Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
195119PRTArtificialLi62 VH Variant 195Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Trp Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
196119PRTArtificialLi62 VH Variant 196Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Val Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
197119PRTArtificialLi62 VH Variant 197Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp His Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
198119PRTArtificialLi62 VH Variant 198Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Ser Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
199119PRTArtificialLi62 VH Variant 199Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Gln Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
200119PRTArtificialLi62 VH Variant 200Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Gly Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
201119PRTArtificialLi62 VH Variant 201Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Met Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
202119PRTArtificialLi62 VH Variant 202Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Asn Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
203119PRTArtificialLi62 VH Variant 203Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
204119PRTArtificialLi62 VH Variant 204Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
205119PRTArtificialLi62 VH Variant 205Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Phe Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
206119PRTArtificialLi62 VH Variant 206Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Gly Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
207119PRTArtificialLi62 VH Variant 207Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ile Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
208119PRTArtificialLi62 VH Variant 208Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Asp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
209119PRTArtificialLi62 VH Variant 209Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Met Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
210119PRTArtificialLi62 VH Variant 210Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser
Ile Tyr 20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr
Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly
His Asn Asp Leu Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu
Val Thr Val Ser Ser 115 211119PRTArtificialLi62 VH Variant 211Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn
Asp Thr Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr
Val Ser Ser 115 212119PRTArtificialLi62 VH Variant 212Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25
30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Ile
Tyr Phe Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 213119PRTArtificialLi62 VH Variant 213Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Trp Ile Gly Gly Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
214119PRTArtificialLi62 VH Variant 214Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Trp Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
215119PRTArtificialLi62 VH Variant 215Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Tyr Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
216119PRTArtificialLi62 VH Variant 216Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
217119PRTArtificialLi62 VH Variant 217Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Pro Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
218119PRTArtificialLi62 VH Variant 218Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Val Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
219119PRTArtificialLi62 VH Variant 219Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Thr Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Trp Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
220119PRTArtificialLi62 VH Variant 220Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly His Asn Asp Gln Tyr Phe Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
221108PRTArtificialLi81 VL Variant 221Asp Ile Gln Met Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Leu 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 222108PRTArtificialLi81 VL Variant 222Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Ile 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 223108PRTArtificialLi81 VL Variant 223Asp Ile Gln
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Trp Pro Gln 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 224108PRTArtificialLi81 VL Variant 224Asp
Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Trp Pro Lys 85 90 95 Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 225108PRTArtificialLi81 VL Variant
225Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ala 85 90 95 Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 226108PRTArtificialLi81 VL
Variant 226Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Val 85 90 95 Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
227108PRTArtificialLi81 VL Variant 227Asp Ile Gln Met Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Tyr 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 228108PRTArtificialLi81 VL Variant 228Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Trp Pro Phe 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 229118PRTArtificialLi81 VH Variant 229Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25
30
Glu Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Val Ile Gly Leu Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe
Asp Ile Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
230118PRTArtificialLi81 VH Variant 230Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Ser Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
231118PRTArtificialLi81 VH Variant 231Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Thr Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
232118PRTArtificialLi81 VH Variant 232Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Trp Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
233118PRTArtificialLi81 VH Variant 233Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Glu Met Lys
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Val Ile Gly Gly Ser Gly Gly Phe Thr Phe Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Thr Glu Gly Asp Asn Asp Ala Phe Asp Ile Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
234108PRTArtificialLi81 VL Variant 234Asp Ile Gln Met Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Ala
Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 235108PRTArtificialLi81 VL Variant 235Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Asp Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 236108PRTArtificialLi81 VL Variant 236Asp Ile Gln
Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Arg Ser Asn Leu Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 237108PRTArtificialLi81 VL Variant 237Asp
Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Asn Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 238108PRTArtificialLi81 VL Variant
238Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser
Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile
Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Arg Ser Asn Gly Pro Met 85 90 95 Tyr Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105 239108PRTArtificialLi81 VL
Variant 239Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Ser Asn Gln Pro Met 85 90 95 Tyr Thr
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
240108PRTArtificialLi81 VL Variant 240Asp Ile Gln Met Thr Gln Ser
Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr
Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55
60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Val
Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 241108PRTArtificialLi81 VL Variant 241Asp Ile Gln Met Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser
Asn Ser Pro Met 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
Ile Lys 100 105 242119PRTArtificialLi113 VH Variant 242Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25
30 Pro Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser His Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp
Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys
Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp
Tyr Leu Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 243119PRTArtificialLi113 VH Variant 243Glu Val Gln Leu Leu
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro
Met Phe Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Phe Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Thr Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu
Asp Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
244119PRTArtificialLi113 VH Variant 244Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Leu Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
245119PRTArtificialLi113 VH Variant 245Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Met Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
246119PRTArtificialLi113 VH Variant 246Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Leu Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
247119PRTArtificialLi113 VH Variant 247Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Ser Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
248119PRTArtificialLi113 VH Variant 248Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Trp Ile Gly Thr Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr
Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp
Leu Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
249119PRTArtificialLi113 VH Variant 249Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Trp Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
250119PRTArtificialLi113 VH Variant 250Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Val Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
251119PRTArtificialLi113 VH Variant 251Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp His Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
252119PRTArtificialLi113 VH Variant 252Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Ser Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
253119PRTArtificialLi113 VH Variant 253Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Gln Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
254119PRTArtificialLi113 VH Variant 254Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Gly Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
255119PRTArtificialLi113 VH Variant 255Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Met Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
256119PRTArtificialLi113 VH Variant 256Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Asn Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
257119PRTArtificialLi113 VH Variant 257Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly His Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
258119PRTArtificialLi113 VH Variant 258Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Leu Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
259119PRTArtificialLi113 VH Variant 259Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Phe Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
260119PRTArtificialLi113 VH Variant 260Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Gly Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
261119PRTArtificialLi113 VH Variant 261Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ile Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
262119PRTArtificialLi113 VH Variant 262Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Asp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
263119PRTArtificialLi113 VH Variant 263Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Met Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
264119PRTArtificialLi113 VH Variant 264Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Leu Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
265119PRTArtificialLi113 VH Variant 265Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Thr Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
266119PRTArtificialLi113 VH Variant 266Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90
95 Ala Arg Glu Gly Thr Tyr Asp Ile Tyr Leu Asp Leu Trp Gly Arg Gly
100 105 110 Thr Leu Val Thr Val Ser Ser 115
267119PRTArtificialLi113 VH Variant 267Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Gly Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
268119PRTArtificialLi113 VH Variant 268Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Trp Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
269119PRTArtificialLi113 VH Variant 269Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Tyr Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
270119PRTArtificialLi113 VH Variant 270Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ser Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
271119PRTArtificialLi113 VH Variant 271Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Pro Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
272119PRTArtificialLi113 VH Variant 272Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Val Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
273119PRTArtificialLi113 VH Variant 273Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Thr Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Trp Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
274119PRTArtificialLi113 VH Variant 274Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Phe
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Trp Ile Gly Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr
Tyr Cys 85 90 95 Ala Arg Glu Gly Thr Tyr Asp Gln Tyr Leu Asp Leu
Trp Gly Arg Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
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