Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNE MEDIATORS AND RELATED METHODS
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is related to U.S. Serial No. 60/191,274, filed March 22,
2000; U.S. Serial No. 60/204,249, filed May 15, 2000; and U.S. Serial No.
60/264,003,
filed January 23, 2001. Each of the aforementioned applications are herein
incorporated
by reference in their entirety.
This application is also related to U.S. Serial No. 09/261,811, filed March
3, 1999; which is a continuation of U.S. Serial No. 08/657,581, filed June 7,
1996, now
abandoned; which is a continuation in part of U.S. Serial No. 08/480,002,
filed June 7,
1995, now abandoned; U.S. Serial No 09/184,692, filed November 2, 1998, now
abandoned; U.S. Serial No. 08/483,241, filed June 7, 1995; U.S. Serial No.
08/482,133,
filed June 7, 1995; and U.S. Provisional Application No. 60/005,964, filed
October 27,
1995.
BACKGROUND OF THE INVENTION
T cells, unlike B cells, do not directly recognize antigens. Instead, an
accessory cell must first process an antigen and present it in association
with an MHC
molecule in order to elicit a T cell-mediated immunological response. The
major function
of MHC glycoproteins appears to be the binding and presentation of processed
antigen in
the form of short antigenic peptides. '
In addition to binding foreign or "non-self antigenic peptides," MHC
molecules can also bind "self' peptides. If T lymphocytes then respond to
cells
presenting "self" or autoantigenic peptides, a condition of autoimmunity
results. Over 30
autoimmune diseases are presently known, including myasthenia gravis (MG),
multiple
sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA),
insulin-
dependent diabetes mellitus (IDDM) , etc. Characteristic of these diseases is
an attack by
the immune system on the tissues of the host. In non-diseased individuals,
such attack
does not occur because the immune system recognizes these tissues as "self."
Autoimmunity occurs when a specific adaptive immune response is mounted
against self
tissue antigens.
There is therefore currently a great interest in developing pharmaceuticals
based on the growing understanding of the structure and function of the major
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2
histocompatibility complex (MHC) antigens. Identification of synthetic
autoantigenic
peptides, and demonstration that these peptides bind selectively to MHC
molecules
associated with disease and that stimulates T cells would help to implicate a
particular
peptide or peptide:MHC complex in susceptibility to an autoimmune disease. In
particular, the development of single chain MHC class II complexes would be
particularly
useful in treatment of a number of diseases associated with antigen
presentation by MHC
molecules. Furthermore, the development of single chain, multimeric complexes
would
be of interest (see, e.g., WO 93/10220, WO 98/05684, WO 97/35991, WO 98/03552,
WO
99/13095, WO 98/06749, WO 99/09064, and U.S. Patent 5,869,270).
SUMMARY OF THE INVENTION
The present invention provides recombinant nucleic acid constructs that
encode single chain, recombinant MHC class II molecules comprising a [31
domain and
an al domain that may or may not be further linked to an antigenic peptide. In
one
embodiment, the single chain polypeptide is a /31 domain and an al domain. In
another
embodiment, the single chain polypeptide is a (31 domain-[32 domain (a (3
chain) and an
al domain- a2 domain (an a chain). The single chain constructs of the
invention can be
further dimerized or multimerized by inter-chain fusion. The fusion sequence
(also
referred to as a dimerization or multimerization sequence) can be any sequence
that
allows for covalent or non-covalent linkages between the molecules of the
invention. As
shown below, a preferred means for carrying this out is through use of
segments from
immunoglobulin family proteins (e.g., antibodies, MHC molecules, T cell
receptors and
the like) that have cysteine residues capable of forming interchain disulfide
bonds (e.g.,
constant regions from Ig light chains, e.g. Cx or C~,, or constant regions
from Ig heavy
chains, e.g., CH1, hinge, CH2, or CH3). In another embodiment, a leucine
zipper domain
forms a non-covalent linkage. One of skill will recognize that any of a number
of
polypeptide sequences can be used for this purpose. The single chain molecules
of the
invention thus can be multimers wherein each single chain molecule is from a
different
MHC class II allele. In addition, each single chain molecule in the multimer
can be
bound to a different antigen.
In one embodiment, monomeric and dimeric forms of recombinant single
chain mouse I-AS-peptide complexes, fused to an antigenic MBP 90-101 peptide
with
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3
flexible linkers were constructed. The recombinant single chain I-AS proteins
share
structural similarity to that of crystallized native human MHC class II
protein as
determined by protein modeling. The recombinant single chain proteins were
expressed
in E. coli and in an insect expression system and purified by affinity
chromatography and
FPLC. The purified single chain recombinant I-AS proteins showed ih vitro
biological
activity as assayed using an antigen-specific mouse T cell clone. The in vivo
activity of
the recombinant single chain I-AS fusion proteins in the EAE model using
susceptible
SJL mice shows that treatment with the recombinant single chain I-AS proteins
prevents
mortality and significantly reduces paralysis induced by myelin homogenate.
Histological examination of sections from animal spinal cord reveals that
these treatments
also reduce the inflammatory lesions. These results demonstrate that the
single chain
MHC class II molecules have therapeutic benefit as antigen-specific drugs for
the
treatment of autoimmune diseases.
In another embodiment, novel linkers are provided for forming single
chain MHC class II molecules. These linkers can be used with the multimer
constructs
described above. In another embodiment, the constructs of the invention are
optimized
for prokaryotic expression, using codons adjusted for E. coli codon bias.
The present invention also provides MHC class II heterodimers, wherein a
recombinant (3 chain and a recombinant a chain are covalently linked using
polypeptide
fusion segments, e.g., from immunoglobulin family proteins (e.g., antibodies,
MHC
molecules, T cell receptors and the like) that have cysteine residues capable
of forming
interchain disulfide bonds (e.g., constant regions from Ig light chains, e.g.
Cx or C7~, or
constant regions from Ig heavy chains, e.g., CH1, hinge, CH2, or CH3). Such a
heterodimer can also be "dimerized" or "multimerized" by the use of additional
fusion
domains, such as leucine zipper domains or immunoglobulin domains (see Figure
10).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Schematic structure of the recombinant single chain I-AS
.MBP.(3lal (monomer) and I-AS.MBP.(31(32a1a2.CK (dimer) proteins.
Figure 2. In vitro biological activities of the recombinant I-AS proteins
compared with APC+ antigenic peptide in the mouse T cell clone, HS-1.
A. I-AS.MBP.(31a1, monomer, B. I-AS.MBP.Ck, dimer, and C. APC+ antigenic
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4
peptide. The positive (anti-CD3) and the negative (HS-1 cell alone) controls
are also
shown in each panel.
Figure 3. Diagram of the EAE model and standard for EAE scoring.
Figure 4. The EAE model studies. The recombinant I-AS proteins were
administered to SJL mice on day 1, 4, 7, and II by i.v. injection after
inducing the disease
with myelin emulsified in CFA. The animals were evaluated for neurological
dysfunction. Panel A: Untreated, injected with same amount of PBS solution.
Panel B.
Treated with the recombinant I-AS.MBP.Ck protein, a dimer form. Panel C:
Treated
with the recombinant I-AS.(3lal. This recombinant molecule does not carry the
antigenic
peptide. Panel D: Treated with the recombinant I-AS.MBP.(31a1, a monomer form.
Figure 5 shows a schematic representation of a (il-al single chain MHC
class II peptide complex that is a dimer with two peptide specificities.
Figure 6 shows a schematic representation of a j31 J32-ala2 single chain
MHC class II peptide complex that is a dimer with two peptide specificities.
Figure 7 shows a schematic representation of a (31 /32-al a2 single chain
MHC class II peptide complex that is a tetramer with four peptide
specificities.
Figure 8 shows a schematic representation of a (31 (32-a 1 a2 single chain
MHC class II peptide complex that is a tetrarner with two peptide
specificities and two
different MHC class II alleles.
Figure 9 shows the effect of different recombinant MHC class II molecules
on the development of EAD (day 60+).
Figure 10. Diagram of [31-al single chain MHC class II peptide complex;
diagram of recombinant (3132 chains fused to recombinant ala2 chains via a
fusion
domain from an immunoglobulin; and diagram of multimerized MHC class II
molecules.
Figure 11. Sequence comparison of mouse C0608 single chain molecules.
Figure 12. Sequence comparison of mouse C0561 single chain molecules.
DETAILED DESCRIPTION OF THE INVENTION
Introduction
The present invention provides recombinant DNA constructs that encode
single chain MHC class II molecules that may or may not be further linked to
an antigenic
peptide. Typically, the constructs comprise a first DNA segment encoding a (31
domain
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of a selected MHC class II molecule; a second DNA segment encoding a al domain
of
the selected MHC class II molecule; and a first linker DNA segment connecting
in-frame
the first and second DNA segments; wherein linkage of the first DNA segment to
the
second DNA segment by the first linker DNA segment results in a fused first
DNA-first
5 linker-second DNA polysegment. The constructs of the invention may also
comprise a
third DNA segment encoding an antigenic peptide capable of associating with a
peptide
binding groove of the selected MHC class II molecule and a second linker DNA
segment
connecting in-frame the third DNA segment to the fused first DNA-first linker-
second
DNA polysegment.
In another embodiment, the present invention also provides recombinant
components of an MHC class II heterodimer, which comprise a fusion domain. One
recombinant component comprises a (31 domain, or optionally a (31 domain-(32
domain
(i.e., a (3 chain). One recombinant component comprises an al domain, or
optionally an
al domain- a2 domain (i.e., an a chain). The two recombinant chains are
linked, either
covalently, e.g., via a disulfide bond, or non covalently, using the fusion
domain. Such
molecules can also be made into multimers using additional fusion or
multimerization
domains. In one embodiment, the invention provides the following recombinant
components of an MHC class II heterodimer: pCB220, which is an IAS.MBP.alpha
chain
fused to an IgG2a CH1 and truncated hinge region; pCB223, which is an
IAS.MBP.alpha
chain fused to an IgG2a CH1.H.CH2.CH3; and pCB229, which is an IAS.MBP.beta
chain
fused to a mouse CK domain. These recombinant components can be fused via the
fusion
domain to form a MHC class II heterodimer molecule that is covalently linked
via a
disulfide bond at the fusion domain.
The present invention provides single chain MHC class II molecules that
comprise an additional polypeptide sequence that allows for inter-chain
dimerization of
the single chain molecules of the invention. The additional polypeptide allows
multimerization of the single chain MHC class II molecules, to produce, e.g.,
dimers and
tetramers. The sequence can be any sequence that allows for covalent or non-
covalent
linkages between the molecules of the invention. In one embodiment, the single
chain
molecules axe covalently linked using chemical methods known to those of skill
in the art,
e.g., photoaffinity methods or homo-bifunctional protein cross-linkers (see,
e.g.,
Hermanson et al., Bioconjugate Techniques, (1996)). In one embodiment, the
molecules
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6
are covalently linked using heterobifunctional protein cross-linkers. As shown
below,
one means for carrying this out is through use of segments form immunoglobulin
family
proteins (e.g., antibodies, MHC molecules, T cell receptors and the like) that
have
cysteine residues capable of forming interchain disulfide bonds. An example
shown
below is the use of the constant region of the kappa chain of an antibody
(CK), from either
a heavy or a light chain. Other dimerization sequences include a leucine
zipper, a STAT
protein N-terminal domain, or the FK506 binding protein (see, e.g., O'Shea,
Science 254:
539 (1991), Barahmand-Pour et al., C'urr. Top. Microbiol. Immunol. 211:121-128
(1996);
Klemm et al., Anuu. Rev. Immur~ol. 16:569-592 (1998); Ho et al., Nature
382:822-826
(1996)). One of skill will recognize that any of a number of polypeptide
sequences can
be used for this purpose.
In addition, the multimeric, single chain class II molecules of the invention
comprise at least two different MHC class II alleles that are associated with
an
autoimmune disease state, and/or at least two different autoantigenic peptides
that are
associated with a particular autoimmune disease state. In one example, the
multimeric,
single chain class II molecules have chains from different D1R2 alleles, e.g.,
DRBS*0101
and DItB 1 * 1501. In another embodiment, the autoantigenic peptides are
peptides
associated with multiple sclerosis, e.g., MBP (e.g., amino acid residues 83-
102Y83), PLP
(e.g., amino acid residues 40-60, 89-106, 95-117, and 185-206); and MOG. In
addition,
other antigens associated with autoimmune disease, such as acetylcholine
receptor and
type II collagen, can be linked to the single chain molecules of the
invention..
In a further embodiment, the single chain class II molecules of the
invention have novel linkers, as described herein. The mammalian MHC class II
single
chain constructs of the invention may also be constructed to use preferred
prokaryotic
codons, for expression, e.g., in E. coli, using codon preference tables and
methods known
to those of skill in the art.
Definitions
Prior to setting forth the invention, it may be helpful to an understanding
thereof to provide definitions of certain terms to be used hereinafter:
Single chain MHC class II molecule: As used herein this term refers to a
fusion protein such as the recombinant single chain MHC class II complex of
the
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invention, which optionally also is fused to a peptide to form a single chain
MHC
class:peptide complex. The fusion proteins of the invention can also be
multimers,
having two, four or more single chain molecules linked covalently or non-
covalently
through multimerization domains in the single chain molecule. A single chain
molecule
of the invention typically comprises at least an MHC class II (31 domain and
an MHC
class II al domain, optionally (31 (32 al a2 domains or any combination
thereof in any
order. Such molecules are also known as "fused heterodimers." Optionally, the
single
chain molecules are soluble, that is, they lack the naturally occurnng
cytoplasmic and
transmembrane MHC class II domains.
A domain of a selected MHC molecule: A portion of an MHC domain
which is sufficient to form, either alone, or in combination with another
portion of an
MHC domain, a peptide binding site which is capable of presenting an antigenic
peptide
in such a fashion that it is recognized by a T cell receptor. Such MHC domains
would
include the extracellular portion of the two polypeptide chains of Class II
MHC. This
would include the a chain (al and a2 domains) and (3 chain (~31 and (32
domains) of Class
II MHC. This would include (31 and al, (31, (32 and al, a2, a1 or a2
independent of the
other, or al and a2 in tandem (ala2). It would also include (31 or (32
independent of the
other, or (31 and (32 in tandem ((31 (32). This would also include any
suitable combination
of the al, a2, (31, and (32 domains. The domains can be directly linked, or
can be linked
via an amino acid linker.
Linker DNA segment: A segment of DNA encoding from about 1 to about
50, preferably from about 5 to about 25 amino acids, which forms a flexible
link between
two DNA segments. This flexible link allows the two DNA segments to attain a
proper
configuration, such as an MHC peptide binding groove, or allows a peptide to
properly
bind into such a groove.
Anti e~ nic ~e tn ide: The immunological properties of MHC
histocompatibility proteins are largely defined by the antigenic peptide that
is bound to
them. An antigenic peptide is one which contains an epitope (an amino acid
sequence)
recognized by immune cells, e.g., T cells, and is capable of stimulating an
MHC-mediated
immune response.. Antigenic peptides for a number of autoimmune diseases are
known.
For example, in experimentally induced autoimmune diseases, antigens involved
in
pathogenesis have been characterized: in arthritis in rat and mouse, native
type II collagen
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8
is identified in collagen-induced arthritis, and mycobacterial heat shock
protein in
adjuvant arthritis (Stuart et al., Ann. Rev. Immunol. 2:199-218, 1984; and van
Eden et al.,
Nature 331:171-173, 1988); thyroglobulin has been identified in experimental
allergic
thyroiditis (EAT) in mice (Marion et al., J. Exp. Med. 152:1115-1120, 1988);
acetyl-
choline receptor (AChR) in experimental allergic myasthenia gravis (EAMG)
(Lindstrom
et al., Adv. Immunol. 42:233-284, 1988) ; and myelin basic protein (MBP) and
proteolipid
protein (PLP) in experimental allergic encephalomyelitis (EAE) in mouse and
rat (Acha-
Orbea et al., Ann. Rev. Imm. 7:377-405, 1989). In addition, target antigens
have been
identified in humans: type II collagen in human rheumatoid arthritis
(Holoshitz et al.,
Lancet ii:305-309, 1986), acetylcholine receptor in myasthenia gravis
(Lindstrom et al.,
Adv. Immunol. 42.:233-284, 1988), and MBP, PLP, and MOG in multiple sclerosis
in
humans.
MHC: The major histocompatibility complex (NIFiC) is a family of highly
polymorphic proteins, divided into two classes, Class I and Class II, which
are
membrane-associated and present antigen to T lymphocytes (T cells). MHC Class
I and
Class II molecules are distinguished by the types of cells on which they are
expressed,
and by the subsets of T cells which recognize them. Class I MHC molecules
(e.g., HLA-
A, -B and -C molecules in the human system) are expressed on alinost all
nucleated cells
and are recognized by cytotoxic T lymphocytes (CTL), which then destroy the
antigen-
bearing cells. Class II MHC molecules (HLA-DP, -DQ and -DR, for example, in
humans) are expressed primarily on the surface of antigen-presenting cells,
such as B
lymphocytes, dendritic cells, macrophages, and the like. Class II MHC is
recognized by
CD4+ T helper lymphocytes (TH). TH cells induce proliferation of both B and T
lymphocytes, thus amplifying the immune response to the particular antigenic
peptide that
is displayed (Takahashi, Microbiol. Immunol., 37:1-9, 1993).
Two distinct antigen processing pathways are associated with the two
MHC classes. Intracellular antigens, synthesized inside of the cell, such as
from viral or
newly synthesized cellular proteins, for example, are processed and presented
by Class I
MHC. Exogenous antigens, taken up by the antigen-presenting cell (APC) from
outside
of the cell through endocytosis, are processed and presented by Class II MHC.
After the
antigenic material is proteolytically processed by the MHC-bearing cell, the
resulting
antigenic peptide forms a complex with the antigen binding groove of the MHC
molecule
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through various noncovalent associations. The MHC-peptide complex on the cell
surface
is recognized by a specific T cell receptor on a cytotoxic or helper T cell.
The MHC of humans (also referred to as human leukocyte antigens
(HLA)) on chromosome 6 has three loci, HLA-A, HLA-B and HLA-C, the first two
of
which have a large number of alleles encoding alloantigens. An adjacent
region, known
as HLA-D, is subdivided into HLA-DR, HLA-DQ and HLA-DP. The HLA region is now
known as the human MHC region, and is equivalent to the H-2 region in mice.
HLA-A, -
B and -C resemble mouse H-2K, -D, and -L and are the Class I MHC molecules.
HLA-
DP, -DQ and -DR resemble mouse I-A and I-E and are the Class II molecules. MHC
glycoproteins of both classes have been isolated and characterized (see
Fundamental
Immunology, 2d Ed., Paul (ed.), (1989); and Roitt et al., Immunology, 2d Ed.,
(1989) ,
which are both incorporated herein by reference).
Human MHC Class I molecules consist of a polymorphic type I integral
membrane glycoprotein heavy chain of about 46 kD, noncovalently associated
with a
12 kD soluble subunit, (32-microglobulin. The heavy chain consists of two
distinct
extracellular regions, the membrane distal, peptide binding region formed by
the al and
a2 domains, and the membrane proximal, CD8-binding region derived from the a3
domain. (32- microglobulin is a single, compact immunoglobulin-like domain
that lacks a
membrane anchor, and exists either associated with the class I heavy chain or
free in
plasma (Germain and Margulies, Annu. Rev. Immunol. 11:403-50, 1993).
Human MHC Class II is a heterodimeric integral membrane protein. Each
dimer consists of one a and one [3 chain in noncovalent association: The two
chains are
similar to each other, with the a chain having a molecular weight of 32-34 kD
and the
(3 chain having a molecular weight of 29-32 kD. Both polypeptide chains
contain N-
linked oligosaccharide groups and have extracellular amino termini and
intracellular
carboxy termini.
The extracellular portions of the a and (3 chain that comprise the class II
molecule have been subdivided into two domains of about 90 amino acids each,
called
al, a2, and ail, (32, respectively. The a2 and /32 domains each contain a
disulfide-linked
loop. The peptide-binding region of the class II molecule is formed by the
interaction of
the al and al domains. This interaction results in an open-ended, antigenic
peptide-
binding groove made up of two a helices, and an eight-stranded (3-pleated
sheet platform.
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The a and (3 chains of Class II molecules are encoded by different MHC
genes and are polymorphic (see Addas et al., Cellular and Molecular
Immunology, 2d Ed.
(1994), which is incorporated by reference in its entirety). Within the
present invention, a
preferred a chain is DRA*0101 and a preferred (3 chain is DR(31* 1501.
5
MHC Class II alleles
The single chain MHC class II:peptide complexes of the present invention
can incorporate cDNA from any allele that predisposes or increases the
likelihood of
susceptibility to a specific autoimmune disease. Specific autoimmune diseases
are
10 correlated with specific MHC types. Specific haplotypes have been
associated with many
of the autoimmune diseases. For example, HLA-DR2+ and HLA-DR3+ individuals are
at
a higher risk than the general population to develop systemic lupus
erythematosus (SLE)
(Reinertsen et al., N. Engl. J. Med. 299:515-18, 1970). Myasthenia gravis has
been
linked to HLA-D (Safwenberg et al., Tissue Antigens 12:136-42,1978.
Susceptibility to
rheumatoid arthritis is associated with HLA-D/DR in humans. Methods for
identifying
which alleles, and subsequently which MHC-encoded polypeptides, are associated
with
an autoimmune disease are known in the art. Exemplary alleles for H~DM include
DR4,
DQB, DR3, DQ3.2.
Uses of single chain MHC class II molecules
Single chain MHC class II molecules and/or single chain MHC class
II:peptide complexes of the present invention can be used as antagonists to
therapeutically
block the binding of particular T cells and antigen-presenting cells. In
addition, the
molecules can induce anergy, or proliferative nonreponsiveness, and possibly
apoptosis,
in targeted T cells, both in vivo and in vitro. A single chain MHC class
Il:peptide
molecule directed toward a desired autoimmune disease contains the antigenic
peptide
implicated for that autoimmune disease properly positioned in the binding
groove of the
MHC molecule, without need for solublization of MHC or exogenous loading of an
independently manufactured peptide.
Previous methods for producing desirable MHC Class II histocompatibility
proteins have provided material that contains a mixture of antigenic peptides
(Buus et al.,
Science 242:1045-1047, 1988; and Rudensky et al., Nature 353:622-627, 1991),
which
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11
can be only partially loaded with a defined antigenic peptide (Watts and
McConnel, Pro.
Natl. Acad. Sci. USA 83:9660-64, 1986; and Ceppellini et al., Nature 339:392-
94, 1989).
Various methods have been developed to produce heterodimers that do not
present
endogenous antigens (Stern and Wiley, Cell 68:465-77, 1992; Ljunggren et al.,
Nature
346:476-80, 1990; and Schumacher et al., Cell 62:563-67, 1990) that can be
loaded with a
peptide of choice. WO 95123814 and Kozono et al. have described production of
soluble
marine Class II molecules, I-Eak and I-Aa, each with a peptide attached by a
linker to the
N terminus of the (3 chain. Ignatowicz et al. (J. Immunol. 154:38-62, 1995)
have
expressed membrane-bound I-Ad with peptide attached. These methods incorporate
the
use of both membrane-bound heterodimer and soluble heterodimer.
The current invention offers the advantage of a recombinant single chain
MHC class II molecule made up of two or more MHC domains joined together via a
flexible linkage, and onto which is tethered (via an additional flexible
linkage) an
antigenic peptide which is able to bind to the peptide binding groove
presented by the
single chain MHC class II molecule. Such a complex provides an MHC molecule
which
is soluble and, because the MHC class II components and corresponding
antigenic
peptide are permanently linked into a single chain configuration, there is no
need for
complex heterodimer truncation or formation. These complexes eliminate
inefficient and
nonspecific peptide loading. Producing the claimed MHC:peptide complexes by
recombinant methodology results in specific, high yield protein production,
where the
final product contains only the properly configured MHC:peptide complex of
choice.
As used herein, a soluble MHC class II molecules is one that does not
contain the naturally occurring membrane-associated MHC class II sequences.
The
soluble MHC molecules of the present invention has never been membrane-
associated.
Further, the soluble MHC class II molecules do not contain an amino acid
sequence that
acts as a transmerribrane domain or as a cytoplasmic domain.
The present invention therefore provides a single chain MHC class II
molecule which optionally includes an antigenic peptide covalently attached to
the amino
terminal portion of an a or (3 chain of MHC through a peptide linkage, and the
C terminal
of the linked a or j3 chain may be attached to the N terminal portion of
another a or (3
chain, there by creating a two, three, or four domain MHC molecule. The
invention
further provides a multimerization domain to provide a multimeric single chain
MHC
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12
class II molecules. The invention further provides novel linkers, and
multimeric MHC
class II molecules that are bound to different antigenic peptides.
The amino acid sequence of each of a number of Class I and Class II
proteins are known, and the genes or cDNAs have been cloned. Thus, these
nucleic acids
can be used to express MHC polypeptides. If a desired MHC gene or cDNA is not
available, cloning methods known to those skilled in the art may be used to
isolate the
genes. One such method that can be used is to purify the desired MHC
polypeptide,
obtain a partial amino acid sequence, synthesize a nucleotide probe based on
the amino
acid sequence, and use the probe to identify clones that harbor the desired
gene from a
cDNA or genomic library.
Linkers
Linkers of the current invention may be from about 1 to about 50 amino
acids in length, depending on the molecular model of the MHC or MI-IC:peptide
complex.
In one embodiment, flexible linkers are made of repeating Gly residues
separated by one
or more Ser residues to permit a random, flexible motion. In the case of Class
II MHC
complexes this flexibility accommodates positioning of the a and (3 segments
to properly
configure the binding groove, and also allows for maximum positioning of the
peptide in
the groove. In another embodiment, the linker comprises a CD4 binding site, as
described below in the Example section (see also Table 1). In another
embodiment,
longer linkers between the chains contain flexible residues (e.g. alanine or
glycine) and
polar residues (e.g. serine and threonine). To inhibit the continuation of
secondary
structure across the linker, prolines can be added to bracket the linkers.
These prolines
are known to inhibit the formation of alpha helices and beta sheets. In
another
embodiment, flexible regions present in the human MHC and in the marine MHC
could
be used to make a linker by extending the region of interest and ligating the
ends together.
Finally, a combination of these types of linkers could also be used.
Linker position and length can be modeled based on the crystal structure of
MHC Class II molecules (Brown et al., Nature 364:33-39, 1993), where al and
(31 are
assembled to form the peptide binding groove. Linkers joining segments of the
a and (3
chains together are based on the geometry of the region in the hypothetical
binding site
and the distance between the C terminus and the N terminus of the relevant
segments.
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Molecular modeling based on the X-ray crystal structure of Class II MHC (Stern
et al.,
NatuYe 368:215-221, 1994) dictates the length of linkers joining antigenic
peptide, a chain
segments and (3 chain segments. The recombinant portions of the molecules of
the
invention can also be directly linked, without additional amino acids linkers.
Identification of autoantigens
The invention also provides methods for preparing responder T-cell clones
that proliferate when combined with a selected antigenic peptide presented by
a
stimulator cell. Such clones can be used to identify and map antigenic
peptides
associated with autoimmune disease. These peptides can then be incorporated
into the
single chain MHC class II molecule:peptide complexes of the invention. The
method
provides isolation and enrichment of non-adherent, CD56-, CD8- T cells that
are reactive
with a selected antigenic peptide. These cells are herein referred to as
responder cells.
Suitable responder cells can be isolated, for example, from peripheral blood
mononuclear
cells (PBMNC) obtained from patients prior to or after onset of an autoimmune
disease of
interest. For example, PBMNCs can be obtained from prediabetic and new onset
diabetic
patients. These patients can be pre-screened for specific HLA markers, such as
DR3-
DR4 or DQ3.2, which have the highest association with susceptibility to IDDM.
From
the collected PBMNCs, a portion is kept to serve as stimulator cells. From the
remainder,
the desired autoreactive responder cells are purified and isolated by two
rounds of plating,
to remove adherent cells from the population, followed by removal of monocytes
and B
cells with nylon wool. Enrichment for non-adherent CD4~ T cells is completed
by
sequential plating of the cells onto plates coated with anti-CD8 and anti-CDS6
antibodies.
The stimulator cells are pulsed or primed with whole GAD or an
appropriate antigenic peptide. For example, stimulator cells from the PBMNCs
of 1DDM
patients can be stimulated with antigenic GAD peptides then combined with
PBMNCs or
responder cells. After seven or 14 days, responder cell (T cell) clones are
generated
through limiting dilution and tested for antigen reactivity.
These responder cell (T cell) clones can then be used, for example, to map
epitopes which bind to MHC and are recognized by a particular T cell. One such
method
uses overlapping peptide fragments of the autoantigen which are generated by
tryptic
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14
digestion, or more preferably, overlapping peptides are synthesized using
known peptide
synthesis techniques. The peptide fragments are then tested for their ability
to stimulate
the responder T cell clones or lines (for example, Ota et al., Nature 346:183-
187, 1990).
Once such a peptide fragment has been identified, synthetic antigenic
peptides can be specifically designed, for example, to enhance the binding
affinity for
MHC and to out-compete any naturally processed peptides. Such synthetic
peptides,
when combined into a single chain MHC class II molecule:peptide complex, would
allow
manipulation of the immune system in vivo, in order to tolerize or anergize
disease-
associated activated T cells, thereby ameliorating the autoimmune disease.
Dissecting the functional role of individual peptides and peptide clusters in
the interaction of a peptide ligand with an MHC molecule, and also in
subsequent T cell
recognition and reactivity, is a difficult undertaking due to the degeneracy
of peptide
binding to the MHC. Changes in T cell recognition or in the ability of an
altered peptide
to associate with MHC can be used to establish that a particular amino acid or
group of
amino acids comprises part of an MHC or T cell determinant. The interactions
of altered
peptides can be further assessed by competition with the parental peptide for
presentation
to a T cell, or through development of direct peptide-MHC binding assays.
Changes to a
peptide that do not involve MHC binding could well affect T cell recognition.
For
example, in a peptide, specific MHC contact points might only occur within a
central core
of a few consecutive or individual amino acids, whereas those amino acids
involved in
T cell recognition may include a completely different subset of residues.
In a preferred method, residues that alter T cell recognition are determined
by substituting amino acids for each position in the peptide in question, and
by assessing
whether such change in residues alters the peptide's ability to associate with
MHC (Allen
et al., Nature 327:713-15, 1987; Sette et al., Nature 328:395-99, 1987;
O'Sullivan et al.,
J. Immunol. 147:2663-69, 1991; Evavold et al., J. Immunol. 148:347-53, 1992;
Jorgensen
et al., Anrzu. Rev. Immunol. 10:835-73, 1992; Hammer et al., Cell 74:197-203,
1993;
Evavold et al., Immunol. Today 14:602-9, 1993; Hammer et al., Proc. Natl.
Acad. Sci.
USA 91:4456-60, 1994; and Reich et al., J. Immunol. 154:2279-88, 1994). One
method
would involve generating a panel of altered peptides wherein individual or
groups of
amino acid residues are substituted with conservative, semi-conservative or
non
conservative residues. A preferred variant of this method is an alanine scan
(Ala scan)
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where a series of synthetic peptides are synthesized wherein each individual
amino acid is
substituted with L-alanine (L-Ala scan). Alanine is the amino acid of choice
because it is
found in all positions (buried and exposed), in secondary structure, it does
not impose
steric hindrances, or add additional hydrogen bonds or hydrophobic side
chains. Alanine
substitutions can be done independently or in clusters depending on the
information
desired. Where the information pertains to specific residues involved in
binding, each
residue in the peptide under investigation can be converted to alanine and the
binding
affinity compared to the unsubstituted peptide. Additional structural and
conformational
information regarding each residue and the peptide as a whole can be gained,
for
10 example, by synthesizing a series of analogs wherein each residue is
substituted with a D-
amino acid such as D-alanine (D-Ala scan) (Galantino et al., in Smith, J. and
Rivier, J.
(eds.), Peptides Chemistry and Biology (Proceedings of the Twelfth American
Peptide
Symposium), ESCOM, Leiden, 1992, pp. 404-OS). Essential residues can be
identified,
and nonessential residues targeted for modification, deletion or replacement
by other
15 residues that may enhance a desired quality (Cunningham and Wells, Scienee
244:1081-
1085, 1989; Cunningham and WeIIs, Natl. Acad. Sci. USA, 88:3407-3411, 1991;
Ehrlich
et al., J. Biol. Chem. 267:11606-11, 1992; Zhang et al., Proc. Natl. Acad.
Sci. USA
90:4446-50, 1993; see also "Molecular Design and Modeling: Concepts and
Applications
Part A Proteins, Peptides, and Enzymes," Methods in Enzymology, Vol. 202,
Langone
(ed.), Academic Press, San Diego, CA, 1991).
Truncated peptides can be generated from the altered or unaltered peptides
by synthesizing peptides wherein amino acid residues are truncated from the N-
or C-
terminus to determine the shortest active peptide, or between the N- and C-
terminus to
determine the shortest active sequence. Such peptides could be specifically
developed to
stimulate a response when joined to a particular MHC to form a peptide ligand
to induce
anergy in appropriate T cells in vivo or in vitro.
Analysis of single chain MHC class II molecule:peptide complexes
The physical and biological properties of the single chain MHC class II
molecule:peptide complexes may be assessed in a number of ways. Mass spectral
analysis methods such as electrospray and Matrix-Assisted Laser
Desorption/Ionization
Time Of Flight mass spectrometry (MALDI TOF) analysis are routinely used in
the art to
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16
provide such information as molecular weight and confirm disulfide bond
formation.
FACs analysis can be used to determine proper folding of the single chain
complex.
An ELISA (Enzyme-linked Immunosorbent Assay) can be used to
measure concentration and confirm correct folding of the single chain MHC
class II
molecule:peptide complexes. This assay can be used with either whole cells;
solubilized
MHC, removed from the cell surface; or free single chain MHC class II
molecule:peptide
complexes of the current invention. In an exemplary ELISA, an antibody that
detects the
recombinant MHC haplotype is coated onto wells of a microtiter plate. In a
preferred
embodiment, the antibody is L243, a monoclonal antibody that recognizes only
correctly
folded HLA-DR MHC dimers. One of skill in the art will recognize that other
MHC
Class IT-specific antibodies are known and available. Alternatively, there are
numerous
routine techniques and methodologies in the field for producing antibodies
(for example,
Hurrell, (ed)., Monoclonal Hybridoma Antibodies: Techniques and Applications,
CRC
Press Inc., Boca Raton, FL, 1982), if an appropriate antibody for a particular
haplotype
does not exist. Anti-MHC Class II antibodies can also be used to purify Class
II
molecules through techniques such as affinity chromatography, or as a marker
reagent to
detect the presence of Class II molecules on cells or in solution. Such
antibodies are also
useful for Western analysis or immunoblotting, particularly of purified cell-
secreted
material. Polyclonal, affinity purified polyclonal, monoclonal and single
chain antibodies
are suitable for use in this regard. In addition, proteolytic and recombinant
fragments and
epitope binding domains can be used herein. Chimeric, humanized, veneered, CDR-
replaced, reshaped or other recombinant whole or partial antibodies are also
suitable.
In the ELISA format, bound MHC molecules can be detected using an
antibody or other binding moiety capable of binding MHC molecules. This
binding
moiety or antibody may be tagged with a detectable label, or may be detected
using a
detectably labeled secondary antibody or binding reagent. Detectable labels or
tags are
known in the art, and include fluorescent, colorimetric and radiolabels, for
instance.
Other assay strategies can incorporate specific T-cell receptors to screen
for their corresponding MHC-peptide complexes, which can be done either in
vitro or in
vivo. For example, an in vitro anergy assay determines if non-responsiveness
has been
induced in the T cells being tested. Briefly, an MHC molecule containing
antigenic
peptide in the peptide binding groove can be mixed with responder cells,
preferably
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17
peripheral blood mononuclear cells (PBMI~ (a heterogeneous population
including B and
T lymphocytes, monocytes and dendritic cells), PBMNC lymphocytes, freshly
isolated T
lymphocytes, in vivo primed splenocytes, cultured T cells, or established T
cell lines or
clones. Responder cells from mammals immunized with, or having a demonstrable
cellular immune response to, the antigenic peptide are particularly preferred.
Subsequently, these responder cells are combined with stimulator cells
(antigen presenting cells; APCs) that have been pulsed or primed with the same
antigenic
peptide. In a preferred embodiment, the stimulator cells are antigenic peptide-
presenting
cells, such as PBMNCs, PBMNCs that have been depleted of lymphocytes,
appropriate
antigenic peptide-presenting cell lines or clones (such as EBV-transformed B
cells), EBV
transformed autologous and non-autologous PMNCs, genetically engineered
antigen
presenting cells, such as mouse L cells or bare lymphocyte cells BLS-1, in
particular,
DRBI*0401, DRBI*0404 and DRBI*0301 (Kovats et al., J. Exp. Med. 179:2017-22,
1994), or in vivo or in vitro primed or pulsed splenocytes. Stimulator cells
from
mammals immunized with, or having a demonstrable cellular immune response to,
the
antigenic peptide are particularly preferred. For certain assay formats, it is
preferred to
inhibit the proliferation of stimulator cells prior to mixing with responder
cells. This
inhibition may be achieved by exposure to gamma irradiation or to an anti-
mitotic agent,
such as mitomycin C, for instance. Appropriate negative controls are also
included
(nothing; syngeneic APC; experimental peptide; APC + Peptide; MHC:peptide
complex;
control peptide +/- APC). Further, to assure that non-responsiveness
represents anergy,
the proliferation assay may be set up~in duplicate, +/- recombinant IL-2 since
it has been
demonstrated that IL-2, can rescue anergized cells.
After an approximately 72 hour incubation, the activation of responder
cells in response to the stimulator cells is measured. In a preferred
embodiment,
responder cell activation is determined by measuring proliferation using 3H-
thymidine
uptake (Crowley et al., J. Immunol. Meth. 133:55-66, 1990). Alternatively,
responder cell
activation can be measured by the production of cytokines, such as IL-2, or by
determining the presence of responder cell-specific, and particularly T cell-
specific,
activation markers. Cytokine production can be assayed by testing the ability
of the
stimulator + responder cell culture supernatant to stimulate growth of
cytokine-dependent
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18
cells. Responder cell- or T cell-specific activation markers may be detected
using
antibodies specific for such markers.
Preferably, the single chain MHC class II molecule:peptide complex
induces non-responsiveness (for example, anergy) in the antigenic peptide-
reactive
responder cells. In addition to single chain MHC class II molecule:peptide
complex
recognition, responder cell activation requires the involvement of co-
receptors on the
stimulator cell (the APC) that have been stimulated with co-stimulatory
molecules. By
blocking or eliminating stimulation of such co-receptors (for instance, by
exposing
responder cells to purified single chain MHC class II molecule:peptide
complex, by
blocking with anti-receptor or anti-ligand antibodies, or by "knocking out"
the genes)
encoding such receptors), responder cells can be rendered non-responsive to
antigen or to
single chain MHC class II molecule:peptide complex.
In a preferred embodiment, responder cells are obtained from a source
manifesting an autoimmune disease or syndrome. Alternatively, autoantigen-
reactive T
cell clones or lines are preferred responder cells. In another preferred
embodiment,
stimulator cells are obtained from a source manifesting an autoimmune disease
or
syndrome. Alternatively, APC cell lines or clones that are able to
appropriately process
and/or present autoantigen to responder cells are preferred stimulator cells.
In a
particularly preferred embodiment, responder and stimulator cells are obtained
from a
source with diabetes or multiple sclerosis.
At this point, the responder T cells can be selectively amplified and/or
stimulated, thereby producing a subset of T cells that are specific for the
antigenic
peptide. For instance, antigenic peptide-reactive responder cells may be
selected by flow
cytometry, and particularly by fluorescence activated cell sorting. This
subset of
responder cells can be maintained by repetitive stimulation with APCs
presenting the
same antigenic peptide. Alternatively, responder cell clones or lines can be
established
from this responder cell subset. Further, this subset of responder cells can
be used to map
epitopes of the antigenic peptide and the protein from which it is derived.
Other methods to assess the biological activity of the single chain MHC
class II molecule:peptide complexes are known in the art and can be used
herein, such as
using a microphysiometer, to measure production of acidic metabolites in T
cells
following interaction with antigenic peptide. Other assay methods include
competition
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19
assays, comparing single chain MHC class II molecule:complex response with
that to the
normal antigen. Also measurement production of such indicators as cytokines or
y
interferon can provide an indication of complex response.
Animal models of autoimmune disease
Similar assays and methods can be developed for and used in animal
models. For instance, the therapeutic effect of a pharmaceutical composition
of the
single chain molecule or multimer or a polynucleotide encoding the single
chain molecule
or multimer can be tested in vivo in a number of animal models of HLA-DR-
associated
autoimmune disease. These diseases include, but are not limited to, insulin-
dependent
diabetes mellitus, multiple sclerosis, myasthenia gravis, pernicious anemia,
rheumatoid
arthritis, and systemic lupus erythematosus.
For example, NOD mice are a spontaneous model of IDDM. Treatment
with the pharmaceutical compositions prior to or after onset of disease can be
monitored
by assay of urine glucose levels in the NOD mouse, as well as by in vitro T
cell
proliferation assays to assess reactivity to known autoantigens (see, e.g.,
I~aufinan et al.,
Nature 366:69-72 (1993)) for example) . Alternatively, induced models of
autoimmune
disease, such as EAE, can be treated with pharmaceutical composition.
Treatment in a
preventive or intervention mode can be followed by monitoring the clinical
symptoms of
EAE.
Following is a description of several other animal models of HLA-DR-
associated autoimmune disease which can be used to assay in vivo effects of
the peptide.
It will be obvious to one of skill in the art that other suitable animal
models for
autoimmune diseases can be utilized in a similar manner.
Systemic Lunus Er~thematosus fSLEI
F1 hybrids of autoimmune New Zealand black (NZB) mice and the
phenotypically normal New Zealand White (NZV~ mouse strain develop severe
systemic
autoimmune disease, more fulminant than that found in the parental NZB strain.
These
mice manifest several immune abnormalities, including antibodies to nuclear
antigens and
subsequent development of a fatal, immune complex-mediated glomerulonephritis
with
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female predominance, remarkably similar to SLE in humans (Knight et al., J.
Exp. Med.
147:1653 (1978)), which is incorporated hereby by reference.
In both the human and marine forms of the disease, a strong association
with MHC gene products has been reported. HLA-DR2 and HLA-DR3 individuals are
at
5 a higher risk than the general population to develop SLE (Reinertsen et al.,
N. Engl. J.
Med. 299:515 (1970)), while in NZB/W F1 mice (H-2~°), a gene linked to
the h-2°
haplotype .derived from the NZW parent contributes to the development of the
lupus-like
nephritis.
The effect of the invention can be measured by survival rates and by the
10 progress of development of the symptoms, such as protenuria and appearance
of anti-
DNA antibodies.
Proteinuria can be measured by any method known to those of skill in the
art, e.g. colorimetrically by the use of Uristix (Miles Laboratories, Inc.,
Elkhart, III,
giving an approximation of proteinuria as follows: trace, 10 mg/dl; 1+, 30
mg/dl; 100
15 mg/dl; 3+, 300 mg/dl; and 4+, 1000 mg/dl. .
The presence of anti-DNA specific antibodies in NZB/W Fl mice can be
determined by using a modification of a linked immunosorbent assay (ELISA)
described
by Zouali et al., J. Immunol. Methods 90:105 (1986)) which is incorporated
herein by
reference.
Myasthenia Gravis (MGl
Myasthenia gravis is one of several human autoimmune diseases linked to
HLA-D (Safenberg, et al., Tissue Antigens 12:136 (1978); McDevitt et al.,
Arth. Rheum.
20:59 (1977)) which are incorporated herein by reference. In MG antibodies to
the acetyl
choline receptors (AcChoR) impair neuromuscular transmission by mediating loss
of
AcChoR in the postsynaptic membrane.
SJLIJ female mice are a model system for human MG. In these animals,
experimental autoimmune myasthenia gravis (EAMG) can be induced by immunizing
the
mice with soluble AcChoR protein from another species. Susceptibility to EAMG
is
linked in part to the MHC and has been mapped to the region within H-2
(Christadoss et
al., .I. Immunol. 123:2540 (1979)).
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21
AcChoR protein can purified from Torpedo californica and assayed
according to the method of Waldor et al., Proc. Natl. Acad. Sci. USA 80:2713
(1983),
incorporated by reference. For example, emulsified AcChoR, 15 ~,g incomplete
Freund
adjuvant, is injected intradermally among six sites on the back, the hind foot
pads, and the
base of the tail. Animals are reimmunized with this same regimen 4 weeks
later.
Evaluation can be made by measurement of anti-AcChoR antibodies by
any method known to those of skill in the art, e.g., a microtiter ELISA assay
as described
in Waldor et al., supra. In an exemplary assay, the standard reagent volume is
50 p,1 per
well. Reagents are usually incubated in the wells for 2 hr at RT. Five ~,g of
AcChoR
diluted in bicarbonate buffer, pH 9.6, is added to each well. After incubation
with
AcChoR, the plates are rinsed four times with a wash solution consisting of
phosphate-
buffer saline containing 0.05% Tween and 0.05% NaN3. Mouse sera are diluted in
O.O1M
PBS (pH 7.2), 1.5 mfr MgCl2, 2.0 mM 2-mercaptoethanol, 0.05% Tween-80, 0.05%
NaN3 (p-Tween buffer) and incubated on the plate. After the plate is washed,
beta-
galactosidase-conjugated sheep anti-mouse antibody diluted in P-Tween buffer
is added
to each well. After a final washing, the enzyme substrate, p-
nitrophenylgalctopyranoside
is added to the plate, and the degree of substrate catalysis is determined
from the
absorbance at 405 nm after 1 hr.
Anti-AcChoR antibodies are expected to be present in the mice immunized
with AcChoR as compared to nonimmunized mice. Treatment with complex is
expected
to significantly reduce the titer of anti-AcChoR antibodies in the immunized
mice.
The effect of treatment with the invention on clinical EAMG can also be
assessed by any method known to those of skill in the art. Myasthenia symptoms
include
a characteristic hunched posture with drooping of the head and neck,
exaggerated arching
of the back, splayed limbs, abnormal walking, and difficulty in righting. Mild
symptoms
are present after a standard stress test, and should be ameliorated by
administration of
complex.
Rheumatoid Arthritis (RAl
In humans, susceptibility to rheumatoid arthritis is associated with HLA
D/DR. The immune response in mice to native type II collagen has been used to
establish
an experimental model for arthritis with a number of histological and
pathological
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22
features resembling human RA. Susceptibility to collagen-induced arthritis
(CIA) in mice
has been mapped to the H-2 I region, particularly the I-A subregion (Ruse et
al., Fed.
Proc. 43:1820 (1984)).
Mice from a susceptible strain, DEA-1 can be caused to have CIA by
treatment of the mice with native type II collagen, using the technique
described in
Wooley et al., J. Immunol. 134:2366 (1985), incorporated herein by reference.
In another model adjuvant arthritis in rats is an experimental model for
human arthritis, and a prototype of autoimmune arthritis triggered by
bacterial antigens
(Holoschitz et al., Prospects of Immunology (1986); Pearson, Arthritis Rheum.
7:80
(1964)). The disease is the result of a cell-mediated immune response, as
evidenced by its
transmissibility by a clone of T cells which were reactive against the
adjuvant (MT); the
target self antigen in the disease, based upon studies with the same cloned
cells, appears
to be parts) of a proteoglycan molecule of cartilage.
Adjuvant disease in rats is produced as described by Pearson supra, i.e., by
a single injection of Freund's adjuvant (killed tubercle bacilli or chemical
fractions of it,
mineral oil, and an emulsifying agent) given into several depot sites,
preferably
intracutaneously or into a paw or the base of the tail. The adjuvant is given
in the absence
of other antigens.
The effect of the invention treatment on manifestations of the disease can
be monitored by any method known to those of skill in the art. These
manifestations are
histopathological, and include an acute and subacute synovitis with
proliferation of
synovial lining cells, predominantly a mononuclear infiltration of the
articulax and
particular tissues, the invasion of bone and articular cartilage by connective
tissue pannus,
and periosteal new bone formation, especially adjacent to affected joints. In
severe or
chronic cases, destructive changes occur, as do fibrous or bony ankylosis.
These
histopathological symptoms are expected to appear in control animals at about
12 days
after sensitization to the Freund's adjuvant.
Insulin Dependent Diabetes Mellitus (IDDM)
IDDM is observed as a consequence of the selective destruction of insulin-
secreting cells within the Islets of Langerhans of the pancreas. Involvement
of the
immune system in this disease is suggested by morphologic evidence of early
infiltration
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23
of the Islets by mononuclear cells, by the detection of anti-islet cell
antibodies, by the
high frequency of HLA-DR3 and -DR4 alleles in IDDM populations, and by
clinical
associations between IDDM and various autoimmune diseases. An animal model for
spontaneous IDDM and thyroiditis has been developed in the BB rat. As in
humans, the
rat disease is controlled in part by the genes encoding the MHC antigens, is
characterized
by islet infiltration, and is associated with the presence of anti-islet
antibodies. The I-E
equivalent class II MHC antigens appear to be involved in manifestation of the
autoimmune diseases in the BB rat. Biotard et al., Proc. Natl. Acad. Sci. USA
82:6627
(1985).
In morphologic evaluation, insulitis is characterized by the presence of
mononuclear inflammatory cells~within the islets. Thyroiditis is characterized
by focal
interstitial lymphocytic infiltrate within the thyroid gland, as a minimum
criterion. Most
severe cases show diffuse extensive lymphocytic infiltrates, disruption of
acini, fibrosis,
and focal Hurthle call change. See Biotard et al. supra.
Treatment of the BB rats with the invention is expected to ameliorate or
prevent the manifestation of the clinical and morphological symptoms
associated with
IDDM and thyroiditis.
In another model, the NOD mouse strain (H-2Kd Db) is a marine model for
autoimmune IDDM. The disease in these animals is characterized by anti-islet
cell
antibodies, severe insulitis, and evidence for autoimmune destruction of the
beta-cells
(Kanazawa, et al., Diabetolooia 27:113 (1984)). The disease can be passively
transferred
with lymphocytes and prevented by treatment with cyclosporin-A (Ikehara et
al., Proc.
Natl. Acad. Sci. USA 82:7743 (1985)); Mori et al., Diabetolooia 29:244 (1986).
Untreated animals develop profound glucose intolerance and ketosis and succumb
within
weeks of the onset of the disease. Seventy to ninety percent of female and 20-
30% of
male animals develop diabetes within the first six months of life. Breeding
studies have
defined at least two genetic loci responsible for disease susceptibility, one
of which maps
to the MHC. Characterization of NOD Class II antigens at both the serologic
and
molecular level suggest that the susceptibility to autoimmune disease is
linked to I-AB
(Acha-Orbea and McDevitt, Proc. Natl. Acad. Sci. USA 84:235 (1907)).
Treatment of Female NOD mice with complex is expected to lengthen the
time before the onset of diabetes and/or to ameliorate or prevent the disease.
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24
Experimental Allergic Encephalom elf itis fEAEI
Experimental allergic encephalomyelitis (EAE) is an induced autoimmune
disease of the central nervous system which is a model for multiple sclerosis
(MS). The
disease can be induced in many species, including mice and rats.
The disease is characterized by the acute onset of paralysis. Perivascular
infiltration by mononuclear cells in the CNS is observed in both mice and
rats. Methods
of inducing the disease, as well as symptomology, are reviewed in Aranson, The
Autoimmune Diseases (Rose and Mackay, eds., 1985), and in Acha-Orbea et al.,
Ann.
Rev. Imm. 7:377-405 (1989).
One of the genes mediating susceptibility is localized in the MHC class II
region (Moore et al., J. Immunol. 124:1815-1820 (1980)). The best analyzed
encephalitogenic protein is myelin basic protein (MBP), but other
encephalitogenic
antigens are found in the brain. The immunogenic epitopes have been mapped
(see, Acha-
Orbea et al., supra.). In the PL mouse strains (H-2°) two
encephalitogenic peptides in
MBP have been characterized: MBP peptide p35-47 (MBP 35-47), and acetylated
NSF
p1-9 (MBP 1-9).
The effect of the invention on ameliorating disease symptoms in
individuals in which EAE has been induced can be measured by survival rates,
and by the
progress of the development of symptoms.
Methods of making the complexes of the invention
Expression systems suitable for production of appropriate recombinant
single chain MHC class II molecule:peptide complexes are available and known
in the
art. Various prokaryotic, fungal, and eukaryotic host cells are suitable for
expression of
recombinatn, single chain MHC class II molecule:peptide complexes, as well as
for
individual recombinant alpha and beta MHC class II chains.
Prokaryotes that are useful as host cells, according to the present
invention, most frequently are represented by various strains of Escherichia
coli.
However, other microbial strains can also be used, such as bacilli, for
example Bacillus
subtilis, various species of Pseudomonas, or other bacterial strains.
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According to the invention, the single chain MHC class II
molecule:peptide complexes are expressed from recombinantly engineered
nucleotide
sequences that encode the single chain MHC class II molecule:peptide
polypeptides by
operably linking the engineered nucleic acid coding sequence to signals that
direct gene
expression in prokaryotes. A nucleic acid is "operably linked" when it is
placed into a
functional relationship with another nucleic acid sequence. For instance, a
promoter or
enhancer is operably linked to a coding sequence if it effects the
transcription of the
sequence. Generally, operably linked means that the nucleic acid sequences
being linked
are contiguous and, where necessary to join two protein coding regions,
contiguous and in
10 reading frame.
The genes encoding the single chain MHC class II molecule:peptide
complexes may be inserted into an "expression vector," "cloning vector," or
"vector,"
terms which are used interchangeably herein and usually refer to plasmids or
other
nucleic acid molecules that are able to replicate in a chosen host cell.
Expression vectors
15 may replicate autonomously, or they can replicate by being inserted into
the genome of
the host cell, by methods well known in the art. Vectors that replicate
autonomously will
have an origin of replication or autonomous replicating sequence (ARS) that is
functional
in the chosen host cell(s).
Plasmid vectors that contain replication sites and control sequences
20 derived from a species compatible with the chosen host are used. For
example, E. coli is
typically transformed using derivatives of pBR322, a plasmid derived from E.
coli species
by Bolivar et al., Gene 2:95-113, 1977. Often, it is desirable for a vector to
be usable in
more than one host cell, e.g., in E. coli for cloning and construction, and in
a Bacillus cell
for expression.
25 The expression vectors typically contain a transcription unit or expression
cassette that contains all the elements required for the expression of the DNA
encoding
the MHC molecule in the host cells. A typical expression cassette contains a
promoter
operably linked to the DNA sequence encoding a single chain MHC class II
molecule:peptide complex and a ribosome binding site. The promoter is
preferably
positioned about the same distance from the heterologous transcription start
site as it is
from the transcription start site in its natural setting. As is known in the
art, however,
some variation in this distance can be accommodated without loss of promoter
function.
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26
In addition to a promoter sequence, the expression cassette can also contain a
transcription termination region downstream of the structural gene to provide
for efficient
termination. The termination region may be obtained from the same gene as the
promoter
sequence or may be obtained from a different gene.
Commonly used prokaryotic control sequences which are defined herein to
include promoters for transcription initiation, optionally with an operator,
along with
ribosome binding site sequences, include such commonly used promoters as the
beta-
lactamase (penicillinase) and lactose (lac) promoter systems (Change et al.,
Nature
198:1056, 1977) and the tryptophan (trp) promoter system (Goeddel et al.,
Nucleic Acids
Res. 8:4057-74, 1980) and the lambda-derived PL promoter and N-gene ribosome
binding
site (Shimatake et al., Nature 292:128-32, 1981). Any available promoter
system that
functions in prokaryotes can be used.
Either constitutive or regulated promoters can be used in the present
invention. Regulated promoters can be advantageous because the host cells can
be grown
to high densities before expression of the single chain MHC class II
molecule:peptide
complexes is induced. High level expression of heterologous proteins slows
cell growth
in some situations. Regulated promoters especially suitable for use in E. coli
include the
bacteriophage lambda PL promoter, the hybrid trp-lac promoter (Amann et al.,
Gene
25:167-78 1983; and the bacteriophage T7 promoter.
For expression of single chain MHC class II molecule:peptide complexes
in prokaryotic cells other than E. coli, a promoter that functions in the
particular
prokaryotic species is required. Such promoters can be obtained from genes
that have
been cloned from the species, or heterologous promoters can be used. For
example, the
hybrid trp-lac promoter functions in Bacillus in addition to E. coli.
A ribosome binding site (RBS) is also necessary for expression of single
chain MHC class II molecule:peptide complexes in prokaryotes. An RBS in E.
coli, for
example, consists of a nucleotide sequence 3-9 nucleotides in length located 3-
11
nucleotides upstream of the initiation codon (Shine and Dalgarno, Nature,
254:34-40,
1975; Steitz, In Biological regulation and development.' Gene expression (ed.,
Goldberger), vol. 1, p. 349, 1979).
Translational coupling may be used to enhance expression. The strategy
uses a short upstream open reading frame derived from a highly expressed gene
native to
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27
the translational system, which is placed downstream of the promoter, and a
ribosome
binding site followed after a few amino acid codons by a termination codon.
Just prior to
the termination codon is a second ribosome binding site, and following the
termination
codon is a start codon for the initiation of translation. The system dissolves
secondary
structure in the RNA, allowing for the efficient initiation of translation.
See Squires, et.
al., J. Biol. Chem. 263:16297-16302, 1988.
The single chain MHC class II molecule:peptide complexes can be
expressed intracellularly, or can be secreted from the cell. Intracellular
expression often
results in high yields. However, some of the protein may be in the form of
insoluble
inclusion bodies. Although some of the intracellularly produced MHC
polypeptides of
the present invention may be active upon being harvested following cell lysis,
the amount.
of soluble, active MHC polypeptide may be increased by performing refolding
procedures
using methods known to those of skill in the art (see, e.g., Sambrook et al.,
Molecular
Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor, NY, 1989.;
Marston
et al., BiolTechnology 2:800-804, 1985; Schoner et al., BiolTechnology 3:151-
54, 1985).
In one embodiment, for purification and refolding the cell pellet is lysed and
refolded in
urea-borate-DTT buffer followed by urea-borate buffer and reverse phase HPLC
purification using either silica gel based Vydac (Hewlett Packard, Wilmington,
DE) or
polymer based Poros-R2 (PerSeptive Biosystems) resins, with bead size varying
based on
the scale of the culture and is described in further detail below. In one
embodiment, e.g.,
for large scale refolding, the sample can be ultrafiltered into a urea-borate
buffer to which
is then added 0.2 ~.M to 1 mM copper sulfate, preferably 0.2 to 20 ~,M, after
which
folding occurs immediately.
More than one MHC:peptide complex may be expressed in a single
prokaryotic cell by placing multiple transcriptional cassettes in a single
expression vector,
or by utilizing different selectable markers for each of the expression
vectors which are
employed in the cloning strategy.
A second approach for expressing the MHC:peptide complexes of the
invention is to cause the polypeptides to be secreted from the cell, either
into the
periplasm or into the extracellular medium. The DNA sequence encoding the MHC
polypeptide is linked to a cleavable signal peptide sequence. The signal
sequence directs
translocation of the MHC:peptide complex through the cell membrane. An example
of a
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28
suitable vector for use in E. coli that contains a promoter-signal sequence
unit is
pTA1529, which has the E. coli phoA promoter and signal sequence see, e.g.,
Sambrook
et al., supra; Oka et al., Proc. Natl. Acad. Sci. USA 82:7212-16, 1985;
Talinadge et al.,
Proc. Natl. Acad. Sci. USA 77:39892, 1980; Takahara et al., J. Biol. Chem.
260: 2670-74,
1985). Once again, multiple polypeptides can be expressed in a single cell for
periplasmic association. Eukaryotic signal sequences are also well known to
those of
skill in the art, and cause the MHC:peptide complexes of the invention to be
secreted into
the extracellular medium.
The MHC:peptide complexes of the invention can also be produced as
fusion proteins. This approach often results in high yields, because normal
prokaryotic
control sequences direct transcription and translation. In E. coli, lacZ
fusions are often
used to express heterologous proteins. Suitable vectors are readily available,
such as the
pUR, pEX, and pMR100 series (see, e.g., Sambrook et al., supra). For certain
applications, it may be desirable to cleave the non-MHC amino acids from the
fusion
protein after purification. This can be accomplished by any of several methods
known in
the art, including cleavage by cyanogen bromide, a protease, or by Factor X,
(see, e.g.,
Sambrook et al., supra.; Goeddel et al., Proc. Natl. Acad. Sci. USA 76:106-10,
1979;
Nagai et al., Nature 309:810-12, 1984; Sung et al., Proc. Natl. Acad. Sci. USA
83:561-65,
1986). Cleavage sites can be engineered into the gene for the fusion protein
at the desired
point of cleavage.
Foreign genes, such as single chain MHC class II molecule:peptide
complexes, can be expressed in E. coli as fusions with binding partners, such
as
glutathione-S-transferase (GST), maltose binding protein, or thioredoxin.
These binding
partners are highly translated and can be used to overcome inefficient
initiation of
translation of eukaryotic messages in E. coli. Fusion to such binding partner
can result in
high-level expression, and the binding partner is easily purified and then
excised from the
protein of interest. Such expression systems are available from numerous
sources, such
as Invitrogen Inc. (San Diego, CA) and Pharmacia LKB Biotechnology Inc.
(Piscataway, NJ).
A method for obtaining recombinant proteins from E. coli which maintains
the integrity of their N-termini has been described by Miller et al.
Biotechnology 7:698-
704 (1989). In this system, the gene of interest is produced as a C-terminal
fusion to the
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29
first 76 residues of the yeast ubiquitin gene containing a peptidase cleavage
site.
Cleavage at the junction of the two moieties results in production of a
protein having an
intact authentic N-terminal reside.
The vectors containing the nucleic acids that code for the single chain
MHC class II molecule:peptide complexes are transformed into prokaryotic host
cells for
expression. "Transformation" refers to the introduction of vectors containing
the nucleic
acids of interest directly into host cells by well known methods. The
particular procedure
used to introduce the genetic material into the host cell for expression of
the single chain
MHC class II molecule:peptide complex is not particularly critical. Any of the
well
known procedures for introducing foreign nucleotide sequences into host cells
may be
used. It is only necessary that the, particular host cell utilized be capable
of expressing
the gene.
Transformation methods, which vary depending on the type of the
prokaryotic host cell, include electroporation; transfection employing calcium
chloride,
rubidium chloride calcium phosphate, or other substances; microprojectile
bombardment;
infection (where the vector is an infectious agent) ; and other methods. See,
generally,
Sambrook et al, supra, and Ausubel et al., (eds.) Current Protocols in
Molecular Biology,
John Wiley and Sons, Inc., NY, 1987. Reference to cells into which the nucleic
acids
described above have been introduced is meant to also include the progeny of
such cells.
Transformed prokaryotic cells that contain expression vectors for single chain
MIiC class
II molecule:peptide complexes are also included in the invention.
After standard transfection or transformation methods are used to produce
prokaryotic cell lines that express large quantities of the single chain MHC
class II
molecule:peptide complex polypeptide, the polypeptide is then purified using
standard
techniques. See, e.g., Colley et al., J. Chem. 64:17619-22, 1989; and Methods
in
Enzymology, "Guide to Protein Purification", Deutscher, ed., Vol. 182 (1990).
The
recombinant cells are grown and the single chain MHC class II molecule:peptide
complex
is expressed. The purification protocol will depend upon whether single chain
MHC class
II molecule:peptide complex is expressed intracellularly, into the periplasm,
or secreted
from the cell. For intracellular expression, the cells are harvested, lysed,
and the
polypeptide is recovered from the cell lysate (Sambrook et al., supra).
Periplasmic MHC
polypeptide is released from the periplasm by standard techniques (Sambrook et
al.,
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supYa). If the MHC polypeptide is secreted from the cells, the culture medium
is
harvested for purification of the secreted protein. The medium is typically
clarified by
centrifugation or filtration to remove cells and cell debris.
The MHC polypeptides can be concentrated by adsorption to any suitable
5 resin (such as, for example, CDP-Sepharose, Asialoprothrombin-Sepharose 4B,
or Q
Sepharose), or by use of ammonium sulfate fractionation, polyethylene glycol
precipitation, or by ultrafiltration. Other means known in the art may be
equally suitable.
Further purification of the MHC polypeptides can be accomplished by
standard techniques, for example, affinity chromatography, ion exchange
10 chromatography, sizing chromatography, reverse phase HPLC, or other protein
purification techniques used to obtain homogeneity. The purified proteins are
then used
to produce pharmaceutical compositions.
For secretion of a polypeptide or protein of interest, recombinant nucleic
acid constructs of the invention may include sequences that encode signal
sequences or
15 other sequences that direct secretion. Secretory signal sequences, also
called leader
sequences, prepro sequences and/or pre sequences, are amino acid sequences
that play a
role in secretion of mature polypeptides or proteins from a cell. Such
sequences are
characterized by a core of hydrophobic amino acids and are typically (but not
exclusively) found at the amino termini of newly synthesized proteins. The
secretory
20 signal sequence may be that of the protein of interest, or may be derived
from another
secreted protein (e.g., t-PA, a preferred mammalian secretory leader) or
synthesized
de novo. The secretory signal sequence is joined to the DNA sequence encoding
a
protein of the present invention in the correct reading frame. Secretory
signal sequences
are commonly positioned 5' to the DNA sequence encoding the polypeptide of
interest,
25 although certain signal sequences may be positioned elsewhere in the DNA
sequence of
interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al.,
U.S. Patent No.
5,143,30). Very often the secretory peptide is cleaved from the mature protein
during
secretion. Such secretory peptides contain processing sites that allow
cleavage of the
secretory peptide from the mature protein as it passes through the secretory
pathway. An
30 example of such a processing site is a dibasic cleavage site, such as that
recognized by the
Saccharomyces cerevisiae KEX2 gene or a Lys-Arg processing site. Processing
sites
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31
may be encoded within the secretory peptide or may be added to the peptide by,
for
example, in vitro mutagenesis.
Secretory signals include the a factor signal sequence (prepro sequence:
Kurjan & Herskowitz, Cell 3:933-943, 1982; Kurjan et al., U.S. Patent No.
4,546,082;
Brake, EP 116, 201), the PHOS signal sequence (Beck et al., WO 86/00637) , the
BART
secretory signal sequence (MacKay et al., U.S. Patent No. 4,613,572; MacKay,
WO
87/002670), the SUC2 signal sequence (Carlsen et al., Molecular and Cellular
Biology 3:
439-447 , 1983) , the a-1-antitrypsin signal sequence (Kurachi et al., Proc.
Natl. Acad. Sci.
USA 78: 6826-6830, 1981) , the a-2 plasmin inhibitor signal sequence (Tone et
al., J.
Biochem. (Tokyo) 102: 1033-1042, 1987) and the tissue plasminogen activator
signal
sequence (Pennica et al., Nature 301: 214-221, 1983). Alternately, a secretory
signal
sequence may be synthesized according to the rules established, for example,
by von
Heinje (European Journal of Biochemistry 133: 17-21, 1983; Journal of
Molecular
Biology 184: 99-105, 1985; Nucleic Acids Research 14: 4683-4690; 1986).
Another
signal sequence is the synthetic signal LaC212 spx (1-47). ERLE described in
WO
90/10075.
Secretory signal sequences may be used singly or may be combined. For
example, a first secretory signal sequence may be used in combination with a
sequence
encoding the third domain of burner (described in U.S. Patent No. 5,037,243,
which is
incorporated by reference herein in its entirety). The third domain of barrier
may be
positioned in proper reading frame 3' of the DNA segment of interest or 5' to
the DNA
segment and in proper reading frame with both the secretory signal sequence
and a DNA
segment of interest.
The choice of suitable promoters, terminators and secretory signals for all
expression systems, is well within the level of ordinary skill in the art.
Methods for
expressing cloned genes in Saccharomyces cerevisiae are generally known in the
art (see,
"Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel (ed.),
Academic Press, San Diego, CA, 1990 and "Guide to Yeast Genetics and Molecular
Biology, "Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San
Diego,
CA, 1991; which are incorporated herein by reference). Proteins of the present
invention
can also be expressed in filamentous fungi, for example, strains of the fungi
Aspergillus
(McKnight et al., U.S. Patent No. 4,935,349, which is incorporated herein by
reference).
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32
Expression of cloned genes in cultured mammalian cells and in E. coli, for
example, is
discussed in detail in Sambrook et al. (Molecular Cloning: A Laboratory
Manual.
Second Edition, Cold Spring Harbor, NY, 1989; which is incorporated herein by
reference). As would be evident to one skilled in the art, one could express
the proteins
of the instant invention in other host cells such as avian, insect and plant
cells using
regulatory sequences, vectors and methods well established in the literature.
In yeast, suitable yeast vectors for use in the present invention include
YRp7 (Struhl et al., Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YEpl3
(Broach et
al., Gene 8: 121-133, 1979), POT vectors (Kawasaki et al., U.S. Patent No.
4,931,373,
which is incorporated by reference herein), pJDB249 and pJDB219 (Beggs, Nature
275:104-108, 1978) and derivatives thereof. Preferred promoters for use in
yeast include
promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255:
12073-12080,
1980; Alber and Kawasaki, J. Mol. Appl. Genet. 1: 419-434, 1982; Kawasaki,
U.S. Patent
No. 4,599,311) or alcohol dehydrogenase genes (Young et al., in Genetic
Engineering of
Microorganisms for Chemicals, Hollaender et al., (eds.), p. 355, 1982;
Ammerer, Meth.
Enzymol. 101: 192-201, 1983). Other promoters are the TPIl promoter (Kawasaki,
U.S.
Patent No. 4,599,311, 1986) and the ADH2-4° promoter (Russell et al.,
Nature 304: 652-
654, 1983; Irani and Kilgore, U.S. Patent Application Serial No. 07/784,653,
CA
1,304,020 and EP 284 044, which are incorporated herein by reference). The
expression
units may also include a transcriptional terminator such as the TPI1
terminator (Alber and
Kawasaki, ibid.).
Yeast cells, particularly cells of the genus Pichia or Saccharomyces, are a
preferred host for use in producing compound of the current invention. Methods
for
transforming yeast cells with exogenous DNA and producing recombinant proteins
therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311;
Kawasaki
et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et
al., U.S.
Patent No. 5,037,743; and Murray et al., U.S. Patent No. 4,845,075, which are
incorporated herein by reference. Transformed cells are selected by phenotype
determined by a selectable marker, commonly drug resistance or the ability to
grow in the
absence of a particular nutrient (e.g., leucine) . A preferred vector system
for use in yeast
is the POT1 vector system disclosed by Kawasaki et al. (IJ.S. Patent No.
4,931,373),
which allows transformed cells to be selected by growth in glucose-containing
media. A
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33
preferred secretory signal sequence for use in yeast is that of the S.
cerevisiae MFal gene
(Brake, ibid.; Kurjan et al., U.S. Patent No. 4,546,082) . Suitable promoters
and
terminators for use in yeast include those from glycolytic enzyme genes (see,
e.g.,
Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al., U.S. Patent No.
4,615,974; and
Bitter, U.S. Patent No. 4,977,092, which are incorporated herein by reference)
and
alcohol dehydrogenase genes. See also U.S. Patent Nos. 4,990,446; 5,063,154;
5,139,936
and 4,661,454, which are incorporated herein by reference. Transformation
systems for
other yeasts, including Hahsenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia
pastoris, Pichia
methanolica, Pichia guillermohdii and. Candida maltosa are known in the art.
See, for
example, Gleeson et al., J. Geh. Microbiol. 132:3459-65, 1986; Cregg, U.S.
Patent No.
4,882,279; and Stroman et al., U.S. Patent No. 4,879,231.
Other fungal cells are also suitable as host cells. For example, Aspergillus
cells may be utilized according to the methods of McKnight et al., U.S. Patent
No.
4,935,349, which is incorporated herein by reference. Methods for transforming
Acremonium chrysogenum are disclosed by Sumino et al., U.S. Patent No.
5,162,228,
which is incorporated herein by reference. Methods for transforming Neurospora
are
disclosed by Lambowitz, U.S. Patent No. 4,486,533, which is incorporated
herein by
reference.
Host cells containing DNA constructs of the present invention are then
cultured to produce the heterologous proteins. The cells are cultured
according to
standard methods in a culture medium containing nutrients required for growth
of the
particular host cells. A variety of suitable media are known in the art and
generally
include a carbon source, a nitrogen source, essential amino acids, vitamins,
minerals and
growth factors. The growth medium will generally select for cells containing
the DNA
construct by, for example, drug selection or deficiency in an essential
nutrient which is
complemented by a selectable marker on the DNA construct or co-transfected
with the
DNA construct.
Yeast cells, for example, are preferably cultured in a chemically defined
medium, comprising a non-amino acid nitrogen source, inorganic salts, vitamins
and
essential amino acid supplements. The pH of the medium is preferably
maintained at a
pH greater than 2 and less than 8, preferably at pH 6.5. Methods for
maintaining a stable
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34
pH include buffering and constant pH control, preferably through the addition
of sodium
hydroxide. Preferred buffering agents include succinic acid and Bis-Tris
(Sigma
Chemical Co., St. Louis, MO). Yeast cells having a defect in a gene required
for
asparagine-linked glycosylation are preferably grown in a medium containing an
osmotic
stabilizer. A preferred osmotic stabilizer is sorbitol supplemented into the
medium at a
concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M. Cultured
mammalian cells are generally cultured in commercially available serum-
containing or
serum-free media. Selection of a medium appropriate for the particular host
cell used is
within the level of ordinary skill in the art.
Methods for introducing exogenous DNA into mammalian host cells
include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725,
1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb,
Virology 52:456, 1973) , electroporation (Neumann et al., EMBO J. 1:841-45,
1982) and
DEAF-dextran mediated 'transfection (Ausubel et al., (eds), Current Protocols
in
Molecular Biology, John Wiley and Sons, Inc., NY, 1987), which are
incorporated herein
by reference. Cationic Iipid transfection using commercially available
reagents, including
the Boehringer Mannheim TRANSFECTION-REAGENT (N-[1- (2,3-
dioleoyloxy)propyl] -N,N,N-trimethyl ammoniummethylsulfate; Boehringer
Mannheim,
Indianapolis, III or LIPOFECTIN reagent (N-[l-(2,3-dioleoyloxy)propyl~-N,N,N-
trimethylammonium chloride and dioleoyl phosphatidylethanolamine; GIBCO-BRL,
Gaithersburg, MD) using the manufacturer-supplied directions, may also be
used. A
preferred mammalian expression plasmid is Zem229R (deposited under the terms
of the
Budapest Treaty with American Type Culture Collection, 12301 Parklawn Drive,
Rockville, MD on September 28, 1993 as an E. coli HB 101 transformant and
assigned
Accession Number 69447) . The production of recombinant proteins in cultured
mammalian cells is disclosed, for example, by Levinson et al., U.S. Patent No.
4,713,339;
Hagen et al., U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent No.
4,579,821; and
Ringold, U.S. Patent No. 4,656,134, which are incorporated herein by
reference.
Preferred cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-
7
(ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL
10314), DG44, and 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-
72,
1977) cell lines. Additional suitable cell lines are known in the art and
available from
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
public depositories such as the American Type Culture Collection, Rockville,
Maryland.
In general, strong transcription promoters are preferred, such as promoters
from SV-40 or
cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable
promoters include
those from metallothionein genes (U.S. Patents Nos. 4,579,821 and 4,601,978,
which are
5 incorporated herein by reference) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells
into which foreign DNA has been inserted. Such cells are commonly referred to
as
"transfectants." Cells that have been cultured in the presence of the
selective agent and
are able to pass the gene of interest to their progeny are refeiTed to as
"stable
10~ transfectants." A preferred selectable marker is a gene encoding
resistance to the
antibiotic neomycin. Selection is carried out in the presence of a neomycin-
type drug,
such as G-418 or the like. Selection systems may also be used to increase the
expression
level of the gene of interest, a process referred to as "amplification."
Amplification is
carried out by culturing transfectants in the presence of a low level of the
selective agent
15 and then increasing the amount of selective agent to select for cells that
produce high
levels of the products of the introduced genes. A preferred amplifiable
selectable marker
is dihydrofolate reductase, which confers resistance to methotrexate. Other
drug
resistance genes (e.g. hygromycin resistance, mufti-drug resistance, puromycin
acetyltransferase) can also be used.
20 The soluble, fused MHC:peptide complexes of the present invention can
be purified by first isolating the polypeptides from the cells followed by
conventional
purification methods, such as by ion-exchange and partition chromatography as
described
by, for example, Coy et al. (Peptides Structure and Function, Pierce Chemical
Company,
Rockford, IL, pp 369-72, 1983) or by reverse-phase.chromatography as
described, for
25 example, by Andreu and Mernfield (Eur. J. Biochem. 164: 585-90, 1987), or
by HPLC as
described, for example, by Kofod et al. (Int. J. Peptide and Protein Res. 32.:
436-40,
1988). Additional purification can be achieved by additional conventional
purification
means, such as liquid chromatography, gradient centrifugation, and gel
electrophoresis,
among others. Methods of protein purification are known in the art (see
generally,
30 Scopes, R., Protein Purification, Springer-Verlag, NY, 1982, which is
incorporated by
reference herein) and can be applied to the purification of the recombinant
polypeptides
described herein. Single chain MHC class II molecule:peptide complexes of at
least
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36
about 50% purity are preferred, at least about 70-~0% purity more preferred,
and about
95-99% or more purity most preferred, particularly for pharmaceutical uses.
Once
purified, either partially or to homogeneity, as desired, the single chain MHC
class II
molecule:peptide complexes may then be used diagnostically or therapeutically,
as further
described below.
Methods of using single chain MHC class II molecule:peptide complexes
The single chain MHC class II molecule:peptide complexes of the present
invention may be used within methods for down-regulating parts of the immune
system
that are reactive in autoimmune diseases. The single chain MHC class II
molecule:peptide complexes of the present invention are contemplated to be
advantageous for use as immunotherapeutics to induce immunological tolerance
or
nonresponsiveness (anergy) in patients predisposed to mount or already
mounting an
immune response those particular autoantigens. A patient having or predisposed
to a
particular autoimmune disease is identified and MHC type is determined by
methods
known in the art. The patient's T cells can be examined in vitro to determine
autoantigenic peptides) recognized by the patient's autoreactive T cells using
complexes
and methods described herein. The patient can then be treated with complexes
of the
invention. Such methods will generally include administering single chain MHC
class II
molecule:peptide complex in an amount sufficient to lengthen the time period
before
onset of the autoimmune disease and/or to ameliorate or prevent that disease.
Single
chain MHC class II molecule:peptide complexes of the present invention axe
therefore
contemplated to be advantageous for use in both therapeutic and diagnostic
applications
related to auto immune diseases.
Kits can also be supplied for therapeutic or diagnostic uses. Thus, the
subject composition of the present invention may be provided, usually in a
lyophilized
form, in a container. The single chain MHC class II molecule:peptide complex
is
included in the kits with instructions for use, and optionally with buffers,
stabilizers,
biocides, and inert proteins. Generally, these optional materials will be
present at less
than about 5% by weight, based on the amount of single chain MHC class II
molecule:peptide complex, and will usually be present in a total amount of at
least about
0.001 % by weight, based on the single chain MHC class II molecule:peptide
complex
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37
concentration. It may be desirable to include an inert extender or excipient
to dilute the
active ingredients, where the excipient may be present in from about 1 to 99%
weight of
the total composition.
Within one aspect of the present invention, single chain MHC class II
molecule:peptide complexes are utilized to prepare antibodies for diagnostic
or
therapeutic uses. As used herein, the term "antibodies" includes polyclonal
antibodies,
monoclonal antibodies, antigen-binding fragments thereof such as F(ab')2 and
Fab
fragments, as well as recombinantly produced binding partners. These binding
partners
incorporate the variable or CDR regions from a gene which encodes a
specifically
binding antibody. The affinity of a monoclonal antibody or binding partner may
be
readily determined by one of ordinary skill in the art (see, Scatchard, Ann.
lVYAcad. Sci.
51: 660-72, 1949)
Methods for preparing polyclonal and monoclonal antibodies have been
well described in the literature (see, for example, Sambrook et al., Molecular
Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell,
J. G.
R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
Press,
Ind., Boca Raton, FL, 1982, which is incorporated herein by reference) . As
would be
evident to one of ordinary skill in the art, polyclonal antibodies may be
generated from a
variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs,
chickens,
rabbits, mice, or rats, for example. The immunogenicity of the single chain
MHC class II
molecule:peptide complexes may be increased through the use of an adjuvant,
such as
Freund's complete or incomplete adjuvant. A variety of assays known to those
skilled in
the art may be utilized to detect antibodies which specifically bind to a
single chain MHC
class II molecule:peptide complex. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor
Laboratory Press, 1988. Representative examples of such assays include:
concurrent
immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme-
linked immuno-sorbent assays, dot blot assays, inhibition or competition
assays, and
sandwich assays.
Additional techniques for the preparation of monoclonal antibodies may be
utilized to construct and express recombinant monoclonal antibodies. Briefly,
mRNA is
isolated from a B cell population and used to create heavy and light chain
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38
immunoglobulin cDNA expression libraries in a suitable vector such as the
7~,IMMUNOZAP(H) and 7~.IMMUNOZAP(L) vectors, which may be obtained from
Stratagene Cloning Systems (La Jolla, CA). These vectors are then screened
individually
or are co-expressed to form Fab fragments or antibodies (Ruse et al., Science
246 1275-
81, 1989; Sastry et al., Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989).
Positive plaques
are subsequently converted to a non-lytic plasmid which allows high level
expression of
monoclonal antibody fragments in E. coli.
Antibodies of the present invention may be produced by immunizing an
animal selected from a wide variety of warm-blooded animals, such as horses,
cows,
goats, sheep, dogs, chickens, rabbits, mice, and rats, with a recombinant
single chain
MHC class II molecule:peptide complex. Serum from such animals are a source of
polyclonal antibodies. Alternatively antibody producing cells obtained from
the
immunized animals are immortalized and screened. As the generation of human
monoclonal antibodies to a human antigen, such as a single chain MHC class II
molecule:peptide complex, may be difficult with conventional immortalization
techniques, it may be desirable to first make non-human antibodies. Using
recombinant
DNA techniques, the antigen binding regions of the non-human antibody is
transferred to
the corresponding site of a human antibody coding region to produce a
substantially
human antibody molecules. Such methods are generally known in the art and are
described in, for example, U.S. Patent No. 4,816,397, and EP publications
173,494 and
239,400, which are incorporated herein by reference.
In another aspect of the invention, the single chain MHC class II
molecule:peptide complexes can be used to clone T cells which have specific
receptors
for the single chain MHC class II molecule:peptide complex. Once the single
chain MHC
class II molecule:peptide complex-specific T cells are isolated and cloned
using
techniques generally available to the skilled artisan, the T cells or membrane
preparations
thereof can be used to immunize animals to produce antibodies to the single
chain MHC
class II molecule:peptide complex receptors on T cells. The antibodies can be
polyclonal
or monoclonal. If polyclonal, the antibodies can be marine, lagomorph, equine,
ovine, or
from a variety of other mammals. Monoclonal antibodies will typically be
marine in
origin, produced according to known techniques, or human, as described above,
or
combinations thereof, as in chimeric or humanized antibodies. The anti- single
chain
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39
MHC class II molecule:peptide complex receptor antibodies thus obtained can
then be
administered to patients to reduce or eliminate T cell subpopulations that
display such
receptor. This T-cell population recognizes and participates in the
immunological
destruction of cells bearing the autoantigenic peptide in an individual
predisposed to or
already suffering from a disease, such as an autoimmune disease related to the
autoantigenic peptide.
The coupling of antibodies to solid supports and their use in purification of
proteins is well known in the literature (see, for example, Methods in
Molecular Biology.
Yol. 1, Walker (Ed.), Humana Press, New Jersey, 1984, which is incorporated by
reference herein in its entirety). Antibodies of the present invention may be
used as a
marker reagent to detect the presence of MHC heterodimer:peptide complexes on
cells or
in solution. Such antibodies are also useful for Western analysis or
irnmunoblotting,
particularly of purified cell-secreted material. Polyclonal, affinity purified
polyclonal,
monoclonal and single chain antibodies are suitable for use in this regard. In
addition,
proteolytic and recombinant fragments and epitope binding domains can be used
herein.
Chimeric, humanized, veneered, CDR-replaced, reshaped or other recombinant
whole or
partial antibodies are also suitable.
Pharmaceutical compositions
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered (e.g., nucleic acid, protein) as by
the particular
method used to administer the composition. Accordingly, there are a wide
variety of
suitable formulations of pharmaceutical compositions of the present invention
(see, e.g.,
Remington's Pharmaceutical Sciences (17th ed., 1989). Administration can be in
any
convenient manner, e.g., by injection, oral administration, inhalation, or
transdermal
application.
Formulations suitable for oral administration can consist of (a) liquid
solutions, such as an effective amount of the packaged nucleic acid or
polypeptide
suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets
or tablets,
each containing a predetermined amount of the active ingredient, as liquids,
solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and (d)
suitable emulsions.
Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol,
calcium
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phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin,
colloidal silicon
dioxide, talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers,
binders, diluents, buffering agents, moistening agents, preservatives,
flavoring agents,
dyes, disintegrating agents, and pharmaceutically compatible caxriers. Lozenge
forms can
5 comprise the active ingredient in a flavor, e.g., sucrose, as well as
pastilles comprising the
active ingredient in an inert base, such as gelatin and glycerin or sucrose
and acacia
emulsions, gels, and the like containing, in addition to the active
ingredient, Garners
known in the art.
The compound of choice, alone or in combination with other suitable
10 components, can be made into aerosol formulations (i.e., they can be
"nebulized") to be
administered via inhalation. Aerosol formulations can be placed into
pressurized
acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
Formulations suitable for parenteral administration, such as, for example
by intraarticular (in the joints), intravenous, intramuscular, intradermal,
intraperitoneal,
15 and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile
injection
solutions, which can contain antioxidants, buffers, bacteriostats, and solutes
that render
the formulation isotonic with the blood of the intended recipient, and aqueous
and non-
aqueous sterile suspensions that can include suspending agents, solubilizers,
thickening
agents, stabilizers, and preservatives. In the practice of this invention,
compositions can
20 be administered, for example, by intravenous infusion, orally, topically,
intraperitoneally,
intravesically or iritrathecally. Parenteral administration and intravenous
administration
are the preferred methods of administration. The formulations of commends can
be
presented in unit-dose or mufti-dose sealed containers, such as ampules and
vials.
Injection solutions and suspensions can be prepared from sterile powders,
25 granules, and tablets of the kind previously described. Cells transduced by
nucleic acids
for ex vivo therapy can also be administered intravenously or parenterally as
described
above.
The polypeptides of the invention are administered prophylactically or to
an individual already suffering from the disease. The compositions are
administered to a
30 patient in an amount sufficient to elicit an effective immune response. An
amount
adequate to accomplish this is defined as "therapeutically effective dose" or
"immunogenically effective dose." Amounts effective for this use will depend
on, e.g.,
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41
the peptide composition, the manner of administration, the stage and severity
of the
disease being treated, the weight and general state of health of the patient,
and the
judgment of the prescribing physician, but generally range for the initial
immunization
dose (that is for therapeutic or prophylactic administration) from about 0.01
mg to about
50 mg per 70 kilogram patient, more commonly from about 0.5-1 mg to about 10-
15 mg
per 70 kg of body weight. Boosting dosages are typically from about 0.01 mg to
about 50
mg of peptide, more commonly about 0.5-1 mg to about 10-15 mg, using a
boosting
regimen over weeks to months depending upon the patient's response and
condition. A
suitable protocol would include injection at time 0, 2, 6, 8, 10 and 14 weeks,
followed by
booster injections at 24 and 28 weeks. Booster injections can be from one,
two, three,
four, five or more. Initial and booster injection amounts and timing are
determined based
on the judgment of the physician and the antigen being administered. In one
embodiment, the initial and booster dose is 1.3 mg, 4 mg, or 13 mg,
administered via
intramuscular injection, with at least one and up to 3 booster injections at 8
week
intervals, or at least one and up to 4 booster injections at 6 week intervals.
The therapeutic methods of the present invention may involve oral
tolerance (Weiner et al., Nature 376: 177-80, 1995), or intravenous tolerance,
for
example. Tolerance can be induced in mammals, although conditions for inducing
such
tolerance will vary according to a variety of factors. To induce immunological
tolerance
in an adult susceptible to or already suffering from an autoantigen-related
disease such as
IDDM, the precise amounts and frequency of administration will also vary. For
instance
for adults about 20-80 pg/kg can be administered by a variety of routes, such
as
parenterally, orally, by aerosols, intradermal injection, and the like. For
neonates,
tolerance can be induced by parenteral injection or more conveniently by oral
administration in an appropriate formulation. The precise amount
administrated, and the
mode and frequency of dosages, will vary.
The single chain MHC class II molecule:peptide complexes will typically
be more tolerogenic when administered in a soluble form, rather than in an
aggregated or
particulate form. Persistence of a single chain MHC class II molecule:peptide
complex of
the invention is generally needed to maintain tolerance in an adult, and thus
may require
more frequent administration of the complex, or its administration in a form
which
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42
extends the half life of the complex. See for example, Sun et al., Proc. Natl.
Acad. Sci.
USA 91: 10795-99, 1994.
Within another aspect of the invention, a pharmaceutical composition is
provided which comprises a single chain MHC class II molecule:peptide complex
of the
present invention contained in a pharmaceutically acceptable carrier or
vehicle for
parenteral, topical, oral, or local administration, such as by aerosol or
transdermally, for
prophylactic and/or therapeutic treatment, according to conventional methods.
The
composition may typically be in a form suited for systemic injection or
infusion and may,
as such, be formulated with sterile water or an isotonic saline or glucose
solution.
Formulations may further include one or more diluents, fillers, emulsifiers,
preservatives,
buffers, excipients, and the like, and~may be provided in such forms as
liquids, powders,
emulsions, suppositories, liposomes, transdermal patches and tablets, for
example.
Pharmaceutical compositions of the present invention are administered at
daily to weekly intervals. An "effective amount" of such a pharmaceutical
composition is
an amount that provides a clinically significant decrease in a deleterious T
cell-mediated
immune response to an autoantigen, for example, those associated with IDDM, or
provides other pharmacologically beneficial effects. Such amounts will depend,
in part,
on the particular condition to be treated, age, weight, and general health of
the patient,
and other factors evident to those skilled in the art. Preferably the amount
of the single
chain MHC class II molecule:peptide complex administered will be within the
range of
20-80 pg/kg. Compounds having significantly enhanced half lives may be
administered
at lower doses or less frequently.
Ad~uvants
An immunostimulant refers to essentially any substance that enhances or
potentiates an immune response (antibody andlor cell-mediated) to an exogenous
antigen.
One preferred type of immunostimulant comprises an adjuvant. Many adjuvants
contain
a substance designed to protect the antigen from rapid catabolism, such as
aluminum
hydroxide or mineral oil, and a stimulator of immune responses, such as lipid
A,
Bortadella pertussis or Mycobacterium tuberculosis derived proteins. Certain
adjuvants
are commercially available as, for example, Freund's Incomplete Adjuvant and
Complete
Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and
Company,
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43
Inc., Rahway; NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts
such
as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron
or zinc;
an insoluble suspension of acylated tyrosine; acylated sugars; cationically or
anionically
derivatized polysaccharides; polyphosphazenes; biodegradable microspheres;
monophosphoryl lipid A and quil A. Cytokines, such as GM-CSF, interleukin-2, -
7, -12,
and other like growth factors, may also be used as adjuvants.
The compositions may also include a Mycobacterium species CWS
adjuvant, as described above. The effectiveness of an adjuvant may be
determined by
measuring the amount of antibodies directed against the immunogenic peptide.
Certain adjuvants for eliciting a predominantly Thl-type response include,
for example, a combination of monophosphoryl lipid A, preferably 3-de-O-
acylated
monophosphoryl lipid A, together with an aluminum salt. MPL~' adjuvants are
available
from Corixa Corporation (Seattle, WA; see, for example, US Patent Nos.
4,436,727;
4,877,611; 4,866,034 and 4,912,094). CpG-containing oligonucleotides (in which
the
CpG dinucleotide is unmethylated) also induce a predominantly Thl response.
Such
oligonucleotides are well known and are described, for example, in WO
96/02555, WO
99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA
sequences are also described, for example, by Sato et al., Science 273:352,
1996.
Another preferred adjuvant comprises a saponin, such as Quil A, or derivatives
thereof,
including QS2I and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA);
Escin;
Digitonin; or Gypsophila or Chenopodium quinoa saponins . Other preferred
formulations
include~more than one saponin in the adjuvant combinations of the present
invention, for
example combinations of at least two of the following group comprising QS21,
QS7, Quil
A, ~i-escin, or digitonin.
Alternatively the saponin formulations may be combined with vaccine
vehicles composed of chitosan or other polycationic polymers, polylactide and
polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer
matrix,
particles composed of polysaccharides or chemically modified polysaccharides,
liposomes and lipid-based particles, particles composed of glycerol
monoesters, etc. The
saponins may also be formulated in the presence of cholesterol to form
particulate
structures such as liposomes or ISCOMs. Furthermore, the saponins may be
formulated
together with a polyoxyethylene ether or ester, in either a non-particulate
solution or
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44
suspension, or in a particulate structure such as a paucilamelar liposorne or
ISCOM. The
saponins may also be formulated with excipients such as CarbopolR to increase
viscosity,
or may be formulated in a dry powder form with a powder excipient such as
lactose.
In one embodiment, the adjuvant system includes the combination of a
monophosphoryl lipid A and a saponin derivative, such as the combination of
QS21 and
3D-MPL~ adjuvant, as described in WO 94/00153, or a less reactogenic
composition
where the QS21 is quenched with cholesterol, as described in WO 96/33739.
Other
formulations comprise an oil-in-water emulsion and tocopherol. Another
adjuvant
formulation employs QS21, 3D-MPL~ adjuvant and tocopherol in an oil-in-water
emulsion is described in WO 95/17210.
Another enhanced adjuvant system involves the combination of a CpG-
containing oligonucleotide and a saponin derivative particularly the
combination of CpG
and QS21 as disclosed in WO 00/09159. Preferably the formulation additionally
comprises an oil in water emulsion and tocopherol.
Additional illustrative adjuvants for use in the pharmaceutical
compositions of the invention include Montanide ISA 720 (Seppic, France), SAF
(Chiron,
California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of
adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham,
Rixensart,
Belgium), Detox (Enhanzyn~) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton,
MT)
and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those
described in
pending U.S. Patent Application Serial Nos. 08/853,826 and 091074,720, the
disclosures
of which axe incorporated herein by reference in their entireties, and
polyoxyethylene
ether adjuvants such as those described in WO 99/52549A1.
Other preferred adjuvants include adjuvant molecules of the general
formula (I): HO(CH2CHa0)n-A-R,
wherein, n is 1-50, A is a bond or -C(O)-, R is Ci-so alkyl or Phenyl Ci-so
amyl.
One embodiment of the present invention consists of a vaccine formulation
comprising a polyoxyethylene ether of general formula (I), wherein h is
between I and
50, preferably 4-24, most preferably 9; the R component is C1_so, preferably
C4-C2o alkyl
and most preferably C12 alkyl, and A is a bond. The concentration of the
polyoxyethylene
ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most
preferably in
the range 0.1-1%. Preferred polyoxyethylene ethers are selected from the
following
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group: polyoxyethylene-9-lauryl ether, polyoxyethylene-9-steoryl ether,
polyoxyethylene
8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl
ether, and
polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as
polyoxyethylene lauryl
ether are described in the Merck index (12th edition: entry 7717). These
adjuvant
5 molecules are described in WO 99/52549.
The polyoxyethylene ether according to the general formula (I) above
may, if desired, be combined with another adjuvant. For example, a preferred
adjuvant
combination is preferably with CpG as described in the pending UK patent
application
GB 9820956.2.
Liposome- Nanocapsule- and Microparticle-Mediated Delivery
In certain embodiments, the inventors contemplate the use of liposomes,
nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the
like, for the
introduction of the compositions of the present invention into the subjects.
In particular,
the compositions of the present invention may be formulated for delivery
either
encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a
nanoparticle or
the like.
Such formulations may be preferred for the introduction of
pharmaceutically-acceptable formulations of the compositions disclosed herein.
The
formation and use of liposomes is generally known to those of skill in the art
(see, for
example,.Couvreur et al., 1977; Couvreur, 1988; Lasic, 1998; which describes
the use of
liposomes and nanocapsules in the targeted antibiotic therapy for
intracellular bacterial
infections and diseases). Recently, liposomes were developed with improved
serum
stability and circulation half times (Gabizon & Papahadjopoulos, 1988; Allen
and Choun,
1987; U. S. Patent 5,741,516, specifically incorporated herein by reference in
its entirety).
Further, various methods of liposome and liposome-like preparations as
potential drug
carriers have been reviewed (Takakura, 1998; Chandran et al., 1997; Margalit,
1995; U.
S. Patent 5,567,434; U. S. Patent 5,552,157; U. S. Patent 5,565,213; U. S.
Patent
5,738,868 and U. S. Patent 5,795,587, each specifically incorporated herein by
reference
in its entirety).
Liposomes have been used effectively to introduce genes, drugs (Heath &
Martin, 1986; Heath et al., 1986; Balazsovits et al., 1989; Fresta & Puglisi,
1996),
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46
radiotherapeutic agents (Pikul et al., 1987), enzymes (Imaizumi et al., 1990a;
Imaizumi et
al., 1990b), viruses (Falter & Baltimore, 1984), transcription factors and
allosteric
effectors (Nicolau & Gersonde, 1979) into a variety of cultured cell lines and
animals. In
addition, several successful clinical trials examining the effectiveness of
liposome-
mediated drug delivery have been completed (Lopez-Berestein et al., 1985a;
1985b;
Coupe, 1988; Sculier et al., 1988). Furthermore, several studies suggest that
the use of
liposomes is not associated with autoimmune responses, toxicity or gonadal
localization
after systemic delivery (Mori & Fukatsu, 1992).
Liposomes are formed from phospholipids that are dispersed in an aqueous
medium and spontaneously form multilamellar concentric bilayer vesicles (also
termed
multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 run to
4 ~,m.
Sonication of MLVs results in the formation of small unilamellar vesicles
(SUVs) with
diameters in the range of 200 to 500 A, containing an aqueous solution in the
core.
Liposomes bear resemblance to cellular membranes and are contemplated
for use in connection with the present invention as earners for the
compositions. They
are widely suitable as both water- and lipid-soluble substances can be
entrapped, i.e., in
the aqueous spaces and within the bilayer itself, respectively. It is possible
that the drug-
bearing liposomes may even be employed for site-specific delivery of active
agents by
selectively modifying the liposomal formulation.
In addition to the teachings of Couvreur et al. (1977, 1988), the following
information may be utilized in generating liposomal formulations.
Phospholipids can
form a variety of structures other than liposomes when dispersed in water,
depending on
the molar ratio of lipid to water. At low ratios the liposome is the preferred
structure.
The physical characteristics of liposomes depend on pH, ionic strength and the
presence
of divalent cations. Liposomes can show low permeability to ionic and polar
substances,
but at elevated temperatures undergo a phase transition which markedly alters
their
permeability. The phase transition involves a change from a closely packed,
ordered
structure, known as the gel state, to a loosely packed, less-ordered
structure, known as the
fluid state. This occurs at a characteristic phase-transition temperature and
results in an
increase in permeability to ions, sugars and drugs.
In addition to temperature, exposure to proteins can alter the permeability
of liposomes. Certain soluble proteins, such as cytochrome c, bind, deform and
penetrate
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47
the bilayer, thereby causing changes in permeability. Cholesterol inhibits
this penetration
of proteins, apparently by packing the phospholipids more tightly. It is
contemplated that
the most useful liposome formations for antibiotic and inhibitor delivery will
contain
cholesterol.
The ability to trap solutes varies between different types of liposomes. For
example, MLVs are moderately efficient at trapping solutes, but SUVs are
extremely
inefficient. SLTVs offer the advantage of homogeneity and reproducibility in
size
distribution. However, a compromise between size and trapping efficiency is
offered by
large unilamellar vesicles (LWs). These are prepared by ether evaporation and
are three
to four times more efficient at solute entrapment than MLVs.
In addition to liposome characteristics, an important determinant in
entrapping compounds is the physicochemical properties of the compound itself.
Polar
compounds are trapped in the aqueous spaces and nonpolar compounds bind to the
lipid
bilayer of the vesicle. Polar compounds are released through permeation or
when the
bilayer is broken, but nonpolar compounds remain affiliated with the bilayer
unless it is
disrupted by temperature or exposure to lipoproteins. Both types show maximum
efflux
rates at the phase transition temperature.
Liposomes interact with cells via four different mechanisms: endocytosis
by phagocytic cells of the reticuloendothelial system such as macrophages and
neutrophils; adsorption to the cell surface, either by nonspecific weak
hydrophobic or
electrostatic forces, or by specific interactions with cell-surface
components; fusion with
the plasma cell membrane by insertion of the lipid bilayer of the liposome
into the plasma
membrane, with simultaneous release of liposomal contents into the cytoplasm;
and by
transfer of liposomal lipids to cellular or subcellular membranes, or vice
versa, without
any association of the liposome contents. It often is difficult to determine
which
mechanism is operative and more than one may operate at the same time.
The fate and disposition of intravenously injected liposomes depend on
their physical properties, such as size, fluidity, and surface charge. They
may persist in
tissues for hours or days, depending on their composition, and half lives in
the blood
range from minutes to several hours. Larger liposomes, such as MLVs and LUVs,
are
taken up rapidly by phagocytic cells of the reticuloendothelial system, but
physiology of
the circulatory system restrains the exit of such large species at most sites.
They can exit
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48
only in places where large openings or pores exist in the capillary
endothelium, such as
the sinusoids of the liver or spleen. Thus, these organs are the predominant
site of uptake.
On the other hand, SUVs show a broader tissue distribution but still are
sequestered
highly in the liver and spleen. In general, this in vivo behavior limits the
potential
targeting of liposomes to only those organs and tissues accessible to their
large size.
These include the blood, liver, spleen, bone marrow, and lymphoid organs.
Targeting is generally not a limitation in terms of the present invention.
However, should specific targeting be desired, methods are available for this
to be
accomplished. Antibodies may be used to bind to the liposome surface and to
direct the
antibody and its drug contents to specific receptors located on a particular
cell-type
surface. Carbohydrate determinants (glycoprotein or glycolipid cell-surface
components
that play a role in cell-cell recognition, interaction and adhesion) may also
be used as
recognition sites as they have potential in directing liposomes to particular
cell types.
Mostly, it is contemplated that intravenous injection of liposomal
preparations would be
used, but other routes of administration are also conceivable.
Alternatively, the invention provides for pharmaceutically-acceptable
nanocapsule formulations of the compositions of the present invention.
Nanocapsules can
generally entrap compounds in a stable and reproducible way (Henry-Michelland
et al.,
1987; Quintanar-Guerrero et al., 1998; Douglas et al., 1987). To avoid side
effects due to
intracellular polymeric overloading, such ultrafine particles (sized around
0.1 p,m) should
be designed using polymers able to be degraded in vivo. Biodegradable
polyallcyl-
cyanoacrylate nanoparticles that meet these requirements are contemplated for
use in the
present invention. Such particles may be axe easily made, as described
(Couvreur et al.,
1980; 1988; zur Muhlen et al., 1998; Zambaux et al. 1998; Pinto-Alphandry et
al., 1995
and U. S. Patent 5,145,684, specifically incorporated herein by reference in
its entirety).
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or patent
application
were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be readily
apparent to one of ordinary skill in the art in light of the teachings of this
invention that
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49
certain changes and modifications may be made thereto without departing from
the spirit
or scope of the appended claims.
The following example is provided by way of illustration only and not by
way of limitation. Those of skill in the art will readily recognize a variety
of noncritical
parameters that could be changed or modified to yield essentially similar
results.
EXAMPLES
Example 1' Construction of DNA sequences encoding~human single chain MHC class
II
_peptide complexes, HLA-DR4 molecules(C0563 and 005641.
DNA constructs encoding gp39 ~31/al human molecules were prepared
from a cDNA encoding the gp39 peptide fused to the (31/a1 domains of HLA-DR4
according to standard techniques. For the production of "empty" (31/a1 DR4
molecules,
a cDNA encoding the fused [31/a1 domains of HLA-DR4 was prepared using cloned
a
and (3 chains from DR4.
Amino acid sequence of the gp39-(31/al HLA-DR4 human single chain
molecule (linkers are shown in bold)
MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECH
FFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQ
KRAAVDTYCRHNYGVGESFTVQRRGGIKEEHVIIQAEFYLNPDQSGEFMFDFDG
DEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPIT
N*
aal - aa4: leader sequence
aa5 - aal8: gp39 peptide
aal9 - aa28: linker
aa29 - aa122: HLA-DR4 (31 domain
aa123 - aa124: linker
aa125 - as 208: HLA-DR4 al domain
Amino acid sequence of the "empty" (31/a1 HLA-DR4 human single chain
molecule (linkers in bold):
MGDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTEL
GRPDAEYWNSQRDLLEQKR.AAVDTYCRHNYGVGESFTVQRRGGIKEEHVIIQAE
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FYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAV
DKANLEIMTKRSNYTPITN'~
as 1- aa95: HLA-DR4 (31 domain
aa96- aa97: linker
aa98- aa181: HLA-DR4 al domain
For the production of recombinant proteins, the bacteria (pLysS) were
grown in LB (containing ampicillin (50 ~g / ml) and chloramphenicol (5 ~,g J
ml)) at
37°C until OD 600 = 0.5. IPTG was added at the final concentration of
0.5 mM final and
the bacteria were further incubated for 3 hours at 37°C with shaking.
The bacteria were
I O centrifuged at 4°C, 4000xg for 20 min and the pellet was frozen at -
80°C. The following
day, the pellet was resuspended in 40 ml of lysis buffer (50 mM Tris-HCl pH 8,
50, mM
NaCI, 2 mM EDTA, 1 protease inhibitor cocktail tablet, 1% Triton X100 and 1%
deoxycholate), and incubated for 1 hour at 4°C under continuous
agitation with a
magnetic stirrer. The sample was then homogenized using a French Press with a
16,000
15 psi setting, and centrifuged at 4°C, 9000g for 20 min. The pellet
was then resuspended in
30 ml of lysis buffer without Triton and deoxycholate and centrifuged at
4°C, 9000 g for
20 min. The new pellet was resuspended in 10 ml of 20 mM ethanolamine/6 M urea
pH
10, and eventually frozen at -80°C. The recombinant protein was then
purified by FPLC
ion-exchange chromatography using Source 30Q anion-exchange media in an
XK26/20
20 column using a step gradient going from 1 mM to 1 M NaCI in 20 mlVl
ethanolamine/6M
urea pH 10. Fractions were analyzed by SDS/PAGE and those corresponding to the
proteins of interest are pooled and dialyzed against PBS 1X.
Example 2 ~ Production of additional single chain constructs
25 Additional constructs with different composition and length of the 2na
linker (between (31 and al) were engineered by using standard techniques using
00567
as the template. Specifically, PCR primers were designed to replace the old
sequence in
00567 with the new sequence. For example, to make 00581, the primers were
designed
with the following sequences (note these primers were phosphorylated at 5').
30 Primer 1:
5'pCACCAGGAGGAGAGCCGCCCACGCCGGTCTCGCTGG
Primer 2:
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51
5'pGACCACCTGGATCTGGGGACACCCGACCACGTTTC
PCR reaction (100 ~,1) was made of the following components: 2 p,1
C0567 (80 ng) as template, 2 ~,1 each of primer 1 and primer 2 (10 p,M), 2 ~,l
of dNTP
mix (20 mM each), 10 ~.l of l OX pfu buffer, and 80 ~.1 of sterile water.
After all the
components were mixed, 2 ~.1 of Turbo pfu (5U total ) was added, mixed and put
on PCR
machine. The PCR cycles has a pre-denaturation at 95°C for 30 sec, then
10 cycles of
95°C for 30°C, 60°C for 1 min, and 72°C for 7 min.
Then another 22 cycles of 95°C for
30°C, 65°C for 1 min and 72°C for 7 min, followed by a
final 10 min at 72°C.
The PCR mixture was digested with 2 ~,l of DpnI (10U) for 2h at
37°C.
Then the PCR product at ~6 kb was purified from agarose gel after
electrophoresis. The
purified PCR product was ligated by T4 DNA ligase for 1 h at room temperature
then
used to transform into NovaBlue (Novagen)competent cells by standard protocol.
Cells
were plated on LB (+Carb) and grow overnight at 37°C.
Next day, about a dozen single colony from the transformation were
randomly picked for overnight culture in 5 ml LB (+Carb) at 37°C.
Plasmids from these
culture were purified with Wizard Miniprep kit, and analyzed by Xho I
digestion. A few
plasmids that passed the Xho I digestion were further confirmed by DNA
sequencing.
To express the recombinant proteins, a clone with confirmed DNA
sequence was used to transform BL21 (DE3)CodonPlus-RIL (Stratagene) by
standaxd
transformation protocol and plated on LB (Curb+Cam) plates overnight at
37°C. Next
morning,.a single colony was picked to inoculate 100 ml LB (+Carb +Cam) and
the
culture was grown till OD reach between 0.8-1.0, and stored overnight at
4°C. Next day,
the culture was pellet down and used to inoculate into 2xYT (+Caxb+Cam) at
ratio of 25
ml culture per liter rlew media. These large cultures were grown at
37°C till OD=0.5-0.6,
and IPTG was added to induce recombinant protein at 37°C for 3 h. The
induced cultures
were pellet and stored at -80°C till purification.
Table 1 provides a listing of various constructs made according to the
invention.
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TABLE I
Construct peptide upstream linker downstream linker
00523 yes GGGG GG .
00543 none none GG
00563 yes ASGGGSGGG GG
00567 yes ASGGGSGGG TSGGGGSGGGGSSS
00580 ves ASGGGSGGG GSPGGGGSGGGPG-S
0058 ~ yes ASGGGSGGG GSPPGGPPGS
00582 yes ' ASGGGSGGG GSPGGGGPGS
00583 yes ASGGGSGGG TSGGGGS
00584 yes ASGGGSGGG SGGSGGS
00585 yes ASGGGSGGG P17APSPLP
00586 none none TSGGGGSGGGGSSS
00587 none none GSPGGGGSGGGPGS
005 8 8 none none GSPPGGPPGS
00589 none none GSPGGGGPGS
00590 none zone TSGGGGS
00591 none none SGGSGGS
00592 none none FDAPSPLP
00593 yes ASGGGSGGG VYPEVTV
00594 none none VYPEVTV
00595 yes ASGGGSGGG GGGG
00595 yes ASGGGSGGG GGGGS
00597 yes ASGGGSGGG GGGSGG
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S3
Example 3' Four classes of novel linkers for MHC class II single chain
molecules
Inspection of circular dichroism spectra of purified, refolded single chain
constructs indicated that novel linkers could be used for the constructs. The
atomic
structures of the various marine and human MHC class II molecules, as
determined by X-
S ray crystallography, indicated that these molecules have a high degree of
structural
similarity. The circular dichroism results were consistent with two folded
molecules of
clearly different secondary structure. Careful inspection of the structures
reveals that the
human MHC has a longer distance between chains than the equivalent marine
molecule.
This results led to proposing longer linkers between the chains which would
contain
flexible residues (e.g. alanine or glycine) and polar residues (e.g. serine
and threonine).
These constructs make up the first class of linkers. To inhibit the
continuation of
secondary structure across the linker, prolines were added to bracket the
linkers. These
prolines are known to inhibit the formation of alpha helices and beta sheets.
These
linkers make up the second class of linkers disclosed here.
1 S Next, flexible regions present in the human MHC and in the marine MHC
could be used to make a linker by extending the region of interest and
ligating the ends
together. These are the third class of linkers. Finally, a combination of
these types of
linkers could also be used. These are the fourth class of linkers.
Example 4' Human MHC class II single chain molecule with marine linkers
Another linker has been suggested based on a combination of marine and
human MHC class II single chain molecules. This fusion would incorporate
linker
residues from the functional marine single chain MHC class II molecule and the
alpha
and beta chains of the human molecule. The protein sequence of the single
chain
2S molecule is provided below (the linker residues from the mouse construct
are in bold):
MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECHFFNGTERVRF
LDRYFYHQEEYVRFDSDVGEYR.AVTELGRPDAEYWNSQKDLLEQKR.AAVDTYC
RHNYGVGESFTVLRRLGGEDDEADHHVIIQAEFYLNPDQSGEFMFDFDGDEIFH
VDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITN*
Further MHC class II hybrid single chain molecules may be designed by
fusing other portions of the alpha chain and the beta chain together using
linkers as
described elsewhere. A properly folded molecule may be obtained by putting
appropriate
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54
linkers between portions of the human MHC class II which are proximal to each
other as
determined by visual inspection of the atomic coordinates of residues of the
native MHC
available in the publicly accessible protein structure database. These
structures would
predict possible fusion proteins which covalently attach any part of the beta
chain
between residues 82 to 123 or between residues I48 to I64 to portions of the
alpha chain
such as the N-terminal residues, residues 79 to 84, or 92 to 106. The
numbering system
of residues in this example corresponds to those found in the coordinates of
the structure
described in: DESSEN, et al. Immunity 7:473 (1997). Other, homologous residues
could
be used to create equivalent constructs for genotypic and allelic variants of
these
molecules e.g. equivalent residues in DR2 or such. DNAs for such hybrids would
be
prepared and expressed in a recombinant expression system by someone skilled
in the art
and could be assayed for structure and function in appropriate assays.
Example 5 ~ Use of CD4 bindin_,g site(sl of MHC class II molecules as linkers
for the
I S production of bioactive recombinant MHC class Il:peptide complexes
HLA class II molecules present antigenic peptides to the T cell receptor of
the CD4+ T lymphocytes and interact with CD4 during the antigen recognition
process.
Native MHC class II-peptide complexes have been shown to bind to MHC class II
restricted and antigen specific TCRs on a particular T cell and induce T cell
nonresponsiveness. It is proposed that the CD4 binding site is important in
the docking of
MHC class II-peptide complex with the TCR and induce nonresponsiveness. Since
the
binding of CD4 to MHC class II-peptide is important in antigen. presentation
andlor
induction of T cell nonresponsiveness, it is proposed that recombinant MHC
class II-
peptide molecules (truncated or whole) containing CD4 binding site will be
biologically
active. Furthermore, a polypeptide fragment from MHC class II which binds the
CD4,
when used as a linker in preparation of MHC class II-peptide truncated
molecules,
provides resulting recombinant molecules that will be biologically active.
The following describes the concept of different linkers. Peptide is
attached to a linker 1 (L1) which is attached to N-terminus of (31 domain that
is linked to
L2. L2 is linked to L3, which in turn is linked to N-terminus of al chain of
MHC class
II. Here L2 represents the human CD4 binding sequences. It should be noted
that L2
could also be directly linked to N-terminus of al domain by completely
deleting L3.
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Specific examples of L1 and L3 are given in a examples. The sequences of L2
are given
below. These sequences are applicable to most of the DR-Peptide molecules.
RNGQEEKAGVVSTGLI, RNGQETKAGVVSTGLI, YNQQEEKAGGVSTGLI,
FRNGQEEKAGWSTGLI, FRNGQETKAGWSTGLI, FYNQQEEKAGGVSTGLI,
and LNGQEEKAGMVSTGLI.
Example 6' I-As MBP X31 (32a1a2 Cx construct and activity
The "full anergix" single chain molecule, mouse I-As MBP.(31(32a1a2.Cx,
was generated by overlap PCR using standard methodology. The molecules was
expressed in 293T cells and baculoviral cells according to standard
methodology, and
purified according to standard methodology using affinity chromatography using
goat-
anti mouse antibodies. The structure ofI-As MBP.(31~32a1a2.CK is shown in
Figure 1.
The amino acid sequence of the I-As MBP.~31(32a1a2.Cx shown in Figure
1 is as follows:
METDTLLLWVLLLWVPGSTGDFKNIVTPRTPPPASGGGGSGGGGDSERHFVFQF
KGECYFTNGTQRIRSVDRYIYNREEYLRFDSDVGEYRAVTELGRPDPEYYNKQY
LEQTRAELDTVCRHNYEGVETHTSLRRLEQPNVVISLSRTEALNHHNTLVCSVTD
FYPAKIKVRWFRNGQEETVGVSSTQLIRNGDWTFQVLVMLEMTPRRGEVYTCH
VEHPSLKSPITVEWTSGGGGSGGGGSGGGGSGGGGSSSEDDIEADHVGVYGTTV
YQSPGDIGQYTHEFDGDEWFYVDLDKKETIWMLPEFGQLTSFDPQGGLQNIATG
KYTLGILTKRSNSTPATNEAPQATVFPKSPVLLGQPNTLICFVDNIFPPVIhIITWLR
NSKSVTDGVYETSFLVNRDHSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHW
ASGGGGSGGGGADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDG
SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK
SFNRNEC
The in vitro activity of recombinant I-As MBP.(31[32a1a2.Cx was tested
using MBP90-I01 specific IAs restricted mouse T cell clone HS 1. This clone
was
prepared by the immunization of SJL mice with the MBP90-101 peptide, followed
by
cloning out CD4+ T cells by limited dilution techniques. These cells were
maintained by
stimulation every 10 days with irradiated SJF splenocytes and PBP90-101
peptide. The T
cells are activated by a combination of soluble recombinant I-As
MBP.(31~i2ala2.CK and
plate bound anti-CD28 antibody. T cell activation was assayed by 3H-thymidine
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incorporation according to standard methodology. Figure 2 shows the results of
this
assay.
MS is a T cell dependent autoimmune disease caused by localized
demyelination in the central nervous system. Experimental autoimmune
encephalomyelitis is a accepted animal model of MS. The following results
demonstrate
that administration of I-As MBP.(31(32a1a2.Cx reduces the incidence and
severity of
EAE. EAE was induced according to standard methodology according to the myelin
model. Ten p,g of recombinant I-As MBP. (3I (32a 1 a2.Cx was given
intravenously in 100
p,1 of PBS at days l, 4, 7, and 11 after disease induction. 12 days after
immunization,
animals are observed daily for the onset of neurological dysfunction. Disease
is graded
by trained technicians according to standard methods (see Figure 3). Mice are
followed
for up to 60-70 days. The data shown in Figure 4 demonstrate that
administration of the
recombinant I-As MBP.j31/32a1a2.Cx significantly reduced the incidence of
myelin-
induced EAE in SJL mice. In one experiment, 11 out of 20 mice developed EAE in
the
untreated group (55%), whereas only 2 out of 21 animal showed sign of the
disease in the
treated group (16.6%). Similarly, in another experiment, 12 out of 16 mice
developed
EAE in the untreated group (75%), while only 2 out of 16 developed EAE in the
treated
group (12.5%).
Histological examination from SJL mice in the EAE model studies: spinal
cords were removed, f xed in formalin solution and embedded in paraffin.
Sections were
cut, stained with hematoxylin, eosin and graded for inflammatory lesions. A.
Section of
spinal cord from untreated mouse, score = 2.5; B. Section of spinal cord from
mouse
treated with the recombinant I-AS.MBP.Ck, score = 0; C. Section of spinal cord
from
mouse treated with the recombinant I-AS.~ilal without antigenic peptide
fusion, score =
2.0; D. Section of spinal cord from mouse treated with the recombinant I-
AS.MBP.[31a1,
score = 0.5. The histology score for each section is marked.
Example 7' Functional human anergix molecules optimized for E. coli expression
with E.
coli codons
Two human single chain MHC class II molecules (C0528-AC and
C0608-AC) have been optimized for E. coli expression using "artificial
codons," e.g.,
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preferred E. coli codons encoding the mammalian protein. 00528-AC and 00608-AC
were made according to standard PCR overlap technology.
Example 7: Recombinant MHC class II IAs.MBP.I~ multimeric complexes
Recombinant MHC class II (IAs)-peptide Ig fusion complexes were
constructed by fusing the mIgG leader, MBP 90-101, or MBP1-14 (as a control)
to IAs
single chain (blb2ala2), and mIgG.Ck, mIgG.CHI.H, mIgG.CHl.H.CH2, or
mIgG.CHI.H.CH2.CH3 with flexible linkers, according to standard methodology.
The
recombinant IAs fusion proteins were expressed in both mammalian and insect
cells and
detected by western analysis and ELISA. The overexpressed and secreted
recombinant
IAs fusion proteins from both human 293 cell cultures or from insect culture
medium
were purified by affinity chromatography. The purified dimeric and tetrameric
recombinant IAs proteins have in vitro biological activity as assayed using an
antigen-
specific mouse T cell clone. The ih vivo activity of the recombinant IAs
fusion proteins
were studied with the experimental autoimmune encephalomyelitis (EAE) model
using
susceptible SJL mice. In these EAE studies, recombinant IAs fusion protein was
delivered on days l, 4, 7, and 11 by LV. injections after induction of the
disease with
myelin. The animals were then examined for neurological dysfunction. The
results
indicate that treatment with the recombinant IAs fusion proteins prevents
mortality and
~ significantly reduces paralysis induced by myelin homogenate in CFA. In
conclusion,
these studies suggest that the recombinant MHC class II fusion protein has
therapeutic
benefit as antigen-specific drugs for the treatment of autoimmune diseases.
Example 8: Synthesis of mouse model equivalent of 00608
Four forms of the marine MHC Class II IAs (31a1 with MBP peptide
linkers analogous to 00608 human were made. These constructs can be used,
e.g., as
marine clinical control for human 608. m00608 (mouse 00608, lacking the first
five
amino acids, GDSER, as compared to native beta 1 domain), MC0608-A (same as
m608
except lacking four amino acids, GSER, after the methionine as compared to
m608), B.
megaterium-mC0608-A (expressed in Bacillus megaterium), and mC0608-B (same as
m608 except lacking first four amino acids, GSER, after the methionine as
compared to
m608; also lacking second amino acid, D, as compared to native beta 1 domain).
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To make mC0608, the upstream linker of mouse C0521 (GGGS) was
replaced with the human C0608 linker (ASGGGGSGGG) and the downstream linker of
mouse C0521 (GG) was replaced with the downstream linker of C0608 (TSGGGGS),
using PCR according to standard methodology. mC0608-A and m6O8-B were made
from mC0608 using PCR according to standard methodology.
All publications and patent applications cited in this specification are
herein incorporated by reference as if each individual publication or patent
application
were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be readily
apparent to one of ordinary skill in the art in light of the teachings of this
invention that
certain changes and modifications may be made thereto without departing from
the spirit
or scope of the appended claims.
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SEQUENCE LISTING
<210> C0602
$ <211> 558
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacetcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgctgcgccgactcggaggtgaagat300
gacgaggcag atcaccatgtgatcatccaggccgagttctatctgaatcctgaccaatca360
1S ggcgagttta tgtttgactttgatggtgatgagattttccatgtggatatggcaaagaag420
gagacggtct ggcggcttgaagaatttggacgatttgccagctttgaggctcaaggtgca480
ttggccaaca tagctgtggacaaagccaacctggaaatcatgacaaagcgctccaactat540
actccgatca ccaattaa 558
<210> C0601
<211> 558
<212> DNA
<213> Homo Sapiens
2S atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgaggaggtggaagcggc300
30 ggaatcaaag aagaacatgtgatcatccaggccgagttctatctgaatcctgaccaatca360
ggcgagttta tgtttgactttgatggtgatgagattttccatgtggatatggcaaagaag420
gagacggtct ggcggcttgaagaatttggacgatttgccagctttgaggctcaaggtgca480
ttggccaaca tagctgtggacaaagccaacctggaaatcatgacaaagcgctccaactat540
actccgatca ccaattaa 558
35
<210> C0600
<211> 555
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60
gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120
ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180
tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240
agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggagg tggaggcagc 300
atcaaagaag aacatgtgat catccaggcc gagttctatc tgaatcctga ccaatcaggc 360
gagtttatgt ttgactttga tggtgatgag attttccatg tggatatggc aaagaaggag 420
acggtctggc ggcttgaaga atttggacga tttgccagct ttgaggctca aggtgcattg 480
gccaacatag ctgtggacaa agccaacctg gaaatcatga caaagcgctc caactatact 540
ccgatcacca attaa 555
<210> C0599
<211> 552
<212> DNA
<213> Homo sapiens
atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60
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WO 01/70245 PCT/USO1/09616
gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120
ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180
tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240
agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggagg tggaggcatc 300
S aaagaagaac atgtgatcat ccaggccgag ttctatctga atcctgacca atcaggcgag 360
tttatgtttg actttgatgg tgatgagatt ttccatgtgg atatggcaaa gaaggagacg 420
gtctggcggc ttgaagaatt tggacgattt gccagctttg aggctcaagg tgcattggcc 480
aacatagctg tggacaaagc caacctggaa atcatgacaa agcgctccaa ctatactccg 540
atcaccaatt as 552
<210> C0594
<211> 561
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacg.tttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgagtctaccctgaggta300
actgtcatca aagaagaacatgtgatcatccaggccgagttctatctgaatcctgaccaa360
tcaggcgagt ttatgtttgactttgatggtgatgagattttccatgtggatatggcaaag420
aaggagacgg tctggcggcttgaagaatttggacgatttgccagctttgaggctcaaggt480
gcattggcca acatagctgtggacaaagccaacctggaaatcatgacaaagcgctccaac540
tatactccga tcaccaattaa 561
<210> C0592
<211> 564
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgattcgacgcacctagc300
ccactcccaa tcaaagaagaacatgtgatcatccaggccgagttctatctgaatcctgac360
caatcaggcg agtttatgtttgactttgatggtgatgagattttccatgtggatatggca420
aagaaggaga cggtctggcggcttgaagaatttggacgatttgccagctttgaggctcaa480
ggtgcattgg ccaacatagctgtggacaaagccaacctggaaatcatgacaaagcgctcc540
aactatactc cgatcaccaattaa 564
<210> C0591
<211> 561
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
S0 ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgaagtggcggtagtggc300
ggtagtatca aagaagaacatgtgatcatccaggccgagttctatctgaatcctgaccaa360
tcaggcgagt ttatgtttgactttgatggtgatgagattttccatgtggatatggcaaag420
S$ aaggagacgg tctggcggcttgaagaatttggacgatttgccagctttgaggctcaaggt480
gcattggcca acatagctgtggacaaagccaacctggaaatcatgacaaagcgctccaac540
tatactccga tcaccaattaa 561
<210> C0590
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
61
<211> 561
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgaactagtggtggcggt300
ggcagcatca aagaagaacatgtgatcatccaggccgagttctatctgaatoctgaccaa360
tcaggcgagt ttatgtttgactttgatggtgatgagattttccatgtggatatggcaaag420
aaggagacgg tctggcggcttgaagaatttggacgatttgccagctttgaggctcaaggt480
gcattggcca acatagctgtggacaaagccaacctggaaatcatgacaaagcgctccaac540
tatactccga tcaccaattaa 561
<210> C0589
<211> 570
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
2$ agacacaact acggggttggtgagagcttcacagtgcagcggcgaggctctcctggaggt300
ggaggtcctg gatctatcaaagaagaacatgtgatcatccaggccgagttctatctgaat360
cctgaccaat caggcgagtttatgtttgactttgatggtgatgagattttccatgtggat420
atggcaaaga aggagacggtctggcggcttgaagaatttggacgatttgccagctttgag480
gctcaaggtg cattggccaacatagctgtggacaaagccaacctggaaatcatgacaaag540
cgctccaact atactccgatcaccaattaa 570
<210> C0588
<211> 570
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgaggctctcctcctggt300
ggaccacctg gatctatcaaagaagaacatgtgatcatccaggccgagttctatctgaat360
cctgaccaat caggcgagtttatgtttgactttgatggtgatgagattttccatgtggat420
atggcaaaga aggagacggtctggcggcttgaagaatttggacgatttgccagctttgag480
gctcaaggtg cattggccaacatagctgtggacaaagccaacctggaaatcatgacaaag540
cgctccaact atactccgatcaccaattaa 570
<210> C0587
<211> 582
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacgtttcttggagcaggttaaacatgagtgtcatttcttcaac60
gggacggagc gggtgcggttcctggacagatacttctatcaccaagaggagtacgtgcgc120
ttcgacagcg acgtgggggagtaccgggcggtgacggagctggggcggcctgatgccgag180
tactggaaca gccagaaggacctcctggagcagaagcgggccgcggtggacacctactgc240
agacacaact acggggttggtgagagcttcacagtgcagcggcgagggagtccaggcggt300
ggcgggagcg gcggtggtcctgggagtatcaaagaagaacatgtgatcatccaggccgag360
ttctatctga atcctgaccaatcaggcgagtttatgtttgactttgatggtgatgagatt420
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
62
ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 480
gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 540
atcatgacaa agcgctccaa ctatactccg atcaccaatt as 582
$ <210> C0586
<211> 582
<212> DNA
<213> Homo Sapiens
atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60
gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120
ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180
tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240
agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaactag tggtggcggt 300
ggcagcggcg gtggtggttc ctcgagtatc aaagaagaac atgtgatcat ccaggccgag 360
ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 420
ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 480
gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 540
atcatgacaa agcgctccaa ctatactccg atcaccaatt as 582
<210> C0602
<211> 186
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
40 45
35 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
50 55 60
Gln Lys~Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys
65 70 75 80
Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Leu Arg Arg Leu
85 90 95
Gly Gly Glu Asp Asp Glu Ala Asp His His Val Ile Ile Gln Ala Glu
100 105 110
Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp
115 120 125
Gly Asp Glu Ile Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp
130 135 140
Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala
145 150 155 160
Leu Ala Asn Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys
165 170 175
Arg Ser Asn Tyr Thr Pro Ile Thr Asn
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
63
180 185
<210> C0601
$ <211> 186
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
IS Tyr HisGlnGlu GluTyrVal ArgPheAsp SerAsp ValGlyGlu Tyr
35 40 45
Arg AlaValThr GluLeuGly ArgProAsp AlaGlu TyrTrpAsn Ser
50 55 60
Gln LysAspLeu LeuGluGln LysArgAla AlaVal AspThrTyr Cys
65 70 75 80
Arg HisAsnTyr GlyValGly GluSerPhe ThrVal GlnArgArg Gly
~$ 85 90 95
Gly GlySerGly GlyIleLys GluGluHis ValTle IleGlnAla Glu
100 105 110
Phe TyrLeuAsn ProAspGln SerGlyGlu PheMet PheAspPhe Asp
115 120 125
Gly AspGluIle PheHisVal AspMetAla LysLys GluThrVal Trp
130 135 140
Arg LeuGluGlu PheGlyArg PheAlaSer PheGlu AlaGlnGly Ala
145 150 155 160
Leu AlaAsnIle AlaValAsp LysAlaAsn LeuGlu IleMetThr Lys
165 170 175
Arg SerAsnTyr ThrProIle ThrAsn
180 185
<210> C0600
<211> 185
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
5 ZO 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
35 40 45
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
64
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
50 55 60
Gln Lys Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys
65 70 75 80
Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Gly
85 90 95
1~ Gly Gly Gly Ser Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe
100 105 110
Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly
115 120 125
Asp G1u Ile Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg
130 135 140
Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu
145 150 ~ 155 160
Ala Asn IIe AIa Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg
165 170 175
Ser Asn Tyr Thr Pro Ile Thr Asn
180
<210> C0599
<211> 184
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
4~ Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
55 60
Gln Lys Asp Leu Leu,Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys
65 70 75 80
Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Gly
$~ 85 90 95
Gly Gly Gly Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr
100 105 110
$5 Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp
115 ' 120 125
Glu Ile Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu
130 135 140
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
6$
Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala
145 150 155 160
$ Asn Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser
165 170 175
Asn Tyr Thr Pro Ile Thr Asn
180
<210> C0594
<211> 187
<212> PRT
1$ <213> Homo Sapiens
Met Gly ThrArg ProArg PheLeuGlu GlnValLys HisGluCys
Asp
5 10 15
His Phe AsnGly ThrGlu ArgValArg PheLeuAsp ArgTyrPhe
Phe
20 25 30
Tyr His GluGlu TyrVal ArgPheAsp SerAspVal GlyGluTyr
Gln
35 40 45
2$
Arg Ala ThrGlu LeuGly ArgProAsp AlaGluTyr TrpAsnSer
Val
50 55 60
Gln Lys LeuLeu GluGln LysArgAla AlaValAsp ThrTyrCys
Asp
65 70 75 80
Arg His TyrGly ValGly GluSerPhe ThrValGln ArgArgVal
Asn
85 90 95
3$ Tyr Pro ValThr ValIle LysGluGlu HisValIle IleGlnAla
Glu
100 105 110
Glu Phe'TyrLeuAsn ProAsp GlnSerGly GluPheMet PheAspPhe
115 120 125
40
Asp Gly GluIle PheHis ValAspMet AlaLysLys GluThrVal
Asp
130 135 140
Trp Arg GluGlu PheGly ArgPheAla SerPheGlu AlaGlnGly
Leu
4$ 145 150 155 160
Ala Leu AsnIle AlaVal AspLysIAlaAsnLeuGlu TleMetThr
Ala
165 170 175
$0 Lys Arg AsnTyr ThrPro IleThrAsn
Ser
180 185
<210> C0592
$$ <211> 188
<212> PRT
<213> Homo Sapiens
Met Gly ThrArg ProArg PheLeuGlu GlnValLys HisGluCys
Asp
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
66
10 15
His Phe PheAsn GlyThrGlu ArgValArg PheLeuAsp ArgTyr Phe
20 25 30
$
Tyr His GlnGlu GluTyrVal ArgPheAsp SerAspVal GIyGlu Tyr
35 40 45
Arg Ala ValThr GluLeuGly ArgProAsp AlaGluTyr TrpAsn Ser
50 55 60
Gln Lys AspLeu LeuGluGln LysArgAla AlaValAsp ThrTyr Cys
65 70 75 80
1$ Arg His AsnTyr GlyValGly GluSerPhe ThrValGln ArgArg Phe
85 90 95
Asp Ala ProSer ProLeuPro IleLysGlu GluHisVal IleIle Gln
100 105 110
Ala Glu PheTyr LeuAsnPro AspGlnSer GlyGluPhe MetPhe Asp
115 120 125
Phe Asp GlyAsp GluIlePhe HisValAsp MetAlaLys LysGlu Thr
2$ 130 135 140
Val Trp ArgLeu GluGluPhe GlyArgPhe AlaSerPhe GluAla Gln
145 150 155 160
Gly Ala LeuAla AsnIleAla ValAspLys AlaAsnLeu GluIle Met
165 170 175
Thr Lys ArgSer AsnTyrThr ProIleThr Asn
180 185
3$
<210>
C0591
<211>
187
<212>
PRT
<213> sapiens
Homo
Met Gly AspThr ArgProArg PheLeuGlu GlnValLys HisGlu Cys
5 10 15
4$ His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
35 40 45
$0
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
55 60
Gln Lys Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys
$$ 65 70 75 80
Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg Arg Ser
85 90 95
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
67
Gly Gly SerGly GlySerIle LysGluGlu HisVal IleIleGln Ala
100 105 110
Glu Phe TyrLeu AsnProAsp GlnSerGly GluPhe MetPheAsp Phe
$ 115 120 125
. Gly AspGlu IlePheHis ValAspMet AlaLys LysGluThr Val
Asp
130 135 140
Trp Arg LeuGlu GluPheGly ArgPheAla SerPhe GluAlaGln Gly
145 150 155 160
Ala Leu AlaAsn IleAlaVal AspLysAla AsnLeu GluIleMet Thr
165 170 175
Lys Arg SerAsn TyrThrPro IleThrAsn
180 185
<210> C0590
<211>
187
<212>
PRT
<213>
Homo
Sapiens
Met GlyAspThr ArgProArg PheLeu GluGlnVaI LysHisGlu Cys
5 10 15
His PhePheAsn GlyThrGlu ArgVal ArgPheLeu AspArgTyr Phe
20 25 30
Tyr HisGlnGlu GluTyrVal ArgPhe AspSerAsp ValGlyGlu Tyr
35 40 45
Arg AlaValThr GluLeuGly ArgPro AspAlaGlu TyrTrpAsn Ser
50 55 60
Gln LysAsp.Leu LeuGluGln LysArg AlaAlaVal AspThrTyr Cys
.
65 - 70 75 80
Arg HisAsnTyr GlyValGly GluSer PheThrVal GlnArgArg Thr
85 90 95
Ser GIyGlyGly GlySerIle LysGlu GluHisVal IleIleGln Ala
100 105 110
Glu PheTyrLeu AsnProAsp GlnSer GlyGluPhe MetPheAsp Phe
115 120 125
Asp GlyAspGlu IlePheHis ValAsp MetAlaLys LysGluThr Val
130 135 140
Trp ArgLeuGlu GluPheGly ArgPhe AlaSerPhe GluAlaGln Gly
145 150 155 160
5$ Ala LeuAlaAsn IleAlaVal AspLys AlaAsnLeu GluIleMet Thr
165 170 175
Lys ArgSerAsn TyrThrPro IleThr Asn
180 185
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
68
<210>
C0589
<211>
190
$ <212>
PRT
<213>
Homo
sapiens
Met Gly AspThr ArgProArg PheLeuGlu GlnValLys HisGluCys
5 10 15
His Phe PheAsn GlyThrGlu ArgValArg PheLeuAsp ArgTyrPhe
20 25 30
Tyr His GlnGlu GluTyrVal ArgPheAsp SerAspVal GlyGluTyr
1$ 35 40 45
Arg Ala ValThr GluLeuGly ArgProAsp AlaGluTyr TrpAsnSer
50 55 60
Gln Lys AspLeu LeuGluGln LysArgAla AlaValAsp ThrTyrCys
65 70 75 80
Arg His AsnTyr GlyValGly GluSerPhe ThrValGln ArgArgGly
85 90 95
Ser Pro GlyGly GlyGlyPro GlySerIle LysGluGlu HisValIle
100 105 110
Ile Gln AlaGlu PheTyrLeu AsnProAsp GlnSerGly GluPheMet
115 120 125
Phe Asp PheAsp GlyAspGlu IlePheHis ValAspMet AlaLysLys
130 135 140
Glu Thr ValTrp ArgLeuGlu GluPheGly ArgPheAla SerPheGlu
145 150 155 160
Ala Gln ~GlyAla LeuAlaAsn IleAlaVal AspLysAla AsnLeuGlu
165 170 175
Ile Met ThrLys ArgSerAsn TyrThrPro IleThrAsn
180 185
4S <220> C0588
<211> 190
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
5$
Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr
35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
69
50 55 60
Gln Lys AspLeuLeu GluGlnLys ArgAlaAla ValAspThr TyrCys
65 70 75 80
Arg His AsnTyrGly ValGlyGlu SerPheThr ValGlnArg ArgGly
85 90 95
Ser Pro ProGlyGly ProProGly SerIleLys GluGluHis ValIle
100 105 110
Ile Gln AlaGluPhe TyrLeuAsn ProAspGln SerGlyGlu PheMet
115 120 125
Phe Asp PheAspGly AspGluIle PheHisVal AspMetAla LysLys
130 135 140
Glu Thr ValTrpArg LeuGluGlu PheGlyArg PheAlaSer PheGlu
145 150 155 160
Ala Gln GlyAlaLeu AlaAsnIle AlaValAsp LysAlaAsn LeuGlu
165 170 175
Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn
1ao 1ss
<210>
C0587
<211>
194
<212>
PRT
<2I3> Sapiens
Homo
Met GlyAsp ThrArg ProArgPhe LeuGluGln ValLysHis GluCys
5 10 15
His PhePhe AsnGly ThrGluArg ValArgPhe LeuAspArg TyrPhe
20 25 30
Tyr HisGln GluGlu TyrValArg PheAspSer AspValGly GluTyr
35 40 45
Arg AlaVal ThrGlu LeuGlyArg ProAspAla GluTyrTrp AsnSer
50 55 60
Gln LysAsp LeuLeu GluGlnLys ArgAlaAla ValAspThr TyrCys
65 70 75 80
Arg HisAsn TyrGly ValGlyGlu SerPheThr ValGlnArg ArgGly
85 90 95
Ser ProGly GlyGly GlySerGly GlyGlyPro GlySerIle LysGlu
100 105 110
Glu HisVal IleIle GlnAlaGlu PheTyrLeu AsnProAsp GlnSer
5$ 115 120 125
Gly GluPhe MetPhe AspPheAsp GlyAspGlu TlePheHis ValAsp
130 135 140
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe
145 150 155 160
Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala Val Asp Lys
$ 165 170 175
Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr
180 185 190
10 Asn
<210> C0586
IS <211> 194
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys
20 5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
2$ Tyr His GlnGluGlu TyrVal ArgPheAsp SerAspVal GlyGluTyr
35 40 45
Arg Ala ValThrGlu LeuGly ArgProAsp AlaGluTyr TrpAsnSer
50 55 ~ 60
30
Gln Lys AspLeuLeu GluGln LysArgAla AlaValAsp ThrTyrCys
65 70 ' 75 80
Arg His AsnTyrGly ValGly GluSerPhe ThrValGln ArgArgThr
35 85 90 95
Ser Gly GlyGlyGly SerGly GlyGlyGly SerSerSer IleLysGlu
100 105 110
40 Glu His ValIleIle GlnAla GluPheTyr LeuAsnPro AspGlnSer
115 120 125
Gly Glu PheMetPhe AspPhe AspGlyAsp GluIlePhe HisValAsp
130 135 140
45
Met Ala LysLysGlu ThrVal TrpArgLeu GluGluPhe GlyArgPhe
145 150 155 160
Ala Ser PheGluAla GlnGly AlaLeuAla AsnIleAla ValAspLys
$0 165 170 175
Ala Asn LeuGluIle MetThr LysArgSer AsnTyrThr ProIIeThr
180 185 190
5$ Asn
<210> C0598
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
71
<211> 639
<212> DNA
<213> Homo Sapiens
S atgggggaca ccggtcgcagcttcaccctggcctccagcgagaccggcgtgggcgctagc60
ggagggggcg gaagcggcggagggggggacacccgaccacgtttcttggagcaggttaaa120
catgagtgtc atttcttcaacgggacggagcgggtgcggttcctggacagatacttctat180
caccaagagg agtacgtgcgcttcgacagcgacgtgggggagtaccgggcggtgacggag240
ctggggcggc ctgatgccgagtactggaacagccagaaggacctcctggagcagaagcgg300
gccgcggtgg acacctactgcagacacaactacggggttggtgagagcttcacagtgctg360
cgccgactcg gaggtgaagatgacgaggcagatcaccatgtgatcatccaggccgagttc420
tatctgaatc ctgaccaatcaggcgagtttatgtttgactttgatggtgatgagattttc480
catgtggata tggcaaagaaggagacggtctggcggcttgaagaatttggacgatttgcc540
agctttgagg ctcaaggtgcattggccaacatagctgtggacaaagccaacctggaaatc600
atgacaaagc gctccaactatactccgatcaccaattaa 639
<210> C0597
<211> 639
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcagcttcaccctggcctccagcgagaccggcgtgggcgctagc60
ggagggggcg gaagcggcggagggggggacacccgaccacgtttcttggagcaggttaaa120
catgagtgtc atttcttcaacgggacggagcgggtgcggttcctggacagatacttctat180
caccaagagg agtacgtgcgcttcgacagcgacgtgggggagtaccgggcggtgacggag240
ctggggcggc ctgatgccgagtactggaacagccagaaggacctcctggagcagaagcgg300
gccgcggtgg acacctactgcagacacaactacggggttggtgagagcttcacagtgcag360
cggcgaggag gtggaagcggcggaatcaaagaagaacatgtgatcatccaggccgagttc420
tatctgaatc ctgaccaatcaggcgagtttatgtttgactttgatggtgatgagattttc480
catgtggata tggcaaagaaggagacggtctggcggcttgaagaatttggacgatttgcc540
agctttgagg ctcaaggtgcattggccaacatagctgtggacaaagccaacctggaaatc600
atgacaaagc gctccaactatactccgatcaccaattaa 639
<210> C0596
<211> 636
<212 > DNA
<213> Homo Sapiens
atgggggaca ccggtcgcagcttcaccctggcctccagcgagaccggcgtgggcgctagc60
ggagggggcg gaagcggcggagggggggacacccgaccacgtttcttggagcaggttaaa120
catgagtgtc atttcttcaacgggacggagcgggtgcggttcctggacagatacttctat180
caccaagagg agtacgtgcgcttcgacagcgacgtgggggagtaccgggcggtgacggag240
ctggggcggc ctgatgccgagtactggaacagccagaaggacctcctggagcagaagcgg300
gccgcggtgg acacctactgcagacacaactacggggttggtgagagcttcacagtgcag360
cggcgaggag gtggaggcagcatcaaagaagaacatgtgatcatccaggccgagttctat420
ctgaatcctg accaatcaggcgagtttatgtttgactttgatggtgatgagattttccat480
gtggatatgg caaagaaggagacggtctggcggcttgaagaatttggacgatttgccagc540
tttgaggctc aaggtgcattggccaacatagctgtggacaaagccaacctggaaatcatg600
acaaagcgct ccaactatactccgatcaccaattaa 636
<210> C0595
<211> 633
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
72
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgaggag gtggaggcat caaagaagaa catgtgatca tccaggccga gttctatctg 420
aatcctgacc aatcaggcga gtttatgttt gactttgatg gtgatgagat tttccatgtg 480
S gatatggcaa agaaggagac ggtctggcgg cttgaagaat ttggacgatt tgccagcttt 540
gaggctcaag gtgcattggc caacatagct gtggacaaag ccaacctgga aatcatgaca 600
aagcgctcca actatactcc gatcaccaat taa 633
<2l0> C0593
<211> 642
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgagtct accctgaggt aactgtcatc aaagaagaac atgtgatcat ccaggccgag 420
ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 480
ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 540
gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 600
atcatgacaa agcgctccaa ctatactccg atcaccaatt as 642
<210> C0585
<211> 645
<212> DNA
<213> Homo sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgattcg acgcacctag cccactccca atcaaagaag aacatgtgat catccaggcc 420
gagttctatc tgaatcctga ccaatcaggc gagtttatgt ttgactttga tggtgatgag 480
attttccatg tggatatggc aaagaaggag acggtctggc ggcttgaaga atttggacga 540
tttgccagct ttgaggctca aggtgcattg gccaacatag ctgtggacaa agccaacctg 600
gaaatcatga caaagcgctc caactatact ccgatcacca attaa 645
<210> C0584
<211> 642
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
'catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgaagtg gcggtagtgg cggtagtatc aaagaagaac atgtgatcat ccaggccgag 420
ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 480
ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 540
gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 600
atcatgacaa agcgctccaa ctatactccg atcaccaatt as 642
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
73
<210> C0583
<211> 642
<212> DNA
$ <213> HomoSapiens
atgggggacaccggtcgcagcttcaccctggcctccagcgagaccggcgtgggcgctagc60
ggagggggcggaagcggcggagggggggacacccgaccacgtttcttggagcaggttaaa120
catgagtgtcatttcttcaacgggacggagcgggtgcggttcctggacagatacttctat180
caccaagaggagtacgtgcgcttcgacagcgacgtgggggagtaccgggcggtgacggag240
ctggggcggcctgatgccgagtactggaacagccagaaggacctcctggagcagaagcgg300
gccgcggtggacacctactgcagacacaactacggggttggtgagagcttcacagtgcag360
cggcgaactagtggtggcggtggcagcatcaaagaagaacatgtgatcatccaggccgag420
ttctatctgaatcctgaccaatcaggcgagtttatgtttgactttgatggtgatgagatt480
ttccatgtggatatggcaaagaaggagacggtctggcggcttgaagaatttggacgattt540
gccagctttgaggctcaaggtgcattggccaacatagctgtggacaaagccaacctggaa600
atcatgacaaagcgctccaactatactccgatcaccaattas 642
<210> C0582
<211> 651
<212> DNA
<213> HomoSapiens
atgggggacaccggtcgcagcttcaccctggcctccagcgagaccggcgtgggcgctagc60
2S ggagggggcggaagcggcggagggggggacacccgaccacgtttcttggagcaggttaaa120
catgagtgtcatttcttcaacgggacggagcgggtgcggttcctggacagatacttctat180
caccaagaggagtacgtgcgcttcgacagcgacgtgggggagtaccgggcggtgacggag240
ctggggcggcctgatgccgagtactggaacagccagaaggacctcctggagcagaagcgg300
gccgcggtggacacctactgcagacacaactacggggttggtgagagcttcacagtgcag360
3~ cggcgaggctctcctggaggtggaggtcctggatctatcaaagaagaacatgtgatcatc420
caggccgagttctatctgaatcctgaccaatcaggcgagtttatgtttgactttgatggt480
gatgagattttccatgtggatatggcaaagaaggagacggtctggcggcttgaagaattt540
ggacgatttgccagctttgaggctcaaggtgcattggccaacatagctgtggacaaagcc600
aacctggaaatcatgacaaagcgctccaactatactccgatcaccaattaa 651
35
<210> C0581
<211> 651
<212> DNA
40 <213> HomoSapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
45 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgaggct ctcctcctgg tggaccacct ggatctatca aagaagaaca tgtgatcatc 420
caggccgagt tctatctgaa tcctgaccaa tcaggcgagt ttatgtttga ctttgatggt 480
5~ gatgagattt tccatgtgga tatggcaaag aaggagacgg tctggcggct tgaagaattt 540
ggacgatttg ccagctttga ggctcaaggt gcattggcca acatagctgt ggacaaagcc 600
aacctggaaa tcatgacaaa gcgctccaac tatactccga tcaccaatta a 651
55 <210> C0580
<211> 663
<212> DNA
<213> Homo Sapiens
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
74
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
$ ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgaggga gtccaggcgg tggcgggagc ggcggtggtc ctgggagtat caaagaagaa 420
catgtgatca tccaggccga gttctatctg aatcctgacc~aatcaggcga gtttatgttt 480
gactttgatg gtgatgagat tttccatgtg gatatggcaa agaaggagac ggtctggcgg 540
cttgaagaat ttggacgatt tgccagcttt gaggctcaag gtgcattggc caacatagct 600
gtggacaaag ccaacctgga aatcatgaca aagcgctcca actatactcc gatcaccaat 660
taa 663
1$ <210> C0567
<211> 663
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60
ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120
catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180
caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240
ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300
2,$ gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360
cggcgaacta gtggtggcgg tggcagcggc ggtggtggtt cctcgagtat caaagaagaa 420
catgtgatca tccaggccga gttctatctg aatcctgacc aatcaggcga gtttatgttt 480
gactttgatg gtgatgagat tttccatgtg gatatggcaa agaaggagac ggtctggcgg 540
cttgaagaat ttggacgatt tgccagcttt gaggctcaag gtgcattggc caacatagct 600
gtggacaaag ccaacctgga aatcatgaca aagcgctcca actatactcc gatcaccaat 660
taa 663
<210> C0598
3$ <211> 213
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
4$ Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
35 40 45
$0
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
$$ 85 90 95
Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His,Asn Tyr Gly
100 105 110
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
~$
Val Gly Glu Ser Phe Thr Val Leu Arg Arg Leu Gly Gly Glu Asp Asp
115 120 125
Glu Ala Asp His His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
$ 130 135 140
Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
145 150 155 160
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
165 170 175
1$
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
180 185 190
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
I95 200 ' 205
Pro Ile Thr Asn
210
<210> C0597
<211> 213
2$ <212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
3$ 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
50- 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
65 70 75 80
4$
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
85 90 95
Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly
100 105 110
Val Gly Glu Ser Phe Thr Val Gln Arg Arg Gly Gly Gly Ser Gly Gly
$0 115 120 125
Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn Pro
130 135 140
$$ Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile Phe
145 150 155 160
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg.Leu Glu Glu Phe
165 170 175
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
76
Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile Ala
180 185 190
Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr Thr
195 200 205
Pro Ile Thr Asn
210
<210>
C0596
<211>
212
<212>
PRT
IS <213>
Homo
sapiens
Met Gly AspThrGly ArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
Val Gly AlaSerGly GlyGly GlySerGly GlyGlyGly AspThrArg
20 25 30
Pro Arg PheLeuGlu GlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
Thr Glu ArgValArg PheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPheAsp SerAsp ValGlyGlu TyrArgAla ValThrGlu
65 70 75 80
Leu Gly ArgProAsp AlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
85 90 95
3$ Glu Gln LysArgAla AlaVal AspThrTyr CysArgHis AsnTyrGly
100 105 110
Val Gly GluSerPhe ThrVal GlnArgArg GlyGlyGly GlySerIle
115 120 125
Lys Glu GluHisVal IleIle GlnAlaGlu PheTyrLeu AsnProAsp
13 13 14'0
0 5
Gln Ser GlyGluPhe MetPhe AspPheAsp GlyAspGlu IlePheHis
145 150 155 160
Val Asp MetAlaLys LysGlu ThrValTrp ArgLeuGlu GluPheGly
165 170 175
$0 Arg Phe AlaSerPhe GluAla GlnGlyAla LeuAlaAsn IleAlaVal
180 185 190
Asp ,LysAlaAsnLeu GluIle MetThrLys ArgSerAsn TyrThrPro
195 200 205
Ile Thr Asn
210
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
77
<210>
C0595
<211>
ZI1
<212>
PRT
<213>
Homo
Sapiens
Met Gly AspThrGly ArgSer PheThrLeuAla SerSer GluThrGly
5 10 15
Val Gly AlaSerGly GlyGly GlySerGlyGly GlyGly AspThrArg
20 25 30
Pro Arg PheLeuGlu GlnVal LysHisGluCys HisPhe PheAsnGly
35 40 ~ 45
Thr Glu ArgValArg PheLeu AspArgTyrPhe TyrHis GlnGluGlu
50 55 60
Tyr ~ValArgPheAsp SerAsp ValGlyGluTyr ArgAla ValThrGlu
65 70 75 80
Leu Gly ArgProAsp AlaGlu TyrTrpAsnSer GlnLys AspLeuLeu
85 90 95
Glu Gln LysArgAla AlaVal AspThrTyrCys ArgHis AsnTyrGly
100 105 110
Val Gly GluSerPhe ThrVal GlnArgArgGly GlyGly GlyIleLys
115 120 125
3~ Glu Glu HisValIle IleGln AlaGluPheTyr LeuAsn ProAspGln
130 135 140
Ser Gly GluPheMet PheAsp PheAspGlyAsp GluIle PheHisVal
145 150 155 160
35
Asp Met AlaLysLys GluThr ValTrpArgLeu GluGlu PheGlyArg
165 170 175
Phe Ala SerPheGlu AlaGln GlyAlaLeuAla AsnIle AlaValAsp
180 185 190
Lys Ala AsnLeuGlu IleMet ThrLysArgSer AsnTyr ThrProIle
195 200 '205
45 Thr Asn
210
<210> C0593
50 <211> 214
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5$ 5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
25 30
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
78
Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
65 70 7S 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
85 90 95
Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly
100 105 110
Val Gly Glu Ser Phe Thr Val Gln Arg Arg Val Tyr Pro Glu Val Thr
115 120 125
Val Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn
130 135 140
Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile
145 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu
165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile
180 185 190
Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr
195 200 205
Thr Pro Ile Thr Asn
210
<210> C0585
<211> 215
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
$0 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
70 ' 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
85 90 9S
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
79
Glu Gln LysArg AlaAlaVal AspThrTyr CysArgHis AsnTyrGly
100 105 110
Val Gly GluSer PheThrVal GlnArgArg PheAspAla ProSerPro
115 120 125
Leu Pro IleLys GluGluHis ValIleIle GlnAlaGlu PheTyrLeu
130 135 140
Asn Pro AspGln SerGlyGlu PheMetPhe AspPheAsp GlyAspGlu
145 150 155 160
Ile Phe HisVal AspMetAla LysLysGlu ThrValTrp ArgLeuGlu
1$ 165 170 175
Glu Phe GlyArg PheAlaSer PheGluAla GlnGlyAla LeuAlaAsn
180 185 190
Ile Ala ValAsp LysAlaAsn LeuGluIle MetThrLys ArgSerAsn
195 200 205
Tyr Thr ProIle ThrAsn
210
~$
<210>
C0584
<211>
214
<212>
PRT
<213> Sapiens
Homo
Met Gly AspThr GlyArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
Val Gly AlaSer GlyGlyGly GlySerGly GlyGlyGly AspThrArg
20 25 30
Pro Arg ~PheLeu GluGlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
Thr Glu ArgVal ArgPheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPhe AspSerAsp ValGlyGlu TyrArgAla ValThrGlu
4S 65 70 75 80
Leu Gly ArgPro AspAlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
85 90 95
Glu Gln LysArg AlaAlaVal AspThrT,yrCysArgHis AsnTyrGly
100 105 110
Val Gly Glu Ser Phe Thr Val Gln Arg Arg Ser Gly Gly Ser Gly Gly
115 120 125
Ser Ile Lys G1u Glu His Val Tle Ile Gln Ala Glu Phe Tyr Leu Asn
130 135 140
Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
145 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu
165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile
180 185 190
Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr
10 195 200 205
Thr Pro Ile Thr Asn
210
1$
<210>
C0583
<211>
214
<212>
PRT
<213> Sapiens
Homo
20
Met Gly AspThr GlyArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
Val Gly AlaSer GlyGlyGly GlySerGly GlyGlyGly AspThrArg
2$ 20 25 30
Pro Arg PheLeu GluGlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
30 Thr Glu ArgVal ArgPheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPhe AspSerAsp ValGlyGlu TyrArgAla ValThrGlu
65 ~ 70 75 80
3$
Leu Gly ArgPro AspAlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
85 90 95
Glu Gln LysArg AlaAlaVal AspThrTyr CysArgHis AsnTyrGly
40 100 105 110
Val Gly GluSer PheThrVal GlnArgArg ThrSerGly GlyGlyGly
115 120 125
4$ Ser Ile LysGlu GluHisVal IleIleGln AlaGluPhe TyrLeuAsn
_ 130 ' 135 140
Pro Asp GlnSer GlyGluPhe MetPheAsp PheAspGly AspGluIle
145 150 155 160
$0
Phe His ValAsp MetAlaLys LysGluThr ValTrpArg LeuGluGlu
165 170 175
Phe Gly ArgPhe AlaSerPhe GluAlaGln GlyAlaLeu AlaAsnIle
$$ 180 185 190
Ala Val AspLys AlaAsnLeu GluIleMet ThrLysArg SerAsnTyr
195 200 205
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81
Thr Pro Ile Thr Asn
210
$ <210>
00582
<211>
217
<212>
PRT
<213> sapiens
Homo
Met Gly AspThrGly ArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
Val Gly AlaSerGly GlyGly GlySerGly GlyGlyGly AspThrArg
20 25 30
1$
Pro Arg PheLeuGlu GlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
Thr Glu ArgValArg PheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPheAsp SerAsp ValGlyGlu TyrArgAla ValThrGlu
65 70 75 80
Leu Gly ArgProAsp AlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
85 90 95
Glu Gln LysArgAla AlaVal AspThrTyr CysArgHis AsnTyrGly
100 105 110
Val Gly GluSerPhe ThrVal GlnArgArg GlySerPro GlyGlyGly
115 120 125
Gly Pro GlySerTle LysGlu GluHisVal IleIleGln AlaGluPhe
3$ 130 135 140
Tyr Leu AsnProAsp GlnSer GlyGluPhe MetPheAsp PheAspGly
145 - 150 155 160
Asp Glu IlePheHis ValAsp MetAlaLys LysGluThr ValTrpArg
165 170 175
Leu Glu GluPheGly ArgPhe AlaSerPhe GluAlaGln GlyAlaLeu
180 185 190
4$
Ala Asn IleAlaVal AspLys AlaAsnLeu GluIleMet ThrLysArg
l95 200 205
Ser Asn TyrThrPro IleThr Asn
$0 210 215
<210>
00581
<211>
217
$$ <212>
PRT
<213> Sapiens
Homo
Met Gly AspThrGly ArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
CA 02403432 2002-09-23
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82
Val Gly AlaSerGly GlyGly GlySerGly GlyGlyGly AspThrArg
20 25 30
$ Pro Arg PheLeuGlu GlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
Thr Glu ArgValArg PheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPheAsp SerAsp ValGlyGlu TyrArgAla ValThrGlu
65 70 75 80
Leu Gly ArgProAsp AlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
1$ 85 90 95
Glu Gln LysArgAla AlaVal AspThrTyr CysArgHis AsnTyrGly
100 105 110
Val Gly GluSerPhe ThrVal GlnArgArg GlySerPro ProGlyGly
115 120 125
Pro Pro GlySerIle LysGlu GluHisVal IleIleGln AlaGluPhe
130 135 140
2$
Tyr Leu AsnProAsp GlnSer GlyGluPhe MetPheAsp PheAspGly
145 150 155 160
Asp Glu IlePheHis ValAsp MetAlaLys LysGluThr ValTrpArg
165 170 175
Leu Glu GluPheGly ArgPhe AlaSerPhe GluAlaGln GlyAlaLeu
180 185 190
3$ Ala Asn IleAlaVal AspLys AlaAsnLeu GluIleMet ThrLysArg
195 200 205
Ser Asn'Tyr Thr Pro Ile Thr Asn
210 215
<210> C0580
<211> 221
<212> PRT
4$ <213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
$0 Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
35 40 45
$$
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
CA 02403432 2002-09-23
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65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
85 90 95
Glu Gln LysArgAla AlaVal AspThrTyr CysArgHis AsnTyrGly
100 105 110
Val Gly GluSerPhe ThrVal GlnArgArg GlySerPro GlyGlyGly
115 120 125
Gly Ser GlyGlyGly ProGly SerIleLys GluGluHis ValIleIle
130 135 140
IS Gln AIa GluPheTyr LeuAsn ProAspGln SerGlyGlu PheMetPhe
145 150 155 160
Asp Phe AspGlyAsp GluIle PheHisVal AspMetAla LysLysGlu
165 170 175
Thr Val TrpArgLeu GluGlu PheGlyArg PheAlaSer PheGluAla
180 185 190
Gln Gly AlaLeuAla AsnIle AlaValAsp LysAlaAsn LeuGluIle
195 200 205
Met Thr LysArgSer AsnTyr ThrProIle ThrAsn
210 215 220
<210>
C0567
<211>
221
<212>
PRT
<213>
Homo
sapiens
Met Gly AspThrGly ArgSer PheThrLeu AlaSerSer GluThrGly
5 10 15
Val Gly AlaSerGly GlyGly GlySerGly GlyGlyGly AspThrArg
20 25 30
Pro Arg PheLeuGlu GlnVal LysHisGlu CysHisPhe PheAsnGly
35 40 45
Thr Glu ArgValArg PheLeu AspArgTyr PheTyrHis GlnGluGlu
50 55 60
Tyr Val ArgPheAsp SerAsp ValGlyGlu TyrArgAla ValThrGlu
65 70 75 80
Leu Gly ArgProAsp AlaGlu TyrTrpAsn SerGlnLys AspLeuLeu
85 90 95
Glu Gln LysArgAla AlaVal AspThrTyr CysArgHis AsnTyrGly
5$ 100 105 110
Val Gly GluSerPhe ThrVal GlnArgArg ThrSerGly GlyGlyGly
115 120 125
CA 02403432 2002-09-23
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84
Ser Gly Gly Gly Gly Ser Ser Ser Ile Lys Glu Glu His Val Ile IIe
130 135 140
Gln Ala Glu Phe Tyr Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe
$ 145 150 155 160
Asp Phe Asp Gly Asp Glu Ile Phe His VaI Asp Met AIa Lys Lys Glu
165 170. 175
1~ Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala
' 180 185 190
Gln Gly Ala Leu Ala Asn Ile AIa Val Asp Lys A7.a Asn Leu Glu Ile
195 200 205
1$
Met Thr Lys Arg Ser Asn Tyr Thr Pro Ile Thr Asn
210 215 220
I-As MBP. (31(32a1a~ . Cx nucleotide sequence
ao
gcggccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttcca60
ggttccactggtgacttcaagaacattgtgacacctcgaacaccacctccagctagcgga120
gggggcggaagcggcggagggggagactccgaaaggcatttcgtgttccagttcaagggc180
gagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatctacaac240
2$ cgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgaccgagctg300
gggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggccgagctg360
gacacggtgtgcagacacaactacgagggggtggagacccacacctccctgcggcggctt420
gaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccacaacact480
ctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttccggaat540
3~ ggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggactggacc600
ttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacctgccac660
gtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggcggtggc720
agcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagtgaagac780
gacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcctggagac840
3$ attggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttggataag900
aaggagactatctggatgcttcctgagtttggccaattgacaagctttgacccccaaggt960
ggactgcaaaacatagctacaggaaaatacaccttgggaatcttgactaagaggtcaaat1020
tccaccccagctaccaatgaggctcctcaagcgactgtgttccccaagtcccctgtgctg1080
ctgggtcagcccaacaccctcatctgctttgtggacaacatcttccctcctgtgatcaac1140
4~ atcacatggctcagaaatagtaagtcagtcacagacggcgtttatgagaccagcttcctt1200
gtcaaccgtgaccattccttccacaagctgtcttatctcaccttcatcccttctgacgat1260
gatatttatgactgcaaggtggagcactggggcctggaggagccggttctgaaacactgg1320
gctagcggagggggcggaagcggcggagggggagctgatgctgcaccaactgtatccatc1380
ttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttgaac1440
4$ aacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaaaat1500
ggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagcagc1560
accctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggccact1620
cacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttagggtacc1680
$0 I-As MBP . (31(32a1a2 . Cx amino acid sequence
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
10 15
$$ Gly Ser Thr Gly Asp Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro
20 25 30
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
8$
Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg
35 40 45
$ His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr
50 55 60
Gln Arg Ile Arg~Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr
65 70 75 80
Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu
85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu Glu Gln Thr
1$ 100 105 lI0
Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu
115 120 125
Thr His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Val Ile Ser
130 135 140
2$
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser
145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn
165 170 175
Gly Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile Arg Asn
180 185 190
Gly Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr Pro Arg
195 200 205
3$ Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser
210 215 220
Pro Ile~Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp
245 250 255
Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gln
4$ 260 265 270
Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly Asp Glu
275 280 285
$0 Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met Leu Pro
290 295 300
Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn
305 310 315 320
$$
Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg Ser Asn
325 330 335
Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe Pro Lys
CA 02403432 2002-09-23
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340 345 350
S
Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe Val Asp
355 360 365
Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn Ser Lys
370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp
385 390 395 400
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile Pro.Ser Asp Asp
405 410 415
1$ Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu GIu Pro Val
420 425 430
Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala
435 440 445
Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu
450 455 460
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro
465 470 475 480
Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn
485 490 495
Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr
500 505 510
Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His
515 520 525
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile
530 535 540
Val Lys Ser Phe Asn Arg Asn Glu Cys
545 550
<210> C0528-AC
<211> 600
<212> DNA
<213> Homo Sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggtggtggc 60
ggttcgcgtc cacgtttctt ggaacaggtt aaacatgagt gtcatttttt caatgggacg 120
gaacgcgtgc gttttctgga tcgttacttt tatcaccaag aggaatacgt acgcttcgac 180
agcgatgtgg gcgaatatcg tgcggtcacg gaactgggtc gtcctgatgc cgaatactgg 240
aacagtcaga aggacttact ggagcagaaa cgtgcagcgg tggataccta ttgccgccac 300
aattacggcg ttggtgaaag cttcacagtc cagcgtcgcg gtggcatcaa agaagagcat 360
gtgattatcc aggcggaatt ctatctgaat ccggatcaat cgggcgaatt catgtttgac 420
ttcgatggtg atgagatttt ccatgttgat atggcaaaga aagaaacggt ctggcgctta 480
gaggaatttg gccgctttgc ctcgttcgaa gctcaaggcg cattggccaa cattgctgtg 540
gataaagcga acctggaaat catgacaaaa cgctccaact atactccgat taccaattaa 600
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
87
<210> C0608-AC
<211> 642
<212> DNA
S <213> Homo sapiens
atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggtgcttct 60
ggcgggggcg gttcgggcgg tgggggtgac acccgtccac gtttcttgga acaggttaaa 120
catgagtgtc attttttcaa tgggacggaa cgcgtgcgtt ttctggatcg ttacttttat 180
1O caccaagagg aatacgtacg cttcgacagc gatgtgggcg aatatcgtgc ggtcacggaa 240
ctgggtcgtc ctgatgccga atactggaac agtcagaagg acttactgga gcagaaacgt 300
gcagcggtgg atacctattg ccgccacaat tacggcgttg gtgaaagctt cacagtccag 360
cgtcgcacta gtggtggcgg tggctctatc aaagaagagc atgtgattat ccaggcggaa 420
ttctatctga atccggatca atcgggcgaa ttcatgtttg acttcgatgg tgatgagatt 480
1S ttccatgttg atatggcaaa gaaagaaacg gtctggcgct tagaggaatt tggccgcttt 540
gcctcgttcg aagctcaagg cgcattggcc aacattgctg tggataaagc gaacctggaa 600
atcatgacaa aacgctccaa ctatactccg attaccaatt as 642
2O <210> 00528
<211> 600
<212> DNA
<213> Homo sapiens
2S atgggggaca ccggaagatc gttcacactc gcatcatcag agacaggagt aggaggagga 60
ggatcgcgac cacgtttctt ggagcaggtt aaacatgagt gtcatttctt caacgggacg 120
gagcgggtgc ggttcctgga cagatacttc tatcaccaag aggagtacgt gcgcttcgac 180
agcgacgtgg gggagtaccg ggcggtgacg gagctggggc ggcctgatgc cgagtactgg 240
aacagccaga gggacctcct ggagcagaag cgggccgcgg tggacaccta ctgcagacac 300
30 aactacgggg ttggtgagag cttcacagtg cagcggcgag gaggtatcaa agaagaacat 360
gtgatcatcc aggccgagtt ctatctgaat cctgaccaat caggcgagtt tatgtttgac 420
tttgatggtg atgagatttt ccatgtggat atggcaaaga aggagacggt ctggcggctt 480
gaagaatttg gacgatttgc cagctttgag gctcaaggtg cattggccaa catagctgtg 540
gacaaagcca acctggaaat catgacaaag cgctccaact atactccgat caccaattaa 600
3S
<210> 00608
<211> 630
<212> DNA
4O <213> Homo Sapiens
ATGGGGGACACCCGCAGCTTCACCCTGGCCTCCAGCGAGACCGGCGTGGGCGCTAGCGGAGGGGGCGGAAGC
GGCGGAGGGGGGCCACGTTTCTTGGAGCAGGTTAAACATGAGTGTCATTTCTTCAACGGGACGGAGCGGGTG
CGGTTCCTGGACAGATACTTCTATCACCAAGAGGAGTACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGG
4S GCGGTGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAAGCGG
GCCGCGGTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAACTAGT
GGTGGCGGTGGCAGCATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAATCCTGACCAATCA
GGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAGGAGACGGTCTGG
CGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATAGCTGTGGACAAA
SO GCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGATCACCAATTAA
<210> CO 608 variation
<211> 642
SS <212> DNA--00608
<213> Homo Sapiens
ATGGGGGACACCGGTCGCAGCTTCACCCTGGCCTCCAGCGAGACCGGCGTGGGCGCTAGCGGAGGGGGCGGA
AGCGGCGGAGGGGGGGACACCCGACCACGTTTCTTGGAGCAGGTTAAACATGAGTGTCATTTCTTCAACGGG
CA 02403432 2002-09-23
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88
ACGGAGCGGGTGCGGTTCCTGGACAGATACTTCTATCACCAAGAGGAGTACGTGCGCTTCGACAGCGACGTG
GGGGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTG
GAGCAGAAGCGGGCCGCGGTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAG
CGGCGAACTAGTGGTGGCGGTGGCAGCATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAAT
S CCTGACCAATCAGGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAG
GAGACGGTCTGGCGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATA
GCTGTGGACAAAGCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGATCACCAATTAA
<210> C0528-AC
<211> 200
<212> PRT
<213> Homo Sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Gly Gly Gly Ser Arg Pro Arg Phe Leu Glu Gln Val Lys His
20 25 30
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg
35 40 45
Tyr Phe Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly
50 55 60
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
65 70 75 80
Asn Ser Gln Lys Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp Thr
85 90 95
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg
100 105 110
Arg Gly Gly Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe.Tyr
115 120 125
Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp
130 135 140
Glu Ile Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu
145 150 155 160
Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala
165 170 175
Asn Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser
180 185 190
Asn Tyr Thr Pro Ile Thr Asn
195
$5 <210> C0608-AC
<211> 214
<212> PRT
<213> Homo Sapiens
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
89
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
$ 20 25 30
Pro Arg Phe Leu Glu Gln Val Lys His Glu Cys His Phe Phe Asn Gly
35 40 45
IO Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gln Glu Glu
50 55 60
IS
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gln Lys Asp Leu Leu
85 90 95
Glu Gln Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly
ZO 100 105 110
Val Gly Glu Ser Phe Thr Val Gln Arg Arg Thr Ser Gly Gly Gly Gly
115 120 125
2$ Ser Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr Leu Asn
130 135 140
Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu Ile
145 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu
165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala Asn Ile
180 185 190
Ala Val Asp Lys AIa Asn Leu Glu Ile Met Thr Lys Arg Ser Asn Tyr
195 200 205
Thr Pro Ile Thr Asn
210
<210> C0608
4S <211> 209
<212> PRT
<213> Homo sapiens
MGDTRSFTLASSETGVGASGGGGSGGGGPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYR
SO AVTELGRPDAEYWNSQKDLLEQKR.AAVDTYCRHNYGVGESFTVQRRTSGGGGSIKEEHVIIQAEFYLNPDQS
GEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIAVDKANLEIMTKRSNYTPITN
<210> C0608-variation
55 <211> 211
<212> PRT
<213> Homo Sapiens
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
10
MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDV
GEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRTSGGGGSIKEEHVIIQAEFYLN
PDQSGEFMFDFDGDEIFHVDMAKKETVWRLEEFGRFASFEAQGALANIADKANLEIMTKRSNYTPIT
<210> C0528
<211> 200
<212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Gly Gly Gly Ser Arg Pro Arg Phe Leu Glu Gln Val Lys His
15 20 25 30
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg
35 40 45
20 Tyr Phe Tyr His Gln Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly
50 55 60
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp
65 70 75 80
Asn Ser Gln Arg Asp Leu Leu Glu Gln Lys Arg Ala Ala Val Asp Thr
85 90 95
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gln Arg
100 105 110
Arg Gly Gly Ile Lys Glu Glu His Val Ile Ile Gln Ala Glu Phe Tyr
115 120 125
Leu Asn Pro Asp Gln Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp
130 135 140
Glu Ile~Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu
145 150 155 160
Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gln Gly Ala Leu Ala
165 170 175
Asn Ile Ala Val Asp Lys Ala Asn Leu Glu Ile Met Thr Lys Arg Ser
180 185 190
Asn Tyr Thr Pro Ile Thr Asn
195
<210> IAS MBPl-l4 CHl.CH2.CH3
<211> 2346
<212> DNA
<213> murine
gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60
ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120
agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
91
aagggcgagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatc240
tacaaccgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgacc300
gagctggggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggcc360
gagctggacacggtgtgcagacacaactacgagggggtggagacccacacctccctgcgg420
cggcttgaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccac480
aacactctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttc540
cggaatggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggac600
tggaccttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacc660
tgccacgtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggc720
ggtggcagcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagt780
gaagacgacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcct840
ggagacattggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttg900
gataagaaggagactatctggatgcttcctgagtttggccaattgacaagctttgacccc960
caaggtggactgcaaaacatagctacaggaaaatacaccttgggaatcttgactaagagg1020
tcaaattccaccccagctaccaatgaggctcctcaagcgactgtgttccccaagtcccct1080
gtgctgctgggtcagcccaacaccctcatctgctttgtggacaacatcttccctcctgtg1140
atcaacatcacatggctcagaaatagtaagtcagtcacagacggcgtttatgagaccagc1200
ttccttgtcaaccgtgaccattccttccacaagctgtcttatctcaccttcatcccttct1260
gacgatgatatttatgactgcaaggtggagcactggggcctggaggagccggttctgaaa1320
cactgggctagcggagggggcggaagcggcggagggggagccaaaacgacacccccatct1380
gtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgc1440
ctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtcc1500
agcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctca1560
gtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccg1620
gccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgc1680
atatgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtg1740
ctcaccattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgat1800
cccgaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaa1860
ccccgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcac1920
caggactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgcc1980
cccatcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacacc2040
attccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataaca2100
gacttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaac2160
tacaagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctc2220
aatgtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgag2280
ggcctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatgatctggt2340
acctgc 2346
<210> IAS
MBP 1-14
CH1.H
<211> 1701
<212> DNA
<213> murine
gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60
ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120
agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180
aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240
tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300
gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360
gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420
cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480
aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540
cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600
SS tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660
tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720
ggtggcagcg gcggtggtgg ttc.cggtggc ggcggttctg gcggtggcgg ttcctcgagt 780
gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840
ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
92
gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960
caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020
tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080
gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140
S atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200
ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260
gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320
cactgggcta gcggaggggg cggaagcggc ggagggggag ccaaaacgac acccccatct 1380
gtctatccac tggcccctgg atctgctgcc.caaactaact ccatggtgac cctgggatgc 1440
ctggtcaagg gctatttccc tgagccagtg acagtgacct ggaactctgg atccctgtcc 1500
agcggtgtgc acaccttccc agctgtcctg cagtctgacc tctacactct gagcagctca 1560
gtgactgtcc cctccagcac ctggcccagc gagaccgtca cctgcaacgt tgcccacccg 1620
gccagcagca ccaaggtgga caagaaaatt gtgcccaggg attgtggttg taagccttgc 1680
atatgtacag tctaaggtac c 1701
<210> IAS MBP 90-101 CH1.H.CH2
<211> 2053
<212> DNA
<213> murine
gcggccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttcca60
ggttccactggtgacttcaagaacattgtgacacctcgaacaccacctccagctagcgga120
gggggcggaagcggcggagggggagactccgaaaggcatttcgtgttccagttcaagggc180
gagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatctacaac240
cgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgaccgagctg300
gggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggccgagctg360
gacacggtgtgcagacacaactacgagggggtggagacccacacctccctgcggcggctt420
gaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccacaacact480
ctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttccggaat'540
ggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggactggacc600
ttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacctgccac660
gtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggcggtggc720
agcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagtgaagac780
gacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcctggagac840
attggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttggataag900
aaggagactatctggatgcttcctgagtttggccaattgacaagctttgacccccaaggt960
ggactgcaaaacatagctacaggaaaatacaccttgggaatcttgactaagaggtcaaat1020
tccaccccagctaccaatgaggctcctcaagcgactgtgttccccaagtcccctgtgctg1080
ctgggtcagcccaacaccctcatctgctttgtggacaaca,tcttccctcctgtgatcaac1140
atcacatggctcagaaatagtaagtcagtcacagacggcgtttatgagaccagcttcctt1200
gtcaaccgtgaccattccttccacaagctgtcttatctcaccttcatcccttctgacgat1260
gatatttatgactgcaaggtggagcactggggcctggaggagccggttctgaaacactgg1320
gctagcggagggggcggaagcggcggagggggagccaaaacaacacccccatcagtctat1380
ccactggcccctgggtgtggagatacaactggttcctccgtgactctgggatgcctggtc1440
aagggctacttccctgagtcagtgactgtgacttggaactctggctccctgtccagcagt1500
gtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcagtgact1560
gtcccctccagcacctggccaagtcagaccgtcacctgcagcgttgctcacccagccagc1620
agcaccacggtggacaaaaaacttgagcccagcgggcccatttcaacaatcaacccctgt1680
SO cctccatgcaaggagtgtcacaaatgcccagctcctaacctggagggtggaccatccgtc1740
ttcatcttccctccaaatatcaaggatgtactcatgatctccctgacacccaaggtcacg1800
tgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggtttgtgaac1860
aacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagtactatc1920
cgggtggtcagcaccctccccatccagcaccaggactggatgagtggcaaggagttcaaa1980
tgcaaggtcaacaacaaagacctcccatcacccatcgagagaaccatctcaaaaattaaa2040
tagggtaccccga 2053
<210> IAS MBP 90-101 CH1.H
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
93
<211> 1707
<212> DNA
<213> murine
gcggccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttcca60
ggttccactggtgacttcaagaacattgtgacacctcgaacaccacctccagctagcgct120
agcggagggggcggaagcggcggagggggagactccgaaaggcatttcgtgttccagttc180
aagggcgagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatc240
tacaaccgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgacc300
gagctggggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggcc360
gagctggacacggtgtgcagacacaactacgagggggtggagacccacacctccctgcgg420
cggcttgaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccac480
aacactctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttc540
cggaatggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggac600
1S tggaccttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacc660
tgccacgtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggc720
ggtggcagcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagt780
gaagacgacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcct840
ggagacattggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttg900
gataagaaggagactatctggatgcttcctgagtttggccaattgacaagcttaagcttt960
gacccccaaggtggactgcaaaacatagctacaggaaaatacaccttgggaatcttgact1020
aagaggtcaaattccaccccagctaccaatgaggctcctcaagcgactgtgttccccaag1080
tcccctgtgctgctgggtcagcccaacaccctcatctgctttgtggacaacatcttccct1140
cctgtgatcaacatcacatggctcagaaatagtaagtcagtcacagacggcgtttatgag1200
2S accagcttccttgtcaaccgtgaccattccttccacaagctgtcttatctcaccttcatc1260
ccttctgacgatgatatttatgactgcaaggtggagcactggggcctggaggagccggtt1320
ctgaaacactgggctagcggagggggcggaagcggcggagggggagccaaaacgacaccc1380
ccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctg1440
ggatgcctggtcaagggctatttccctgagccagtgacagtgacctggaactctggatcc1500
ctgtccagcggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagc1560
agctcagtgactgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcc1620
cacccggccagcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaag1680
ccttgcatatgtacagtctaaggtacc 1707
40
<210> MBP 1-14 CK
<211> 1686
<212> DNA
<213> murine
gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60
ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120
agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180
aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240
tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300
gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360
gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420
cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480
aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540
SO cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600
tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660
tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720
ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780
gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840
ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900
gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960
caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020
tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080
gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
94
atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200
ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260
gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320
cactgggcta gcggaggggg cggaagcggc ggagggggag ctgatgctgc accaactgta 1380
S tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 1440
ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 1500
caaaatggcg'tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 1560
agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 1620
gccactcaca agacatcaac ttcacccatt gtcaagagct'tcaacaggaa tgagtgttag 1680
ggtacc 1686
<210> MBP 1-14 CH1.H.CH2
<211> 2059
1$ <212> DNA
<213> murine
gcggccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttcca60
ggttccactggtgacatggcgtcacagaagagaccctcccagaggcacggatccaaggct120
agcggagggggcggaagcggcggagggggagactccgaaaggcatttcgtgttccagttc180
aagggcgagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatc240
tacaaccgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgacc300
gagctggggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggcc360
gagctggacacggtgtgcagacacaactacgagggggtggagacccacacctccctgcgg420
2S cggcttgaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccac480
aacactctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttc540
cggaatggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggac600
tggaccttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacc660
tgccacgtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggc720
ggtggcagcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagt780
gaagacgacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcct840
ggagacattggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttg900
gataagaaggagactatctggatgcttcctgagtttggccaattgacaagctttgacccc960
caaggtggactgcaaaacatagctacaggaaaatacaccttgggaatcttgactaagagg1020
3S tcaaattccaccccagctaccaatgaggctcctcaagcgactgtgttccccaagtcccct1080
gtgctgctgggtcagcccaacaccctcatctgctttgtggacaacatcttccctcctgtg1140
atcaacatcacatggctcagaaatagtaagtcagtcacagacggcgtttatgagaccagc1200
ttccttgtcaaccgtgaccattccttccacaagctgtcttatctcaccttcatcccttct1260
gacgatgatatttatgactgcaaggtggagcactggggcctggaggagccggttctgaaa1320
cactgggctagcggagggggcggaagcggcggagggggagccaaaacaacacccccatca1380
gtctatccactggcccctgggtgtggagatacaactggttcctccgtgactctgggatgc1440
ctggtcaagggctacttccctgagtcagtgactgtgacttggaactctggctccctgtcc1500
agcagtgtgcacaccttcccagctctcctgcagtctggactctacactatgagcagctcaX560
gtgactgtcccctccagcacctggccaagtcagaccgtcacctgcagcgttgctcaccca1620
gccagcagcaccacggtggacaaaaaacttgagcccagcgggcccatttcaacaatcaac1680
ccctgtcctccatgcaaggagtgtcacaaatgcccagctcctaacctggagggtggacca1740
tccgtcttcatcttccctccaaatatcaaggatgtactcatgatctccctgacacccaag1800
gtcacgtgtgtggtggtggatgtgagcgaggatgacccagacgtccagatcagctggttt1860
gtgaacaacgtggaagtacacacagctcagacacaaacccatagagaggattacaacagt1920
actatccgggtggtcagcaccctccccatccagcaccaggactggatgagtggcaaggag1980
ttcaaatgcaaggtcaacaacaaagacctcccatcacccatcgagagaaccatctcaaaa2040
attaaatagggtaccccga 2059
<210> MBP 90-101
CHl.H.CH2.CH3
<211> 23'43
<212> DNA--
<213> murine
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
gcggccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttcca60
ggttccactggtgacttcaagaacattgtgacacctcgaacaccacctccagctagcgga120
gggggcggaagcggcggagggggagactccgaaaggcatttcgtgttccagttcaagggc180
gagtgctacttcaccaacgggacgcagcgcatacgatctgtggacagatacatctacaac240
S cgggaggagtacctgcgcttcgacagcgacgtgggcgagtaccgcgcggtgaccgagctg300
gggcggccagaccccgagtactacaataagcagtacctggagcaaacgcgggccgagctg360
gacacgacgtgcagacacaactacgagggggtggagacccacacctccctgcggcggctt420
gaacagcccaatgtcgtcatctccctgtccaggacagaggccctcaaccaccacaacact480
ctggtctgctcagtgacagatttctacccagccaagatcaaagtgcgctggttccggaat540
10 ggccaggaggagacggtgggggtctcatccacacagcttattaggaatggggactggacc600
ttccaggtcctggtcatgctggagatgacccctcggcggggagaggtctacacctgccac660
gtggagcatccgagcctgaagagccccatcactgtggagtggactagtggtggcggtggc720
agcggcggtggtggttccggtggcggcggttctggcggtggcggttcctcgagtgaagac780
gacattgaggccgaccacgtaggcgtctatggtacaactgtatatcagtctcctggagac840
15 attggccagtacacacatgaatttgatggtgatgagtggttctatgtggacttggataag900
aaggagactatctggatgcttcctgagtttggccaattgacaagctttgacccccaaggt960
ggactgcaaaacatagctacaggaaaatacaccttgggaatcttgactaagaggtcaaat1020
tccaccccagctaccaatgaggctcctcaagcgactgtgttccccaagtcccctgtgctg1080
ctgggtcagcccaacaccctcatctgctttgtggacaacatcttccctcctgtgatcaac1140
20 atcacatggctcagaaatagtaagtcagtcacagacggcgtttatgagaccagcttcctt1200
gtcaaccgtgaccattecttccacaagctgtcttatctcaccttcatcccttctgacgat1260
gatatttatgactgcaaggtggagcactggggcctggaggagccggttctgaaacactgg1320
gctagcggagggggcggaagcggcggaggaagcttagccaaaacgacacccccatctgtc1380
tatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctg1440
25 gtcaagggctatttccctgagccagtgacagtgacctggaactctggatccctgtccagc1500
ggtgtgcacaccttcccagctgtcctgcagtctgacctctacactctgagcagctcagtg1560
actgtcccctccagcacctggcccagcgagaccgtcacctgcaacgttgcccacccggcc1620
agcagcaccaaggtggacaagaaaattgtgcccagggattgtggttgtaagccttgcata1680
tgtacagtcccagaagtatcatctgtcttcatcttccccccaaagcccaaggatgtgctc1740
30 accattactctgactcctaaggtcacgtgtgttgtggtagacatcagcaaggatgatccc1800
gaggtccagttcagctggtttgtagatgatgtggaggtgcacacagctcagacgcaaccc1860
cgggaggagcagttcaacagcactttccgctcagtcagtgaacttcccatcatgcaccag1920
gactggctcaatggcaaggagttcaaatgcagggtcaacagtgcagctttccctgccccc1980
atcgagaaaaccatctccaaaaccaaaggcagaccgaaggctccacaggtgtacaccatt2040
35 ccacctcccaaggagcagatggccaaggataaagtcagtctgacctgcatgataacagac2100
ttcttccctgaagacattactgtggagtggcagtggaatgggcagccagcggagaactac2160
aagaacactcagcccatcatggacacagatggctcttacttcgtctacagcaagctcaat2220
gtgcagaagagcaactgggaggcaggaaatactttcacctgctctgtgttacatgagggc2280
ctgcacaaccaccatactgagaagagcctctcccactctcctggtaaatgatctggtacc2340
40 tgc 2343
<210> IASMBP 1-14 CH1.CH2.CH3
<211> 679
45 <212> PRT--
<213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His
20 25 30
Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser
SS 35 40 45
Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn
50 55 60
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
96
Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu
65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu Glu
100 105 110
Gln Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly
115 120 125
Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Val
130 135 140
1$
Ile Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val
145 150 155 160
Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe
165 170 175
Arg Asn Gly Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile
180 185 190
2$ Arg Asn Gly Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr
195 200 205
Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu
210 215 220
Lys Ser Pro Ile Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser
3$ 245 250 255
Glu Asp Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val
260 265 270
Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly
275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met
290 295 ~ 300
Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu
305 310 315 320
Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg
$0 325 330 335
Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe
340 345 350
5$ Pro Lys Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe
355 360 365
Val Asp Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn
370 375 380
CA 02403432 2002-09-23
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97
Ser LysSerVal ThrAspGly ValTyrGlu ThrSer PheLeuVal Asn
385 390 395 400
$ Arg AspHisSer PheHisLys LeuSerTyr LeuThr PheIlePro Ser
405 410 415
Asp AspAspIle TyrAspCys LysValGlu HisTrp GlyLeuGlu Glu
420 425 430
Pro ValLeuLys HisTrpAla SerGlyGly GlyGly SerGlyGly Gly
435 440 445
Gly AlaLysThr ThrProPro SerValTyr ProLeu AlaProGly.Cys
IS 450 455 460
Gly AspThrThr GlySerSer ValThrLeu GlyCys LeuValLys Gly
465 470 475 480
Tyr PheProGlu SerValThr ValThrTrp AsnSer GlySerLeu Ser
485 490 495
Ser SerValHis ThrPhePro AlaLeuLeu GlnSer GlyLeuTyr Thr
500 505 510
Met SerSerSer ValThrVal ProSerSer ThrTrp ProSerGln Thr
515 520 525
Val ThrCysSer ValAlaHis ProAlaSer SerThr ThrValAsp Lys
530 535 540
Lys LeuGluPro SerGlyPro IleSerThr IleAsn ProCysPro Pro
.
545 550 555 560
Cys LysGluCys HisLysCys ProAlaPro AsnLeu GluGlyGly Pro
565 570 575
Ser Val~PheIle PheProPro AsnIleLys AspVal LeuMetIle Ser
580 585 590
Leu ThrProLys ValThrCys ValValVal AspVal SerGluAsp Asp
595 600 605
Pro AspValGln IleSerTrp PheValAsn AsnVal GluValHis Thr
610 615 620
Ala GlnThrGln ThrHisArg GluAspTyr AsnSer ThrIleArg Val
625 630 635 640
Val SerThrLeu ProIleGln HisGlnAsp TrpMet SerGlyLys Glu
645 650 655
Phe LysCysLys ValAsnAsn LysAspLeu ProSer ProIleGlu Arg
660 665 670
Thr IleSerLys IleLys
675
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
9~
<210> IAS MBP 1-14 CH1.H
<211>- 677
<212> PRT--
<213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
10 15
Gly Ser Thr Gly Asp Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro
20 25 30
Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg
35 40 45
His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr
50 55 60
Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr
65 70 75 80
Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu
85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu Glu Gln Thr
2$ 100 105 110
Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu
115 120 125
Thr His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Val Ile Ser
130 135 140
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser
145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn
165 l70 175
Gly Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile Arg Asn
4.0 180 185 190
Gly Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr Pro Arg
195 200 205
4S Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser
210 215 220
Pro Ile Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp
245 250 255.
Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gln
$S 260 265 270
Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly Asp Glu
275 280 285
CA 02403432 2002-09-23
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99
Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met Leu Pro
290 295 300
Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn
305 3I0 315 320
Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg Ser Asn
325 330 335
Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe Pro Lys
340 345 350
Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe Val Asp
355 360 365
Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn Ser Lys
370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp
385 390 395 400
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile Pro Ser Asp Asp
405 410 415
Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val
420 425 430
Leu Lys His Trp Ala Ser Gly GIy GIy Gly Ser Gly Gly Gly Gly Ala
435 440 445
Lys Thr Thr Pro Pro Ser Va1 Tyr Pro Leu Ala Pro Gly Cys Gly Asp
450 455 460
Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe
3$ 465 470 475 480
Pro Glu Ser Val Thr Val Thr Trp Asn Ser GIy Ser Leu Ser Ser Ser
485 490 495
Val His Thr Phe Pro Ala Leu Leu Gln Ser Gly Leu Tyr Thr Met Ser
500 505 510
Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gln Thr Val Thr
515 520 525
Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu
530 535 540
Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Cys Pro Pro Cys Lys
545 550 555 560
GIu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val
565 570 575
Phe Ile Phe Pro Pro Asn Ile Lys Asp Val Leu Met Ile Ser Leu Thr
580 585 590
Pro Lys Val Thr Cys Val Val Val Asp Val Ser GIu Asp Asp Pro Asp
595 600 605
CA 02403432 2002-09-23
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100
Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln
610 615 620
$ Thr Gln ThrHisArg GluAspTyr Ser ThrIle ValValSer
Asn Arg
625 630 635 640
Thr Leu ProIleGln HisGlnAsp Met SerGly GluPheLys
Trp Lys
645 650 655
Cys Lys ValAsnAsn LysAspLeu Ser ProIle ArgThrIle
Pro Glu
660 665 670
Ser Lys IleLys
675
<210> CHl.H.CH2
IAS
MBP
90-101
<211>
563
ZO <212>
PRT--
<213>
murine
Met GluThrAsp ThrLeuLeu LeuTrpVal LeuLeuLeu TrpVal Pro
5 10 15
Gly SerThrGly AspPheLys AsnIleVal ThrProArg ThrPro Pro
20 25 30
Pro AlaSerAla SerGlyGly GlyGlySer GlyGlyGly GlyAsp Ser
35 40 45
Glu ArgHisPhe ValPheGln PheLysGly GluCysTyr PheThr Asn
50 55 60
Gly ThrGlnArg IleArgSer ValAspArg TyrIleTyr AsnArg Glu
65 70 75 80
Glu Tyr~LeuArg PheAspSer AspValGly GluTyrArg AlaVal Thr
85 90 95
Glu LeuGlyArg ProAspPro GluTyrTyr AsnLysGln TyrLeu Glu
100 105 110
Gln ThrArgAla GluLeuAsp ThrValCys ArgHisAsn TyrGlu Gly
115 120 125
Val GluThrHis ThrSerLeu ArgArgLeu GluGlnPro AsnVal Val
130 135 140
SO Ile SerLeuSer ArgThrGlu AlaLeuAsn HisHisAsn ThrLeu Val
145 150 155 160
Cys SerValThr AspPheTyr ProAlaLys IleLysVal ArgTrp Phe
165 170 175
Arg AsnGlyGln GluGluThr ValGlyVal SerSerThr GlnLeu Ile
180 185 190
Arg AsnGlyAsp TrpThrPhe GlnValLeu ValMetLeu GluMet Thr
CA 02403432 2002-09-23
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101
195 200 205
Pro Arg ArgGly GluValTyr ThrCysHis ValGluHis ProSer Leu
210 215 220
Lys Ser ProIle ThrValGlu TrpThrSer GlyGlyGly GlySer Gly
225 230 235 240
Gly Gly GlySer GlyGlyGly GlySerGly GlyGlyGly SerSer Ser
~
1~ 245 250 255
Glu Asp AspIle GluAlaAsp HisValGly ValTyrGly ThrThr Val
260 265 270
Tyr Gln SerPro GlyAspIle GlyGlnTyr ThrHisGlu PheAsp Gly
275 280 285
Asp Glu TrpPhe TyrValAsp LeuAspLys LysGluThr IleTrp Met
290 295 300
Leu Pro GluPhe GlyGlnLeu ThrSerLeu SerPheAsp ProGln Gly
305 310 315 320
Gly Leu GlnAsn IleAlaThr GlyLysTyr ThrLeuGly IleLeu Thr
325 330 335
Lys Arg SerAsn SerThrPro AlaThrAsn GluAlaPro GlnAla Thr
340 345 350
3~ Val Phe ProLys SerProVal LeuLeuGly GlnProAsn ThrLeu Ile
355 360 365
Cys Phe ValAsp AsnIlePhe ProProVal IleAsnIle ThrTrp Leu
370 375 380
Arg Asn SerLys SerValThr AspGlyVal TyrGluThr SerPhe Leu
385 390 395 400
Val Asn ArgAsp HisSerPhe HisLysLeu SerTyrLeu ThrPhe Ile
405 410 415
Pro Ser AspAsp AspIleTyr AspCysLys ValGluHis TrpGly Leu
420 425 430
Glu Glu ProVal LeuLysHis TrpAlaSer GlyGlyGly GlySer Gly
435 440 445
Gly Gly GlyAla LysThrThr ProProSer ValTyrPro LeuAla Pro
450 455 460
Gly Ser AlaAla GlnThrAsn SerMetVal ThrLeuGly CysLeu Val
465 470 475 480
Lys Gly TyrPhe ProGluPro ValThrVal ThrTrpAsn SerGly Ser
$$ 485 490 495
Leu Ser SerGly ValHisThr PheProAla ValLeuGln SerAsp Leu
500 505 510
CA 02403432 2002-09-23
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102
Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser
515 520 525
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val
530 535 540
Asp Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys
545 550 555 560
Thr Val
<210> IAS MBP 90-101 CH1.H
<211> 556
<212> PRT
<213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu.Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His
20 25 30
Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser
40 45
Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn
50 55 60
Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu
65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu Glu
100 105 110
Gln Thr ArgAla GluLeuAsp ThrValCys ArgHis AsnTyrGlu Gly
115 120 125
Val Glu ThrHis ThrSerLeu ArgArgLeu GluGln ~ProAsnVal Val
130 135 140
Ile Ser LeuSer ArgThrGlu AlaLeuAsn HisHis AsnThrLeu Val
145 150 155 160
Cys Ser ValThr AspPheTyr ProAlaLys IleLys ValArgTrp Phe
$0 165 170 175
Arg Asn GlyGln GluGluThr ValGlyVal SerSer ThrGlnLeu Ile
180 185 190
$$ Arg Asn GlyAsp TrpThrPhe GInValLeu ValMet LeuGluMet Thr
195 200 205
Pro Arg ArgGly GluValTyr ThrCysHis ValGlu HisProSer Leu
210 215 220
CA 02403432 2002-09-23
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103
Lys Ser Pro Ile Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly
225 230 235 240
$ Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser
245 250 255
Glu Asp Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val
260 265 270
Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly
275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met
1$ 290 295 300
Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu
305 310 315 320
Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg
325 330 335
Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe
340 345 350
2$
Pro Lys Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe
355 360 365
Val Asp Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn
370 375 38.0
Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn
385 390 395 400
3$ Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile Pro Ser
405 410 415
Asp Asp'Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu
420 425 430
Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu
4$ 450 455 460
Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe
465 470 475 480
$0 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg
485 490 495
Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser
500 505 510
$$
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu
515 520 525
Arg His Asn Ser Tyr Thr Cys GIu Ala Thr His Lys Thr Ser Thr Ser
CA 02403432 2002-09-23
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104
530 535 540
Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
545 550 555
<210>
IAS
MBP
1-14
CK
<211>
775
<212>
PRT
<213>
murine
Thr Met GluThrAsp ThrLeu LeuLeuTrpVal LeuLeu LeuTrpVal
5 10 15
1$ Pro Gly SerThrGly AspMet AlaSerGlnLys ArgPro SerGlnArg
20 25 30
His Gly SerLysAla SerGly GlyGlyGlySer GlyGly GlyGlyAsp
35 40 45
Ser Glu ArgHisPhe ValPhe GlnPheLysGly GluCys TyrPheThr
50 ' S5 60
Asn Gly ThrGlnArg IleArg SerValAspArg TyrIle TyrAsnArg
2$ 65 70 75 80
Glu Glu TyrLeuArg PheAsp SerAspValGly GluTyr ArgAlaVal
g5 90 95
Thr Glu LeuGlyArg ProAsp ProGlu.TyrTyr AsnLys GlnTyrLeu
100 105 110
Glu Gln ThrArgAla GluLeu AspThrValCys ArgHis AsnTyrGlu
115 120 125
Gly Val GluThrHis ThrSer LeuArgArgLeu GluGln ProAsnVal
130 135 140
Val Ile SerLeuSer ArgThr GluAlaLeuAsn HisHis AsnThrLeu
145 150 155 160
Val Cys SerValThr AspPhe TyrProAlaLys IleLys ValArgTrp
165 170 175
Phe Arg AsnGlyGln GluGlu ThrValGlyVal SerSer ThrGlnLeu
180 185 190
Ile Arg AsnGlyAsp TrpThr PheGlnValLeu ValMet LeuGluMet
195 200 205
Thr Pro ArgArgGly GluVal TyrThrCysHis ValGlu HisProSer
210 215 ~ 220
Leu Lys SerProIle ThrVal GluTrpThrSer GlyGly GlyGlySer
5$ 225 230 235 240
Gly Gly GlyGlySer GlyGly GlyGlySerGly GlyGly GlySerSer
245 250 255
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
105
Ser Glu Asp Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr
260 265 270
Val Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp
$ 275 280 285
Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp
290 295 300
Met Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly
305 310 315 320
Leu Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys
325 330 335
Arg Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val
340 345 350
Phe Pro Lys Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile ,Cys
355 360 365
Phe Val Asp Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg
370 375 380
2$ Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val
385 390 395 400
Asn Arg Asp His Ser Phe~His Lys Leu Ser Tyr Leu Thr Phe Ile Pro
405 410 415
Ser Asp Asp Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu
420 425 430
Glu Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly
3$ 435 440 445
Gly Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly
450 455 460
Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys
465 470 475 480
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu
485 490 495
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr
500 505 510
Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu
$0 515 520 525
Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp
530 535 540
Lys Lys Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr
545 550 555 560
Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp
565 570 575
CA 02403432 2002-09-23
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106
Val Leu ThrIleThr LeuThr ProLysVal ThrCysVal ValValAsp
580 585 590
$ Ile Ser LysAspAsp ProGlu ValGlnPhe SerTrpPhe ValAspAsp
595 600 605
Val Glu ValHisThr AlaGln ThrGlnPro ArgGluGlu GlnPheAsn
610 615 620
Ser Thr PheArgSer ValSer GluLeuPro IleMetHis GlnAspTrp
625 630 635 640
Leu Asn GlyLysGlu PheLys CysArgVal AsnSerAla AlaPhePro
1$ 645 650 655
Ala Pro IleGluLys ThrIle SerLys.ThrLysGlyArg ProLysAla
660 665 670
Pro Gln ValTyrThr IlePro ProProLys GluGlnMet AlaLysAsp
675 680 685
Lys Val SerLeuThr CysMet IleThrAsp PhePhePro GluAspIle
690 695 700
2$
Thr Val GluTrpGln TrpAsn GlyGlnPro AlaGluAsn TyrLysAsn
705 710 715 720
Thr Gln ProIleMet AspThr AspGlySer TyrPheVal TyrSerLys
725 730 735
Leu Asn ValGlnLys SerAsn TrpGluAla GlyAsnThr PheThrCys
740 745 750
3$ Ser Val LeuHisGlu GlyLeu HisAsnHis HisThrGlu LysSerLeu
755 760 765
Ser His~Ser Pro Gly Lys
770
<210> IAS MBP 1-14 CH1.H.CH2
<211> 561
<212> PRT
4$' <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
$0 Gly Ser Thr Gly Asp Met Ala Ser Gln Lys Arg Pro Ser Gln Arg His
20 25 30
Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser
35 40 45
$$
Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn
55 60
Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu
CA 02403432 2002-09-23
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107
65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr
g5 90 95
Glu Leu GlyArgPro AspProGlu TyrTyrAsn LysGln TyrLeuGlu
100 105 110
Gln Thr ArgAlaGlu LeuAspThr ValCysArg HisAsn TyrGluGly
115 120 125
Val Glu ThrHisThr SerLeuArg ArgLeuGlu GlnPro AsnValVal
130 135 140
1$ Ile Ser LeuSerArg ThrGluAla LeuAsnHis HisAsn ThrLeuVal
145 150 155 160
Cys Ser ValThrAsp PheTyrPro AlaLysIle LysVal ArgTrpPhe
165 170 175
Arg Asn GlyGlnGlu GluThrVal GlyValSer SerThr GlnLeuIle
180 185 190
Arg Asn GlyAspTrp ThrPheGln ValLeuVal MetLeu GluMetThr
2$ 195 200 205
Pro Arg ArgGlyGlu ValTyrThr CysHisVal GluHis ProSerLeu
210 215 220
Lys Ser ProIleThr ValGluTrp ThrSerGly GlyGly GlySerGly
225 230 235 240
Gly Gly GlySerGly GlyGlyGly SerGlyGly GlyGly SerSerSer
245 250 255
Glu Asp AspIleGlu AlaAspHis ValGlyVal TyrGly ThrThrVal
260 265 270
Tyr Gln SerProGly AspIleGly GlnTyrThr HisGlu PheAspGly
275 280 285
Asp Glu TrpPheTyr ValAspLeu AspLysLys GluThr IleTrpMet
290 295 300
Leu Pro GluPheGly GlnLeuThr SerPheAsp ProGln GlyGlyLeu
305 310 315 320
Gln Asn IleAlaThr GlyLysTyr ThrLeuGly IleLeu ThrLysArg
325 330 335
Ser Asn SerThrPro AlaThrAsn GluAlaPro GlnAla ThrValPhe
340 345 350
Pro Lys SerProVal LeuLeuGly GlnProAsn ThrLeu IleCysPhe
355 360 365
Val Asp AsnIlePhe ProProVal IleAsnIle ThrTrp LeuArgAsn
370 375 380
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
10~
Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn
385 390 395 400
Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile Pro Ser
405 410 415
Asp Asp Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu
420 425 430
Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly
435 440 445
Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser
450 455 460
Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly
465 470 475 480
Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
485 490 495
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr
500 505 510
Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr
515 520 525
Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys
530 535 540
Lys Ile Val.Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val
545 550 555 560
<210> IAS MBP 90-101 CH1.H.CH2.CH3
<211> 773
<212> PRT
<213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro
20 25 30
Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg
35 40 . 45
His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr
50 55 60
Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr
65 70 75 80
Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu
85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu Glu Gln Thr
100 105 110
CA 02403432 2002-09-23
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109
Arg Ala Glu Leu Asp Thr Thr Cys Arg His Asn Tyr Glu Gly Val Glu
115 120 125
$ Thr His Thr Ser Leu Arg Arg Leu Glu Gln Pro Asn Val Val Ile Ser
130 135 140
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser
145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys Ile Lys Val Arg Trp Phe Arg Asn
165 170 175
Gly Gln Glu Glu Thr Val Gly Val Ser Ser Thr Gln Leu Ile Arg Asn
1$ 180 185 190
Gly Asp Trp Thr Phe Gln Val Leu Val Met Leu Glu Met Thr Pro Arg
195 200 205
Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser
210 215 220
2$
Pro Ile Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly
225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp
245 250 255
Asp Ile Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gln
260 265 270
Ser Pro Gly Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly Asp Glu
275 280 285
3$ Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met Leu Pro
290 295 300
Glu Phe~Gly G1n Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn
305 310 315 320
Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg Ser Asn
325 330 335
Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe Pro Lys
4$ 340 345 350
Ser Pro Val Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe Val Asp
355 360 365
$0 Asn Ile Phe Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn Ser Lys
370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp
385 390 395 400
$$
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe Ile Pro Ser Asp Asp
405 410 415
Asp Ile Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
110
420 425 430
Leu Lys HisTrpAla SerGly GlyGlyGly SerGlyGly GlySerLeu
435 440 445
Ala Lys ThrThrPro ProSer ValTyrPro LeuAlaPro GlySerAla
450 455 460
Ala Gln ThrAsnSer MetVal ThrLeuGly CysLeuVal LysGlyTyr
465 470 475 480
Phe Pro GluProVal ThrVal ThrTrpAsn SerGlySer LeuSerSer
485 490 495
1$ Gly Val HisThrPhe ProAla ValLeuGln SerAspLeu TyrThrLeu
500 505 510
Ser Ser SerValThr ValPro SerSerThr TrpProSer GluThrVal
515 520 525
Thr Cys AsnValAla HisPro AlaSerSer ThrLysVal AspLysLys
530 535 540
Ile Val ProArgAsp CysGly CysLysPro CysIleCys ThrValPro
545 550 555 560
Glu Val SerSerVal PheIle PheProPro LysProLys AspValLeu
565 570 ~ 575
Thr Ile ThrLeuThr ProLys ValThrCys ValValVal AspIleSer
580 585 590
Lys Asp AspProGlu ValGln PheSerTrp PheValAsp AspValGlu
595 600 605
Val His ThrAlaGln ThrGln ProArgGlu GluGlnPhe AsnSerThr
610 615 620
Phe Arg SerValSer GluLeu ProIleMet HisGlnAsp TrpLeuAsn
625 630 635 640
Gly Lys GluPheLys CysArg ValAsnSer AlaAlaPhe ProAlaPro
645 650 655
Ile Glu LysThrIle SerLys ThrLysGly ArgProLys AlaProGln
660 665 670
Val Tyr ThrIlePro ProPro LysGluGln MetAlaLys AspLysVal
675 680 685
Ser Leu ThrCysMet IleThr AspPhePhe ProGluAsp IleThrVal
690 695 700
Glu Trp GlnTrpAsn GlyGln ProAlaGlu AsnTyrLys AsnThrGln
$5 705 710 715 720
Pro Ile MetAspThr AspGly SerTyrPhe ValTyrSer LysLeuAsn
725 730 735
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
111
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
740 745 750
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
755 760 765
Ser Pro Gly Lys
770
<210> pCRC203
<211> 609
<212> DNA
<213> Murine
1$ <400> 1
catatgttca agaacattgt gacacctcga acaccacctc caggaggagg atccggagac 60
tcggaaaggc atttcgtgtt ccagttcaag ggcgagtgct acttcaccaa cgggacgcag 120
cgcatacgat ctgtggacag atacatctac aaccgggagg agtacctgcg cttcgacagc 180
gacgtgggcg agtaccgcgc ggtgaccgag ctggggcggc cagaccccga gtactacaat 240
aagcagtacc tggagcaaac gcgggccgag ctggacacgg tgtgcagaca caactacgag 300
ggggtggaga cccacacctc cctgcggcgg cttggaggtg aagacgacat tgaggccgac 360
cacgtaggcg tctatggtac aactgtatat cagtctcctg gagacattgg ccagtacaca 420
catgaatttg atggtgatga gtggttctat gtggacttgg ataagaagga gactatctgg 480
atgcttcctg agtttggcca attgacaagc tttgaccccc aaggtggact gcaaaacata 540
gctacaggaa aatacacctt gggaatcttg actaagaggt caaattccac cccagctacc 600
aatctcgag 609
<210> pCRC201
<211> 614
<212> DNA
<213> Murine
<400> 2
catatgttca agaacattgt gacacctcga acaccacctc caggaggagg atccggagac 60
tcggaaaggc atttcgtgtt ccagttcaag ggcgagtgct acttcaccaa cgggacgcag 120
cgcatacgat ctgtggacag atacatctac aaccgggagg agtacctgcg cttcgacagc 180
gacgtgggcg agtaccgcgc ggtgaccgag ctggggcggc cagaccccga gtactacaat 240
aagcagtacc tggagcaaac gcgggccgag ctggacacgg tgtgcagaca caactacgag 300
ggggtggaga cccacacctc cctgcggcgg cttggaggtg aagacgacat tgaggccgac 360
cacgtaggcg tctatggtac aactgtatat cagtctcctg gagacattgg ccagtacaca 420
catgaatttg atggtgatga gtggttctat gtggacttgg ataagaagga gactatctgg 480
atgcttcctg agtttggcca attgacaagc tttgaccccc aaggtggact gcaaaacata 540
gctacaggaa aatacacctt gggaatcttg actaagaggt caaattccac cccagctacc 600
aattaagcgg ccgc 614
<210> pCRCl99
<211> 642
<212> DNA
<213> Murine
<400> 3
catatgttca agaacattgt gacacctcga acaccacctc cagctagcgg agggggcgga 60
agcggcggag ggggagactc cgaaaggcat ttcgtgttcc agtttaaagg cgagtgctac 120
ttcaccaacg ggacgcagcg catacgatct gtggacagat acatctacaa ccgggaggag 180
tacctgcgct tcgacagcga cgtgggcgag taccgcgcgg tgaccgagct ggggcggcca 240
gaccccgagt actacaataa gcagtacctg gagcaaacgc gggccgagct ggacacggtg 300
tgcagacaca actacgaggg ggtggagacc cacacctccc tgcggcggct tactagtggt 360
ggcggtggca gcgaagacga cattgaggcc gaccacgtag gcgtctatgg tacaactgta 420
tatcagtctc ctggagacat tggccagtac acacatgaat ttgatggtga tgagtggttc 480
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
112
tatgtggact tggataagaa ggagactatc tggatgcttc ctgagtttgg ccaattgaca 540
agctttgacc cccaaggtgg actgcaaaac atagctacag gaaaatacac cttgggaatc 600
ttgactaaga ggtcaaattc caccccagct accaatctcg ag 642
$ <210> pCRC197
<211> 647
<212> DNA
<213> Murine
<400> 4
catatgttca agaacattgt gacacctcga acaccacctc cagctagcgg agggggcgga 60
agcggcggag ggggagactc cgaaaggcat ttcgtgttcc agtttaaagg cgagtgctac 120
ttcaccaacg ggacgcagcg catacgatct gtggacagat acatctacaa ccgggaggag 180
tacctgcgct tcgacagcga cgtgggcgag taccgcgcgg tgaccgagct ggggcggcca 240
1S gaccccgagt actacaataa gcagtacctg gagcaaacgc gggccgagct ggacacggtg 300
tgcagacaca actacgaggg ggtggagacc cacacctccc tgcggcggct tactagtggt 360
ggcggtggca gcgaagacga cattgaggcc gaccacgtag gcgtctatgg tacaactgta 420
tatcagtctc ctggagacat tggccagtac acacatgaat ttgatggtga tgagtggttc 480
tatgtggact tggataagaa ggagactatc tggatgcttc ctgagtttgg ccaattgaca 540
agctttgacc cccaaggtgg actgcaaaac atagctacag gaaaatacac cttgggaatc 600
ttgactaaga ggtcaaattc caccccagct accaattaag cggccgc 647
<210> pCRC188
<211> 1698
2S <212> DNA
<213> Murine
<400> 5
gcggccgccaccatggctctgcagatccccagcctcctcctctcggctgctgtggtggtg60
ctgatggtgctgagcagcccagggactgagggcttcaagaacattgtgacacctcgaaca120
ccacctccagctagcggagggggcggaagcggcggagggggagactccgaaaggcatttc180
gtgttccagttcaagggcgagtgctacttcaccaacgggacgcagcgcatacgatctgtg240
gacagatacatctacaaccgggaggagtacctgcgcttcgacagcgacgtgggcgagtac300
cgcgcggtgaccgagctggggcggccagaccccgagtactacaataagcagtacctggag360
3S caaacgcgggccgagctggacacggtgtgcagacacaactacgagggggtggagacccac420
acctccctgcggcggcttgaacagcccaatgtcgtcatctccctgtccaggacagaggcc480
ctcaaccaccacaacactctggtctgctcagtgacagatttctacccagccaagatcaaa540
gtgcgctggttccggaatggccaggaggagacggtgggggtctcatccacacagcttatt600
aggaatggggactggaccttccaggtcctggtcatgctggagatgacccctcggcgggga660
gaggtctacacctgccacgtggagcatccgagcctgaagagccccatcactgtggagtgg720
actagtggtggcggtggcagcggcggtggtggttccggtggcggcggttctggcggtggc780
ggttcctcgagtgaagacgacattgaggccgaccacgtaggcgtctatggtacaactgta840
tatcagtctcctggagacattggccagtacacacatgaatttgatggtgatgagtggttc900
tatgtggacttggataagaaggagactatctggatgcttcctgagtttggccaattgaca960
agctttgacccccaaggtggactgcaaaacatagctacaggaaaatacaccttgggaatc1020
ttgactaagaggtcaaattccaccccagctaccaatgaggctcctcaagcgactgtgttc1080
cccaagtcccctgtgctgctgggtcagcccaacaccctcatctgctttgtggacaacatc1140
ttccctcctgtgatcaacatcacatggctcagaaatagtaagtcagtcacagacggcgtt1200
tatgagaccagcttccttgtcaaccgtgaccattccttccacaagctgtcttatctcacc1260
ttcatcccttctgacgatgatatttatgactgcaaggtggagcactggggcctggaggag1320
ccggttctgaaacactgggctagcggagggggcggaagcggcggagggggagctgatgct1380
gcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctca1440
gtcgtgtgcttcttgaacaacttctaccccaaagacatcaatgtcaagtggaagattgat1500
ggcagtgaacgacaaaatggcgtcctgaacagttggactgatcaggacagcaaagacagc1560
S$ acctacagcatgagcagcaccctca.cgttgaccaaggacgagtatgaacgacataacagc1620
tatacctgtgaggccactcacaagacatcaacttcacccattgtcaagagcttcaacagg1680
aatgagtgttagggtacc 1698
<210> pCRC187
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
113
<211> 1662
<212> DNA
<213> Murine
<400> 6
gcggccgccaccatggctctgcagatccccagcctcctcctctcggctgctgtggtggtg60
ctgatggtgctgagcagcccagggactgagggcgctagcggagggggcggaagcggcgga120
gggggagactccgaaaggcatttcgtgttccagttcaagggcgagtgctacttcaccaac180
gggacgcagcgcatacgatctgtggacagatacatctacaaccgggaggagtacctgcgc240
ttcgacagcgacgtgggcgagtaccgcgcggtgaccgagctggggcggccagaccccgag300
tactacaataagcagtacctggagcaaacgcgggccgagctggacacggtgtgcagacac360
aactacgagggggtggagacccacacctccctgcggcggcttgaacagcccaatgtcgtc420
atctccctgtccaggacagaggccctcaaccaccacaacactctggtctgctcagtgaca480
gatttctacccagccaagatcaaagtgcgctggttccggaatggccaggaggagacggtg540
1S ggggtctcatccacacagcttattaggaatggggactggaccttccaggtcctggtcatg600
ctggagatgacccctcggcggggagaggtctacacctgccacgtggagcatccgagcctg660
aagagccccatcactgtggagtggactagtggtggcggtggcagcggcggtggtggttcc720
ggtggcggcggttctggcggtggcggttcctcgagtgaagacgacattgaggccgaccac780
gtaggcgtctatggtacaactgtatatcagtctcctggagacattggccagtacacacat840
gaatttgatggtgatgagtggttctatgtggacttggataagaaggagactatctggatg900
cttcctgagtttggccaattgacaagctttgacccccaaggtggactgcaaaacatagct960
acaggaaaatacaccttgggaatcttgactaagaggtcaaattccaccccagctaccaat1020
gaggctcctcaagcgactgtgttccccaagtcccctgtgctgctgggtcagcccaacacc1080
ctcatctgctttgtggacaacatcttccctcctgtgatcaacatcacatggctcagaaat1140
agtaagtcagtcacagacggcgtttatgagaccagcttccttgtcaaccgtgaccattcc1200
ttccacaagctgtcttatctcaccttcatcccttctgacgatgatatttatgactgcaag1260
gtggagcactggggcctggaggagccggttctgaaacactgggctagcggagggggcgga1320
agcggcggagggggagctgatgctgcaccaactgtatccatcttcccaccatccagtgag1380
cagttaacatctggaggtgcctcagtcgtgtgcttcttgaacaacttctaccccaaagac1440
atcaatgtcaagtggaagattgatggcagtgaacgacaaaatggcgtcctgaacagttgg1500
actgatcaggacagcaaagacagcacctacagcatgagcagcaccctcacgttgaccaag1560
gacgagtatgaacgacataacagctatacctgtgaggccactcacaagacatcaacttca1620
cccattgtcaagagcttcaacaggaatgagtgttagggtacc 1662
<210> pCB229
<211> 1085
<212> DNA
<213> Murine
<400> 7
gaattcggtaccaccatggctctgcagatccccagcctcctcctctcggctgctgtggtg60
gtgctgatggtgctgagcagcccagggactgagggcttcaagaacattgtgacacctcga120
acaccacctccagctagcggagggggcggaagcggcggagggggagactccgaaaggcat180
ttcgtgttccagttcaagggcgagtgctacttcaccaacgggacgcagcgcatacgatct240
gtggacagatacatctacaaccgggaggagtacctgcgcttcgacagcgacgtgggcgag300
taccgcgcggtgaccgagctggggcggccagaccccgagtactacaataagcagtacctg360
gagcaaacgcgggccgagctggacacggtgtgcagacacaactacgagggggtggagacc420
cacacctccctgcggcggcttgaacagcccaatgtcgtcatctccctgtccaggacagag480
gccctcaaccaccacaacactctggtctgctcagtgacagatttctacccagccaagatc540
aaagtgcgctggttccggaatggccaggaggagacggtgggggtctcatccacacagctt600
attaggaatggggactggaccttccaggtcctggtcatgctggagatgacccctcggcgg660
ggagaggtctacacctgccacgtggagcatccgagcctgaagagccccatcactgtggag720
tggagggcacagtctgagtctgcccggagcggatccgctgatgctgcaccaactgtatcc780
atcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttcttg840
aacaacttctaccccaaagacatcaatgtcaagtggaagattgatggcagtgaacgacaa900
aatggcgtcctgaacagttggactgatcaggacagcaaagacagcacctacagcatgagc960
agcaccctcacgttgaccaaggacgagtatgaacgacataacagctatacctgtgaggcc1020
actcacaagacatcaacttcacccattgtcaagagcttcaacaggaatgagtgttaggcg1080
gccgc 1085
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
114
<210> pCB223
<211> 1676
<212> DNA
$ <213> Murine
<400> 8
gaattcggtaccatggctctgcagatccccagcctcctcctctcggctgctgtggtggtg60
ctgatggtgctgagcagcccagggactgagggcgaagacgacattgaggccgaccacgta120
ggcgtctatggtacaactgtatatcagtctcctggagacattggccagtacacacatgaa180
tttgatggtgatgagtggttctatgtggacttggataagaaggagactatctggatgctt240
cctgagtttggccaattgacaagctttgacccccaaggtggactgcaaaacatagctaca300
ggaaaatacaccttgggaatcttgactaagaggtcaaattccaccccagctaccaatgag360
gctcctcaagcgactgtgttccccaagtcccctgtgctgctgggtcagcccaacaccctc420
atctgctttgtggacaacatcttccctcctgtgatcaacatcacatggctcagaaatagt480
aagtcagtcacagacggcgtttatgagaccagcttccttgtcaaccgtgaccattccttc540
cacaagctgtcttatctcaccttcatcccttctgacgatgatatttatgactgcaaggtg600
gagcactggggcctggaggagccggttctgaaacactgggaacctgagattccagccccc660
atgtcagaaggatctgccaaaacaacagccccatcggtctatccactggcccctgtgtgt720
ggagatacaactggctcctcggtgactctaggatgcctggtcaagggttatttccctgag780
ccagtgaccttgacctggaactctggatccctgtccagtggtgtgcacaccttcccagct840
gtcctgcagtctgacctctacaccctcagcagctcagtgactgtaacctcgagcacctgg900
cccagccagtccatcacctgcaatgtggcccacccggcaagcagcaccaaggtggacaag960
aaaattgagcccagagggcccacaatcaagccctgtcctccatgcaaatgcccagcacct1020
aacctcttgggtggaccatccgtcttcatcttccctccaaagatcaaggatgtactcatg1080
atctccctgagccccatagtcacatgtgtggtggtggatgtgagcgaggatgacccagat1140
gtccagatcagctggtttgtgaacaacgtggaagtacacacagctcagacacaaacccat1200
agagaggattacaacagtactctccgggtggtcagtgccctccccatccagcaccaggac1260
tggatgagtggcaaggagttcaaatgcaaggtcaacaacaaagacctcccagcgcccatc1320
gagagaaccatctcaaaacccaaagggtcagtaagagctccacaggtatatgtcttgcct1380
ccaccagaagaagagatgactaagaaacaggtcactctgacctgcatggtcacagacttc1440
atgcctgaagacatttacgtggagtggaccaacaacgggaaaacagagctaaactacaag1500
aacactgaaccagtcctggactctgatggttcttacttcatgtacagcaagctgagagtg1560
gaaaagaagaactgggtggaaagaaatagctactcctgttcagtggtccacgagggtctg1620
cacaatcaccacacgactaagagcttctcccggactccgggtaaatgagcggccgc 1676
<210> pCB212
<211> 773
<212> DNA
<213> Murine
<400> 9
ccatgggtaa gaaacagacc gctgttgcat tcgctctggc gctcctggct ctttctatga 60
ccccggcgta cgctttcaag aacattgtga cacctcgaac accacctcca gctagcggag 120
ggggcggaag cggcggaggg ggagactccg aaaggcattt cgtgttccag tttaaaggcg 180
agtgctactt caccaacggg acgcagcgca tacgatctgt ggacagatac atctacaacc 240
gggaggagta cctgcgcttc gacagcgacg tgggcgagta ccgcgcggtg accgagctgg 300
ggcggccaga ccccgagtac tacaataagc agtacctgga gcaaacgcgg gccgagctgg 360
acacggtgtg cagacacaac tacgaggggg tggagaccca cacctccctg cggcggcttg 420
gtggcggtgg cagcggcggt ggtggttccg gtggcggcgg ttctggcggt ggcggttccg 480
gtggcggtgg cagcgaagac gacattgagg ccgaccacgt aggcgtctat ggtacaactg 540
tatatcagtc tcctggagac attggccagt acacacatga atttgatggt gatgagtggt 600
tctatgtgga cttggataag aaggagacta tctggatgct tcctgagttt ggccaattga 660
caagctttga cccccaaggt ggactgcaaa acatagctac aggaaaatac accttgggaa 720
SS tcttgactaa gaggtcaaat tccaccccag ctaccaatta aggtaccgga tcc 773
<210> pCB214
<211> 702
<212> DNA
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
115
<213> Murine
<400> 10
atgttcaaga acattgtgac acctcgaaca ccacctccag ctagcggagg gggcggaagc 60
ggcggagggg gagactccga aaggcatttc gtgttccagt ttaaaggcga gtgctacttc 120
accaacggga cgcagcgcat acgatctgtg gacagataca tctacaaccg ggaggagtac 180
ctgcgcttcg acagcgacgt gggcgagtac cgcgcggtga ccgagctggg gcggccagac 240
cccgagtact acaataagca gtacctggag caaacgcggg ccgagctgga cacggtgtgc 300
agacacaact acgagggggt ggagacccac acctccctgc ggcggcttgg tggcggtggc 360
agcggcggtg gtggttccgg tggcggcggt tctggcggtg gcggttccgg tggcggtggc 420
agcgaagacg acattgaggc cgaccacgta ggcgtctatg gtacaactgt atatcagtct 480
cctggagaca ttggccagta cacacatgaa tttgatggtg atgagtggtt ctatgtggac 540
ttggataaga aggagactat ctggatgctt cctgagtttg gccaattgac aagctttgac 600
ccccaaggtg gactgcaaaa catagctaca ggaaaataca ccttgggaat cttgactaag 660
1S aggtcaaatt ccaccccagc taccaattaa ggtaccggat cc 702
<210> pCB220
<211> 1013
<212> DNA
<213> Murine
<400> 11
gaattcggtaccatggctctgcagatccccagcctcctcctctcggctgctgtggtggtg60
ctgatggtgctgagcagcccagggactgagggcgaagacgacattgaggccgaccacgta120
ggcgtctatggtacaactgtatatcagtctcctggagacattggccagtacacacatgaa180
tttgatggtgatgagtggttctatgtggacttggataagaaggagactatctggatgctt240
cctgagtttggccaattgacaagctttgacccccaaggtggactgcaaaacatagctaca300
ggaaaatacaccttgggaatcttgactaagaggtcaaattccaccccagctaccaatgag360
gctcctcaagcgactgtgttccccaagtcccctgtgctgctgggtcagcccaacaccctc420
atctgctttgtggacaacatcttccctcctgtgatcaacatcacatggctcagaaatagt480
aagtcagtcacagacggcgtttatgagaccagcttccttgtcaaccgtgaccattccttc540
cacaagctgtcttatctcaccttcatcccttctgacgatgatatttatgactgcaaggtg600
gagcactggggcctggaggagccggttctgaaacactgggaacctgagattccagccccc660
atgtcagaaggatccgccaaaacaacagccccatcggtctatccactggcccctgtgtgt720
ggagatacaactggctcctcggtgactctaggatgcctggtcaagggttatttccctgag780
ccagtgaccttgacctggaactctggatctctgtccagtggtgtgcacaccttcccagct840
gtcctgcagtctgacctctacaccctcagcagctcagtgactgtaacctcgagcacctgg900
cccagccagtccatcacctgcaatgtggcccacccggcaagcagcaccaaggtggacaag960
aaaattgagcccagagggcccacaatcaagccctgtgctgcataggcggccgc 1013
<210> pCRC203
<211> 207
<212> PRT
<213> Murine
<400> 12
Met Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Gly Gly Gly
5 10 15
SO Ser Gly Asp Ser Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys
20 25 30
Tyr Phe Thr Asn Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr 21e
35 40 45
Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr
50 55 60
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
116
65 70 75 80
Gln Tyr Leu Glu Gln Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His
85 90 95
$
Asn Tyr GluGlyVal GluThrHis ThrSerLeu ArgArg LeuGlyGly
100 105 110
Glu Asp AspIleGlu AlaAspHis ValGlyVal TyrGly ThrThrVal
115 120 125
Tyr Gln SerProGly AspIleGly GlnTyrThr HisGlu PheAspGly
13 13 14
0 5 0
1$ Asp Glu TrpPheTyr ValAspLeu AspLysLys GluThr IleTrpMet
145 150 155 160
Leu Pro GluPheGly GlnLeuThr SerPheAsp ProGln GlyGlyLeu
165 170 175
Gln Asn IleAlaThr GlyLysTyr ThrLeuGly IleLeu ThrLysArg
180 185 190
Ser Asn SerThrPro AlaThrAsn HisHisHis HisHis His
2$ 195 200 205
<210> pCRC201
<211> 201
<212> PRT
<213> Murine
<400>
13
Met Phe LysAsnIle ValThr ProArgThr ProProPro GlyGlyGly
3$ 5 10 15
Ser Gly AspSerGlu ArgHis PheValPhe GlnPheLys GlyGluCys
20 25 30
Tyr Phe ThrAsnGly ThrGln ArgIleArg SerValAsp ArgTyrIle
35 40 45
Tyr Asn ArgGluGlu TyrLeu ArgPheAsp SerAspVal GlyGluTyr
50 55 60
4$
Arg Ala ValThrGlu LeuGly ArgProAsp ProGluTyr TyrAsriLys
65 70 75 80
Gln Tyr LeuGluGln ThrArg AlaGluLeu AspThrVal CysArgHis
$0 85 90 95
Asn Tyr GluGlyVal GluThr HisThrSer LeuArgArg LeuGlyGly
100 105 110
$$ Glu Asp AspIleGlu AlaAsp HisValGly ValTyrGly ThrThrVal
115 120 125
Tyr Gln SerProGly AspIle GlyGlnTyr ThrHisGlu PheAspGly
130 135 140
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Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met
145 150 155 160
$ Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu
165 170 175
Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg
180 185 190
1~
5er Asn Ser Thr Pro Ala Thr Asn
195 200
1$ <210> pCRC199
<211> 218
<212> PRT
<213> Murine
<400> 14
Met Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly
10 15
Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe
20 25 30
Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gln Arg Ile'Arg
35 40 45
30 Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp
50 55 60
Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp
65 70 75 80
3$
Pro Glu TyrTyrAsn LysGln TyrLeuGlu GlnThrArg AlaGlu Leu
85 90 95
Asp Thr ValCysArg HisAsn TyrGluGly ValGluThr HisThr Ser
100 105 110
Leu Arg ArgLeuThr SerGly GlyGlyGly SerGluAsp AspIle Glu
115 120 125
4$ Ala Asp HisValGly ValTyr GlyThrThr ValTyrGln SerPro Gly
130 135 140
Asp Ile GlyGlnTyr ThrHis GluPheAsp GlyAspGlu TrpPhe Tyr
145 150 155 160
$~
Val Asp LeuAspLys LysGlu ThrIleTrp MetLeuPro GluPhe Gly
165 170 175
Gln Leu ThrSerPhe AspPro GlnGlyGly LeuGlnAsn IleAla Thr
$$ 180 185 190
Gly Lys TyrThrLeu GlyIle LeuThrLys ArgSerAsn SerThr Pro
195 200 205
CA 02403432 2002-09-23
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11~
Ala Thr Asn His His His His His His
210 215
$ <210> pCRC197
<211> 212
<212> PRT
<213> Murine
<400> 15
Met Phe Lys Asn Ile Val Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly
5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe
1$ 20 25 30
Gln Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gln Arg Ile Arg
35 40 45
Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp
50 55 60
Ser Asp ValGly GluTyrArg AlaValThr GluLeuGly ArgProAsp
65 70 75 80
2$
Pro Glu TyrTyr AsnLysGln TyrLeuGlu GlnThrArg AlaGluLeu
85 90 95
Asp Thr ValCys ArgHisAsn TyrGluGly ValGluThr HisThrSer
100 105 110
Leu Arg ArgLeu ThrSerGly GlyGlyGly SerGluAsp AspIleGlu
115 120 125
3$ Ala Asp HisVal GlyValTyr GlyThrThr ValTyrGln SerProGly
130 135 140
Asp Ile ~GlyGln TyrThrHis GluPheAsp GlyAspGlu TrpPheTyr
145 150 155 160
Val Asp LeuAsp LysLysGlu ThrIleTrp MetLeuPro GluPheGly
165 170 175
Gln Leu ThrSer PheAspPro GlnGlyGly LeuGlnAsn IleAlaThr
4$ 180 185 190
Gly Lys TyrThr LeuGlyIle LeuThrLys ArgSerAsn SerThrPro
.
195 200 205
$0 Ala Thr Asn
210
<210> pCRC188
$$ <211> 548
<212> PRT
<213> Murine
<400> 16
CA 02403432 2002-09-23
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119
Met Ala L2u Gln Ile Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Ala Ser Gly Gly Gly
20 25 30
Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe Gln.Phe
35 40 45
Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gln Arg Ile Arg Ser Val
50 55 60
Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp
65 70 75 80
Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu
85 90 95
Tyr Tyr Asn Lys Gln Tyr Leu Glu Gln Thr Arg Ala Glu Leu Asp Thr
100 105 110
Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg
115 120 125
Arg Leu Glu Gln Pro Asn Val Val Ile Ser Leu Ser Arg Thr Glu Ala
130 135 140
Leu Asn His His Asn Thr Leu Val Cys Ser Val Thr Asp Phe Tyr Pro
145 150 155 160
Ala Lys Ile Lys Val Arg Trp Phe Arg Asn Gly Gln Glu Glu Thr Val
165 170 175
Gly Val Ser Ser Thr Gln Leu Ile Arg Asn Gly Asp Trp Thr Phe Gln
' 180 185 190
Val Leu Val Met Leu Glu Met Thr Pro Arg Arg Gly GIu Val Tyr Thr
'195 200 205
Cys His Val Glu His Pro Ser Leu Lys Ser Pro Ile Thr Val Glu Trp
2I0 215 220
Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
225 230 235 240
Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp Asp Ile Glu Ala Asp His
245 250 255
Val Gly Val Tyr Gly Thr Thr Val Tyr Gln Ser Pro Gly Asp Ile Gly
260 265 270
Gln Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu
275 280 285
SS Asp Lys Lys Glu Thr Ile Trp Met Leu Pro Glu Phe Gly Gln Leu Thr
290 295 300
Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn Ile Ala Thr Gly Lys Tyr
305 310 315 320
CA 02403432 2002-09-23
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120
Thr Leu GlyIleLeu ThrLys ArgSerAsn SerThrPro AlaThrAsn
325 330 335
Glu Ala ProGlnAla ThrVal PheProLys SerProVal LeuLeuGly
340 345 350
Gln Pro AsnThrLeu IleCys PheValAsp AsnIlePhe ProProVal
355 360 365
Ile Asn IleThrTrp LeuArg AsnSerLys SerValThr AspGlyVal
370 375 380
Tyr Glu ThrSerPhe LeuVal AsnArgAsp HisSerPhe HisLysLeu
1$ 385 390 395 400
Ser Tyr LeuThrPhe IlePro SerAspAsp AspIleTyr AspCysLys
405 410 415
Val Glu HisTrpGly LeuGlu GluProVal LeuLysHis TrpAlaSer
420 425 430
Gly Gly GlyGlySer GlyGly GlyGlyAla AspAlaAla ProThrVal
435 440 445
Ser Ile PheProPro SerSer GluGlnLeu ThrSerGly GlyAlaSer
450 455 460
Val Val CysPheLeu AsnAsn PheTyrPro LysAspIle AsnValLys
465 470 475 480
Trp Lys IleAspGly SerGlu ArgGlnAsn GlyValLeu AsnSerTrp
485 490 495
Thr Asp GlnAspSer LysAsp SerThrTyr SerMetSer SerThrLeu
500 505 510
Thr Leu-ThrLysAsp GluTyr GluArgHis AsnSerTyr ThrCysGlu
515 520 525
Ala Thr HisLysThr SerThr SerProIle ValLysSer PheAsnArg
530 535 540
Asn Glu Cys
545
<210>
pCRC187
<211>
253
<212>
PRT
<213>
Murine
<400>
17
Met Gly LysLysGln ThrAla ValAlaPhe AlaLeuAla LeuLeuAla
5$ 5 10 15
Leu Ser Met Thr Pro Ala Tyr Ala Phe Lys Asn Ile Val Thr Pro Arg
20 25 30
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Thr Pro Pro Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp
35 40 45
Ser Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys Tyr Phe Thr
$ 50 ~ 55 . 60
Asn Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile Tyr Asn Arg
65 70 75 80
Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val
85 90 95
Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gln Tyr Leu
100 105 110
Glu Gln Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu
115 120 125
Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Gly Gly Gly Gly Ser
130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Glu Asp Asp Ile Glu Ala Asp His Val Gly Val Tyr
165 170 175
Gly Thr Thr Val Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His'
180 185 190
Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu
195 200 205
Thr Ile Trp Met Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro
3$ 210 215 220
Gln Gly Gly Leu Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile
225 - 230 235 240
Leu Thr Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn
245 250
<210> pCB229
<211> 354
<212> PRT
<213> Murine
<400> 18
$0 Met Ala Leu Gln Ile Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Phe Lys Asn Ile Val
20 25 30
$$
Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly
35 40 45
Gly Gly Asp Ser Glu Arg His Phe Val Phe Gln Phe Lys Gly Glu Cys
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50 55 60
Tyr Phe Thr Asn Gly Thr Gln Arg Ile Arg Ser Val Asp Arg Tyr Ile
65 70 75 80
Tyr Asn ArgGlu GluTyrLeu ArgPheAsp SerAspVal GlyGluTyr
85 90 95
Arg Ala ValThr GluLeuGly ArgProAsp ProGluTyr TyrAsnLys
100 105 110
Gln Tyr LeuGlu GlnThrArg AlaGluLeu AspThrVal CysArgHis
115 120 125
1$ Asn Tyr GluGly ValGluThr HisThrSer LeuArgArg LeuGluGln
130 135 . 140
Pro Asn ValVal IleSerLeu SerArgThr GluAlaLeu AsriHisHis
145 150 155 160
Asn Thr LeuVal CysSerVal ThrAspPhe TyrProAla LysIleLys
165 170 175
Val Arg TrpPhe ArgAsnGly GlnGluGlu ThrValGly ValSerSer
2S 180 185 190
Thr Gln LeuIle ArgAsnGly AspTrpThr PheGlriVal LeuValMet
195 200 205
Leu Glu MetThr ProArgArg GlyGluVal TyrThrCys HisValGlu
210 215 220
His Pro SerLeu LysSerPro IleThrVal GluTrpArg AlaGlnSer
225 230 235 240
Glu Ser AlaArg SerGlySer AlaAspAla AlaProThr ValSerIle
245 250 255
Phe Pro ProSer SerGluGln LeuThrSer GlyGlyAla SerValVal
260 265 270
Cys Phe Leu,AsnAsnPheTyr ProLysAsp IleAsnVal LysTrpLys
275 280 285
Ile Asp GlySer GluArgGln AsnGlyVal LeuAsnSer TrpThrAsp
290 295 300
Gln Asp SerLys AspSerThr TyrSerMet SerSexThr LeuThrLeu
305 310 315 320
Thr Lys AspGlu TyrGluArg HisAsnSer TyrThrCys GluAlaThr
325 330 335,
His Lys ThrSer ThrSerPro IleValLys SerPheAsn ArgAsnGlu
$$ 340 345 350
Cys
CA 02403432 2002-09-23
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<210>
p/cb223
<211> 2
55
<212>
PRT
<213>
Murine
<400>
19
Met Ala LeuGlnIle ProSer LeuLeuLeu SerAlaAla ValVal Val
5 10 15
Leu Met ValLeuSer SerPro GlyThrGlu GlyGluAsp AspIle Glu
20 25 30
Ala Asp HisValGly ValTyr GlyThrThr ValTyrGln SerPro Gly
35 40 45
Asp Ile GlyGlnTyr ThrHis GluPheAsp GlyAspGlitTrpPhe Tyr
50 55 60
Val Asp LeuAspLys LysGlu ThrIleTrp MetLeuPro GluPhe Gly
65 70 75 80
Gln Leu ThrSerPhe AspPro GlnGlyGly LeuGlnAsn IleAla Thr
85 90 95
Gly Lys TyrThrLeu GlyIle LeuThrLys ArgSerAsn SerThr Pro
100 105 110
Ala Thr AsnGluAla ProGln AlaThrVal PheProLys SerPro Val
115 120 125
Leu Leu GlyGlnPro AsnThr LeuIleCys PheValAsp AsnIle Phe
130 135 140
Pro Pro ValIleAsn IleThr TrpLeuArg AsnSerLys SerVal Thr
145 150 155 160
Asp Gly-ValTyrGlu ThrSer PheLeuVal AsnArgAsp HisSer Phe
165 170 175
His Lys LeuSerTyr LeuThr PheIlePro SerAspAsp AspIle Tyr
180 185 190
Asp Cys LysValGlu HisTrp GlyLeuGlu GluProVal LeuLys His
195 200 205
Trp Glu ProGluIle ProAla ProMetSer GluGlySer AlaLys Thr
210 215 220
Thr Ala ProSerVal TyrPro LeuAlaPro ValCysGly AspThr Thr
225 230 235 240
Gly Ser SerValThr LeuGly CysLeuVal LysGlyTyr PhePro Glu
245 250. 255
$5
Pro Val ThrLeuThr TrpAsn SerGlySer LeuSerSer GlyVal His
260 265 270
Thr Phe ProAlaVal LeuGln SerAspLeu TyrThrLeu SerSer Ser
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275 280 285
Val Thr ValThrSer SerThrTrp ProSerGln SexIle ThrCysAsn
290 295 300
Val Ala HisProAla SerSerThr LysValAsp LysLys IleGluPro
305 310 315 , 320
Arg Gly ProThrIle LysProCys ProProCys LysCys ProAlaPro
325 330. 335
Asn Leu LeuGlyGly ProSerVal PheIlePhe ProPro LysIleLys
340 345 350
1$ Asp Val LeuMetIle SerLeuSer ProIleVal ThrCys ValValVal
355 360 365
Asp Val SerGluAsp AspProAsp ValGlnIle SerTrp PheValAsn
370 375 380
Asn Val GluValHis ThrAlaGln ThrGln~'hrHisArg GluAspTyr
385 390 395 400
Asn Ser ThrLeuArg ValValSer AlaLeuPro IleGln HisGlnAsp
2$ 405 410 ' 415
Trp Met SerGlyLys GluPheLys CysLysVal AsnAsn LysAspLeu
420 425 430
Pro Ala ProIleGlu ArgThrIle SerLysPro LysGly SerValArg
435 440 445
Ala Pro GlnValTyr ValLeuPro ProProGlu GluGlu MetThrLys
450 455 460
Lys Gln ValThrLeu ThrCysMet ValThrAsp PheMet ProGluAsp
465 470 475 480
Ile Tyr ValGluTrp ThrAsnAsn GlyLysThr GluLeu AsnTyr~Lys
485 490 495
Asn Thr GluProVal LeuAspSer AspGlySer TyrPhe MetTyrSer
500 505 510
4S Lys Leu ArgValGlu LysLysAsn TrpValGlu ArgAsn SerTyrSer
515 520 525
Cys Ser ValValHis GluGlyLeu HisAsnHis HisThr ThrLysSer
530 535 540
Phe Ser ArgThrPro GlyLys
545 550
$5 <210> pCB212
<211> 331
<212> PRT
<213> Murine
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
125
<400> 20
Met Ala Leu Gln Ile Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Glu Asp Asp Ile Glu
20 25 30
Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gln Ser Pro Gly
35 40 45
Asp Ile Gly Gln Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr
50 55 60
Val Asp Leu Asp Lys Lys Glu Thr Ile Trp Met Leu Pro Glu Phe Gly
65 70 75 80
Gln Leu Thr Ser Phe Asp Pro Gln Gly Gly Leu Gln Asn Ile Ala Thr
85 90 95
Gly Lys Tyr Thr Leu Gly Ile Leu Thr Lys Arg Ser Asn Ser Thr Pro
100 105 110
Ala Thr Asn Glu Ala Pro Gln Ala Thr Val Phe Pro Lys Ser Pro Val
115 120 125
Leu Leu Gly Gln Pro Asn Thr Leu Ile Cys Phe Val Asp Asn Ile Phe
130 135 140
Pro Pro Val Ile Asn Ile Thr Trp Leu Arg Asn Ser Lys Ser Val Thr
145 150 155 160
Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp His Ser Phe
165 170 175
3$ His Lys Leu Ser Tyr Leu Thr Phe Ile Pro Ser Asp Asp Asp Ile Tyr
180 185 190
Asp Cys~Lys Val Glu His Trp Gly Leu Glu Glu Pro Val Leu Lys His
195 200 205
Trp Glu Pro Glu Ile Pro Ala Pro Met Ser Glu Gly Ser Ala Lys Thr
210 215 220
Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr
4S 225 230 235 240
Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu
245 250 255
$0 Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His
260 265 270
Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser
275 280 285
$$
Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn
290 295 300
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro
CA 02403432 2002-09-23
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126
'305 310 315 320
Arg Gly Pro Thr Ile Lys Pro Cys Ala Ala
325 330
<210> pCB214
<211> 234
<212> PRT
<213> Murine
<400> 21
Met Phe Lys IleVal Thr ArgThr Pro Pro AlaSer
Asn Pro Pro Gly
5 10 15
1$
Gly Gly Gly GlyGly Gly AspSer Glu Arg PheVal
Ser Gly His Phe
25 30
Gln Phe Lys GluCys Tyr ThrAsn Gly Thr ArgIle
Gly Phe Gln Arg
35 40 45
Ser Val Asp Arg Tyr Ile Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp
50 55 60
2$ Ser Asp ValGly GluTyrArg AlaValThr GluLeuGly ArgPro Asp
65 70 75 80
Pro Glu TyrTyr AsnLysGln TyrLeuGlu GlnThrArg AlaGlu Leu
85 90 95
Asp Thr ValCys ArgHisAsn TyrGluGly ValGluThr HisThr Ser
100 105 110
Leu Arg ArgLeu GlyGlyGly GlySerGly GlyGlyGly SerGly Gly
3$ 115 120 125
Gly Gly SerGly GlyGlyGly SerGlyGly GlyGlySer GluAsp Asp
130 - 135 140
Ile Glu AlaAsp HisValGly ValTyrGly ThrThrVal TyrGln Ser
145 I50 I55 260
Pro Gly AspIle GlyGlnTyr ThrHisGlu PheAspGly AspGlu Trp
165 170 175
4$
Phe Tyr ValAsp LeuAspLys LysGluThr IleTrpMet LeuPro Glu
180 185 190
Phe Gly GlnLeu ThrSerPhe AspProGln GlyGlyLeu GlnAsn Ile
$0 195 200 205
Ala Thr GlyLys TyrThrLeu GlyIleLeu ThrLysArg SerAsn Ser
210 215 220
$$ Thr Pro AlaThr AsnGlyThr GlySer
225
<210> pCB220
CA 02403432 2002-09-23
WO 01/70245 PCT/USO1/09616
I27
<211>
560
<212>
PRT
<213>
Murine
<400>
22
Met Ala LeuGlnIle ProSer LeuLeuLeu SerAla AlaValVal Val
5 10 15
Leu Met ValLeuSer SerPro GlyThrGlu GlyPhe LysAsnIle Val
IO 20 25 30
Thr Pro ArgThrPro ProPro AlaSerGly GlyGly GlySerGly Gly
35 40 45
I5 GIy Gly AspSerGlu ArgHis PheValPhe GlnPhe LysGlyGlu Cys
50 55 60
Tyr Phe ThrAsnGly ThrGln ArgIleArg SerVal AspArgTyr Ile
65 70 75 80
20
Tyr Asn ArgGluGlu TyrLeu ArgPheAsp SerAsp ValGlyGlu Tyr
85 90 95
Arg Ala ValThrGlu LeuGly ArgProAsp ProGlu TyrTyrAsn Lys
25 100 105 110
Gln Tyr LeuGluGln ThrArg AlaGluLeu AspThr ValCysArg His
115 120 125
30 Asn Tyr GluGlyVal .GluThr HisThrSer LeuArg ArgLeuGlu Gln
130 135 140
Pro Asn ValValIle SerLeu SerArgThr GluAla LeuAsnHis His
145 150 155 160
35
Asn Thr LeuValCys SerVal ThrAspPhe TyrPro AlaLysIle Lys
165 170 175
Val Arg TrpPheArg AsnGly GlnGluGlu ThrVal GlyValSer Ser
40 180 185 190
Thr Gln LeuIleArg AsnGly AspTrpThr PheGln ValLeuVal Met
195 200 205
45 Leu Glu MetThrPro ArgArg GlyGluVal TyrThr CysHisVal Glu
210 215 220
His Pro Ser Leu Lys Ser Pro Ile Thr Val Glu Trp Thr Ser Gly Gly
225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
245 250 255
Gly Ser Ser Ser Glu Asp Asp Ile Glu Ala Asp His Val Gly Val Tyr
260 265 270
Gly Thr Thr Val Tyr Gln Ser Pro Gly Asp Ile Gly Gln Tyr Thr His
275 280 285
CA 02403432 2002-09-23
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128
Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu
290 295 300
Thr Ile Trp Met Leu Pro Glu Phe Gly Gln Leu Thr Ser Phe Asp Pro
305 310 315 320
Gln Gly Gly Leu Gln Asn Ile Ala Thr Gly Lys Tyr Thr Leu Gly Ile
325 330 335
Leu Thr Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gln
340 345 350
Ala Thr Val Phe Pro Lys Ser Pro Val Leu Leu Gly Gln Pro Asn Thr
355 360 365
Leu Ile Cys Phe Val Asp Asn Ile Phe Pro Pro Val Ile Asn Ile Thr
370 375 380
Trp Leu Arg Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser
2~ 385 390 395 400
Phe Leu Val Asn Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr
405 410 415
2S Phe Ile Pro Ser Asp Asp Asp Ile Tyr Asp Cys Lys Val Glu His Trp
420 425 430
Gly Leu Glu Glu Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly
435 440 445
Ser Gly Gly Gly Gly Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro
450 455 460
Pro Ser Sex Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe
465 470 475 480
Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp
485 490 495
4~ Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp
500 505 510
Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys
515 520 525
Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys
530 535 540
Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
545 550 555
